Yuling Liu

Transdermal delivery of insulin using microneedle rollers in vivo.

This study characterizes skin perforation by commercially available microneedle rollers and evaluates the efficacy of transdermal delivery of insulin to diabetic rats. Three different needle lengths, 250, 500 and 1000 microm, were used in this work. Creation and resealing of the skin holes that were produced by the needles were observed by Evans blue (EB) staining and transepidermal water loss (TEWL) measurements. EB clearly showed that microchannels were formed in the skin and that the pores created by the longest microneedle (1000 microm) persisted no longer than 8h, while the hypodermic injury was still observed 24h later. TEWL significantly increased after the application of the needles and then decreased with time, which explains the recovery of skin barrier function and agrees well with EB results. The extent of permeation was demonstrated by insulin delivery in vivo. The rapid reduction of blood glucose levels in 1h was caused by the increased permeability of the skin to insulin after applying microneedle rollers. The reduced decrease after 1h is closely associated with hole recovery. In conclusion, microneedle rollers with 500-microm or shorter lengths are safe and useful in transdermal delivery of insulin in vivo.

Self-assembled lecithin/chitosan nanoparticles for oral insulin delivery: preparation and functional evaluation

Purpose Here, we investigated the formation and functional properties of self-assembled lecithin/chitosan nanoparticles (L/C NPs) loaded with insulin following insulin–phospholipid complex preparation, with the aim of developing a method for oral insulin delivery. Methods Using a modified solvent-injection method, insulin-loaded L/C NPs were obtained by combining insulin–phospholipid complexes with L/C NPs. The nanoparticle size distribution was determined by dynamic light scattering, and morphologies were analyzed by cryogenic transmission electron microscopy. Fourier transform infrared spectroscopy analysis was used to disclose the molecular mechanism of prepared insulin-loaded L/C NPs. Fast ultrafiltration and a reversed-phase high-performance liquid chromatography assay were used to separate free insulin from insulin entrapped in the L/C NPs, as well as to measure the insulin-entrapment and drug-loading efficiencies. The in vitro release profile was obtained, and in vivo hypoglycemic effects were evaluated in streptozotocin-induced diabetic rats. Results Our results indicated that insulin-containing L/C NPs had a mean size of 180 nm, an insulin-entrapment efficiency of 94%, and an insulin-loading efficiency of 4.5%. Cryogenic transmission electron microscopy observations of insulin-loaded L/C NPs revealed multilamellar structures with a hollow core, encircled by several bilayers. In vitro analysis revealed that insulin release from L/C NPs depended on the L/C ratio. Insulin-loaded L/C NPs orally administered to streptozotocin-induced diabetic rats exerted a significant hypoglycemic effect. The relative pharmacological bioavailability following oral administration of L/C NPs was 6.01%. Conclusion With the aid of phospholipid-complexation techniques, some hydrophilic peptides, such as insulin, can be successfully entrapped into L/C NPs, which could improve oral bioavailability, time-dependent release, and therapeutic activity.

Targeted Data-Independent Acquisition and Mining Strategy for Trace Drug Metabolite Identification Using Liquid Chromatography Coupled with Tandem Mass Spectrometry.

Detection and identification of unknown or low-level drug-related metabolites in complex biological materials is an ongoing challenge. A highly selective and sensitive method could be a possible solution. Here, we proposed a targeted data-independent acquisition and mining (TDIAM) strategy for the rapid identification of trace drug metabolites using ultra-high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry (UHPLC-HRMS/MS). In this strategy, raw data is acquired by a novel tm-MS scan, which contains an interleaved full MS scan with a targeted mass range and a product ion scan by selecting all ions in the targeted mass range as precursor ions. For efficient discovery of metabolites, raw data are analyzed by a new postacquisition processing method, Molecule- and Fragmentation-driven Mass Defect Filters (MF-MDFs), which was developed based on the fragmentation of parent drug to pick out molecular ions and fragment ions of potential metabolites from the complex matrix. When applying the proposed strategy to paclitaxel metabolism research, we successfully identified 10 metabolites, among which six were not previously reported. The results demonstrated that TDIAM greatly improved throughput, detective sensitivity, and selectivity and, more importantly, yielded almost the same spectrum as traditional HRMS/MS. Therefore, TDIAM provides structure-enriched evidence to confirm the existence and elucidate the structures of metabolites. This strategy is suitable for identification of metabolites present at low concentrations in a complex matrix, and it has the potential to provide an efficient, sensitive, and labor-saving solution for drug metabolite research.

