Mingzhi Zhao

16 Discrimination of Brazilian green propolis and Chinese propolis based on high-performance liquid chromatographic fingerprints and multivariate statistical analysis

Objectives The determination of chemical components is usually used in the quality control of propolis. However, chemical components from different types of propolis are similar. The objective of this investigation was to establish a method based on a specific chemical fingerprint profile and a multivariate mixed model statistical analysis which could easily distinguish propolis of different origins and promote the quality control of propolis. Methods A novel approach using high performance liquid chromatography (HPLC) coupled with multivariate statistical analysis was established for profiling and distinguishing Chinese and Brazilian green propolis. A batch of 22 propolis samples was analyzed, and the datasets on retention time, peak area and sample codes were subjected to mixed multivariate statistical analysis consisting of principal component analysis (PCA) and a self-organization mapping net (SOM). Results The fingerprints were profiled. PCA score plots showed Chinese and Brazilian green propolis clearly classified into two groups. The visualized SOM results showed data from the two groups projected to the adjacent neurons clearly separated from each other. Artepillin C, which contributed greatly to the differentiation, was screened out and identified as the reference compound. Artepillin C is the characteristic component in Brazilian propolis which can be used as chemical marker to distinguish propolis of different origins. Conclusions In this study, fingerprints coupled with multivariate statistical analysis have been successfully applied to distinguish Chinese from Brazilian green propolis. The research identified a chemical marker, and thus helps to investigate and promote the quality control of propolis.

46 Sandwich-type ethylcellulose films for controlled release of anti-restenosis drugs

Objectives Restenosis is a response of the vessel wall to balloon-induced injury and is characterized primarily by elastic recoil of the vessel wall and a series of pathological processes including thrombus, inflammation and vascular smooth muscle cell (VSMC) proliferation. To treat restenosis, appropriate drug delivery vehicles are needed which can release therapeutic agents targeting different symptoms into blood vessels in a controlled manner. The main objective of the present study was to prepare sandwich-type ethyl cellulose films with high performance for efficient drug loading and controlled drug release for restenosis treatment. Methods Sandwich-type ethyl cellulose films loaded with probucol for treating coronary artery disease, or aspirin as an antithrombotic drug, were prepared by casting three individual layers in sequence using an ethyl cellulose/toluene solution. On a glass plate, the first ethyl cellulose layer (bottom layer) was cast without drugs, on to which the middle layer containing probucol or aspirin was then cast. After solvent evaporation at room temperature, a third top layer was cast on to the middle layer. The obtained drug-loading films were further dried at room temperature under vacuum. Results The sandwich-type ethyl cellulose films exhibited a drug loading content (DLC) of 12.1 ± 0.9% and a drug loading efficiency (DLE) of 73.5 ± 3.6% for aspirin, and a DLC of 11.0 ± 0.8% and a DLE of 69.3 ± 3.4% for probucol. Under physiological conditions (37°C, pH 7.4), the release half-life of aspirin from the films was 2.7 ± 0.2 hours, while that of probucol was 7.1 ± 0.6 days. The two drugs showed totally different release behaviors, which can be employed in combination to treat restenosis. Conclusion Sandwich-type ethylcellulose films loaded with probucol or aspirin were successfully prepared and showed ability to release the two drugs in different ways: rapid release of aspirin to treat thrombus and inflammation typical of early-stage restenosis, and sustained release of probucol for inhibition of VSMC proliferation frequently seen in the later stage of restenosis. These drug-loaded ethylcellulose films provide new insight into restenosis therapy.

44 Core-shell nanospheres for pH-responsive release of anticancer drugs and near-infrared imaging

Objectives Nanoscaled drug carriers with pH-responsiveness have attracted extensive interest in view of the acidic environment in cancerous cells. Rapid response to pH changes plays a key role in efficient intracellular drug release. In addition, real-time tracking of drug carriers is important for understanding distribution and targeted accumulation of the drug carriers. This work aims at developing silver selenide quantum dots (Ag2Se QDs)@carboxymethyl chitosan (CMCS) core-shell nanospheres with encapsulated paclitaxel (PTX) for cancer therapy and bioimaging. Methods Oleic acid-capping Ag2 Se QDs were synthesized by a one-pot strategy, washed with ethanol, and obtained by centrifugation. The as-synthesized Ag2Se QDs were reacted with N-hydroxysuccinimide and conjugated with CMCS at the amino sites. In an aqueous solution of PTX, the hydrophobic oleoyl groups tended to aggregate locally and entrap PTX by hydrophobic interaction, spontaneously producing Ag2Se QDs (PTX)@CMCS nanospheres. Results By conjugating the oleic acid-capping Ag2 Se QDs with pH-sensitive CMCS at a degree of substitution (DS) of 13%, biocompatible core-shell nanospheres loaded with PTX were successfully prepared, which had an average size of 36.3 ± 0.2 nm. The drug loading content (DLC) and drug loading efficiency (DLE) for the PTX was 5.01 ± 0.8% and 52.4 ± 3.2%, respectively. The PTX release half-life was 4.1 hours under conditions resembling the intracellular environment of cancerous cells (37°C, pH 5.0). Conclusions Core-shell structured Ag2Se QDs (PTX)@CMCS nanospheres capable of releasing PTX in an acidic environment and emitting NIR fluorescence under NIR laser excitation were synthesized and characterized. The hydrophobic oleoyl groups entrapped PTX via hydrophobic interaction and the oleoyl-CMCS chains were extended at lowered pH to release the otherwise encaged drug. In addition, the encapsulated Ag2Se QDs can emit bright NIR fluorescence for bioimaging by which nanosphere distribution in a patient can be monitored. This study provides a new approach for developing nanocomposite drug carriers for cancer therapy.