Thermal and magnetic dual-responsive liposomes with a cell-penetrating peptide-siRNA conjugate for enhanced and targeted cancer therapy

Due to the absence of effective in vivo delivery systems, the employment of small interfering RNA (siRNA) in the clinic has been hindered. Here, we describe a novel siRNA targeting system that combines features of biological (cell-permeable peptides, CPPs) and physical (magnetic) siRNA targeting for use in magnetic hyperthermia-triggered release. A siRNA-CPPs conjugate (siRNA-CPPs) was loaded into thermal and magnetic dual-responsive liposomes (TML) (siRNA-CPPs/TML), and in vitro siRNA-CPPs thermosensitive release activity, targeted cellular uptake, gene silencing efficiency, in vivo targeted delivery and in vivo antitumor activity were determined. The results demonstrated that siRNA-CPPs/TML exhibited good physicochemical properties, effective cellular uptake, endosomal escape and a significant gene silencing efficiency in MCF-7 cells in vitro. Additionally, in the in vivo study, siRNA-CPPs/TML under an alternating current (AC) magnetic field displayed a superior in vivo targeted delivery efficacy, antitumor efficacy and gene silencing efficiency in a MCF-7 xenograft murine model. In conclusion, the application of siRNA-CPPs/TML under an AC magnetic field represents a new strategy for the selective and efficient delivery of siRNA.

Simultaneous quantification of three active alkaloids from a traditional Chinese medicine Ramulus Mori (Sangzhi) in rat plasma using liquid chromatography-tandem mass spectrometry.

Fagomine, 1-deoxynojirimycin (DNJ) and 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) are the major bioactive constituents in the active fraction of alkaloids from the traditional Chinese medicine mulberry twig (Ramulus Mori, Chinese name Sang Zhi), which has a strong activity on α-glucosidase in vitro and in vivo. A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of DNJ, fagomine and DAB in rat plasma. Plasma samples were prepared using a simple protein precipitation by the addition of 1% volume of Tris and two volumes of methanol-acetonitrile. The analytes and internal standard (IS, miglitol) were chromatographed in an XBridge™ amide column with a gradient mobile phase of acetonitrile-water (0.1% ammonium hydroxide) at a flow rate of 0.7mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI) source in positive ion mode by multiple reaction monitoring (MRM) mode. Linear detection responses were obtained for DNJ ranging from 5.00 to 5000.00ng/mL, 10.00 to 2500.00ng/mL for fagomine and DAB. The lower limits of quantification (LLOQs) were 5.00, 10.00, 10.00ng/mL for DNJ, fagomine and DAB, respectively. Intra-day and inter-day precisions (R.S.D.%) were within 10% for three analytes with accuracies (R.E.%) less than 12%. The mean recoveries of analytes were greater than 85%. All analytes were proved to be stable during the sample storage, preparation and analytic procedures. The method was successfully applied to the pharmacokinetic study of the three alkaloids in rats after oral administration of the active fraction of alkaloids from mulberry twig.

Co-encapsulation of paclitaxel and baicalein in nanoemulsions to overcome multidrug resistance via oxidative stress augmentation and P-glycoprotein inhibition.

Multidrug resistance (MDR) is a major obstacle for clinical application of paclitaxel (PTX). Recent studies have suggested that baicalein (BA) might be a potent MDR reversal agent with the ability of P-glycoprotein inhibition and oxidative stress augmentation. Herein, we co-encapsulated PTX and BA in nanoemulsions (PTX/BA NE) for overcoming MDR in breast cancer. Paclitaxel-cholesterol complex and baicalein-phospholipid complex were prepared to improve the liposolubility of PTX and BA. The cytotoxicity of the combination of PTX and BA with different weight ratios were evaluated and the combination with a weight ratio of 1/1 exhibited the strongest synergistic effect. In vitro cytotoxicity study indicated that PTX/BA NE had a better antitumor efficacy in MCF-7/Tax cells than other PTX formulations. Studies on cellular uptake demonstrated that the PTX/BA NE could effectively accumulate in cancer cells. Mechanism research showed that PTX/BA NE could significantly increase the cellular reactive oxygen species (ROS), decrease cellular glutathione (GSH), and enhance caspase-3 activity in MCF-7/Tax cells. More importantly, in vivo antitumor study demonstrated that PTX/BA NE exhibited a much higher antitumor efficacy than other PTX formulations. These findings suggest that co-delivery of PTX and BA in nanoemulsions might provide us a potential combined therapeutic strategy for overcoming MDR.

The preparation of a complex of insulin-phospholipids and their interaction mechanism.

Subcutaneous injections of insulin remain the standard treatment for insulin‐dependent diabetic patients, and noninvasive routes are studied but with little success. One of the reasons is that insulin is a hydrophilic compounds and is difficult to cross the mucosa barrier. In this paper, we developed a novel technique to fabricate the insulin–phospholipids complex by a solvent evaporation method with the aim of improving the lipophilicity of insulin. A systematic study on the preparation conditions of the insulin–phospholipids complex is reported in the present work. The formation mechanism and the physicochemical properties of the complex are studied. The associated efficiency of the phospholipids and insulin can be up to 100% when their mass ratio is 7.5 : 1 or more, and the solubility of the complex is improved more than 40 000 times compared with that of insulin alone in the n‐octyl alcohol. The results of the insulin content in the complex and hypoglycemic effects in diabetic mice indicated that insulin was able to withstand the preparation procedure. The stability results showed that the complex was stable for 1 year at −20 °C. The interaction mechanism of this formation is that the peptide bonds of insulin interact with the water‐soluble head of phospholipids, forming a reverse micelle‐like structure. This novel complex will be of great importance in the drug delivery system for insulin via noninvasive routes. This method is cost effective, scalable, and can be used in many other peptide drugs. Copyright

Acid Sensitive Polymeric Micelles Combining Folate and Bioreducible Conjugate for Specific Intracellular siRNA Delivery.

An efficiently siRNA transporting nanocarrier still remains to be developed. In this study, utilizing the dual stimulus of acid tumor extracellular environment and redox effect of glutathione in the cytosol, a new siRNA transporting system combining triple effects of folate targeting, acid sensitive polymer micelles, and bio-reducible disulfide bond linked siRNA-cell penetrating peptides (CPPs) conjugate is developed to suppress c-myc gene expression of breast cancer (MCF-7 cells) both in vitro and in vivo. Subsequent research demonstrates that the vesicle has particle size of about 100 nm and siRNA entrapment efficiency of approximately 80%. In vitro studies verified over 90% of encapsulated siRNA-CPPs can be released and the vesicle shows higher cellular uptake in response to the tumorous zone. Determination of gene expression at both mRNA and protein levels indicates the constructed vesicle exhibited enhanced cancer cell apoptosis and improved therapeutic efficacy in vitro and in vivo.

Purification of Houttuynia cordata Thunb. Essential Oil Using Macroporous Resin Followed by Microemulsion Encapsulation to Improve Its Safety and Antiviral Activity

Essential oil extracted from Houttuynia cordata Thunb. (H. cordata) is widely used in traditional Chinese medicine due to its excellent biological activities. However, impurities and deficient preparations of the essential oil limit its safety and effectiveness. Herein, we proposed a strategy to prepare H. cordata essential oil (HEO) safely and effectively by combining the solvent extraction and the macroporous resin purification flexibly, and then encapsulating it using microemulsion. The extraction and purification process were optimized by orthogonal experimental design and adsorption-desorption tests, respectively. The average houttuynin content in pure HEO was then validated at 44.3% ± 2.01%, which presented a great potential for industrial application. Subsequently, pure HEO-loaded microemulsion was prepared by high-pressure homogenization and was then fully characterized. Results showed that the pure HEO-loaded microemulsion was successfully prepared with an average particle size of 179.1 nm and a high encapsulation rate of 94.7%. Furthermore, safety evaluation tests and in vitro antiviral testing indicated that the safety and activity of HEO were significantly improved after purification using D101 resin and were further improved by microemulsion encapsulation. These results demonstrated that the purification of HEO by macroporous resin followed by microemulsion encapsulation would be a promising approach for industrial application of HEO for the antiviral therapies.

A novel matrix dispersion based on phospholipid complex for improving oral bioavailability of baicalein: preparation, in vitro and in vivo evaluations

Abstract Phospholipid complex is one of the most successful approaches for enhancing oral bioavailability of poorly absorbed plant constituents. But the sticky property of phospholipids results in an unsatisfactory dissolution of drugs. In this study, a matrix dispersion of baicalein based on phospholipid complex (BaPC-MD) was first prepared by a discontinuous solvent evaporation method, in which polyvinylpyrrolidone-K30 (PVP-K30) was employed for improving the dispersibility of baicalein phospholipid complex (BaPC) and increasing dissolution of baicalein. The combination ratio of baicalein and phospholipids in BaPC-MD was 99.39% and baicalein was still in a complete complex state with phospholipid in BaPC-MD. Differential scanning calorimetry (DSC), X-ray diffraction (XRD), scanning electron microscopy (SEM) and Fourier Transform Infrared (FTIR) analyzes demonstrated that baicalein was fully transformed to an amorphous state in BaPC-MD and phospholipid complex formed. The water-solubility and n-octanol solubility of baicalein in BaPC-MD significantly increased compared with those of pure baicalein. Compared with baicalein and BaPC, the cumulative dissolution of BaPC-MD at 120 min increased 2.77- and 1.23-fold, respectively. In vitro permeability study in Caco-2 cells indicated that the permeability of BaPC-MD was remarkably higher than those of baicalein and BaPC. Pharmacokinetic study showed that the average Cmax of BaPC-MD was significantly increased compared to baicalein and BaPC. AUC0–14 h of BaPC-MD was 5.01- and 1.91-fold of baicalein and BaPC, respectively. The novel BaPC-MD significantly enhanced the oral bioavailability of baicalein by improving the dissolution and permeability of baicalein without destroying the complexation state of baicalein and phospholipids. The current drug delivery system provided an optimal strategy to significantly enhance oral bioavailability for poorly water-soluble drugs.