47 Gambogic acid delivery using lipid nanoparticles modified with cell-penetrating peptide

Objectives Gambogic acid (GA) is a novel tissue-specific proteasome inhibitor which can potentially be used to treat cancer with low toxicity. However, poor aqueous solubility (∼10 μg/mL) and low tumor cell-specific delivery have limited its clinical application. Clinical application of GA requires the development of delivery vehicles. Methods In this study, we developed a novel nanoparticle GA delivery system. The nanoparticles incorporate a cell-penetrating peptide conjugated to myristic acid (MA-R7W), a folate modified lipid (FA-PEG2000-DSPE), a pH-sensitive lipid (PEG1000-hyd-PE), eggPC and cholesterol. The lipids formed the nanoparticle shells, and GA was loaded into the lipid bilayer of the nanoparticles. PEG on the surface of the nanoparticles provides a long circulation time. Folate is incorporated to enable targeting of tumor cells with amplified folate receptor expression. PEG1000-hyd-PE can shield/unshield R7W on the nanoparticle surface according to the pH difference between normal tissues and cancer. Results In vitro, FA/MA-R7W nanoparticles improved cellular uptake 2.5-fold compared to GA liposomes (without FA-PEG2000-DSPE, AA-R8 and PEG1000-hyd-PE) at pH 5. In vivo, GA encapsulated in FA/MA-R7W nanoparticles induced potent tumor inhibition (62.6%), showed lengthy circulation (Figure 1) and tumor cell targeting.Abstract 47 Figure 1 Plasma concentration-time curves in rats for FA/MA-R7W nanoparticles and free gambogic acid (1 mg/kg) Conclusions In conclusion, FA/MA-R7W nanoparticles are promising vehicles for GA delivery and warrant further investigation. Acknowledgments This research was financially supported by Jilin Province Science and Technology Development Program (Grant No. 20140311072YY) and Jilin Province Science and Technology Development Program (Grant No.20150520141JH).

17 Delivery of betulinic acid lipid nanoparticles assembled by a microfluidic device

Background Microfluidics chip-based approaches (MF) can achieve unique transport properties through laminar flows and vastly increased surface-to-volume ratios, which have been extensively utilized to prepare smaller and homogeneous lipid nanoparticles (LNPs). LNPs have shown potential to carry highly insoluble medicines, especially betulinic acid (BA) which has significant antitumour activity but is difficult to administer for cancer therapy due to its poor water solubility. In this study, we investigated the parameters involved in the continuous production of LNPs encapsulating BA (BA-LNPs) by MF, and the possibility of improving antitumour efficacy and reducing toxicities was described. Methods Briefly, a three-inlet MF system was developed and used to produce the LNPs. EggPC, cholesterol and BA (45:37:18% molar) were dissolved in ethanol. The lipid solution was then loaded into a 1 mL glass syringe and injected into the centre inlet channel, while phosphate-buffered saline (PBS; pH 6.5) was loaded into two 5 mL glass syringes and introduced into the two side inlet channels to establish hydrodynamic focusing. To investigate the physicochemical and biological properties of fabricated liposome at different shear forces, the total flow rate (TFR) is varied from 0.3 to 0.8 mL/min. Results The TFR has a very small effect on particle size distribution, but an increase in TFR causes a progressive decrease in liposome size. The percentage of encapsulating efficiency (EE) was 77–92% and there were no significant changes when BA-LNPs were stored at 4° C. The inhibitory rate of BA-LNPs was significantly higher compared to free BA in vitro. Immunohistochemical analysis showed many damaged tumor cells after BA-LNPs were injected for 15 days. The survival of mice treated with BA-LNPs was apparently prolonged compared to mice treated with free BA. Conclusions This result indicates that much stronger antitumour effects were induced by BA-LNP administration, which is most likely due to the relatively small particle sizes produced by MF and which are suitable for intracellular transportation.