Tanshinone IIA-Loaded Micelles Functionalized with Rosmarinic Acid: A Novel Synergistic Anti-Inflammatory Strategy for Treatment of Atherosclerosis.

Rosmarinic acid (RA) and tanshinone IIA (TA) which are effective components in Salvia miltiorrhiza show anti-inflammatory potential against atherosclerosis. Based on polysulfated propylene-polyethylene glycol (PPS-PEG), RA was grafted onto this polymer via amide bonds to form a micelle carrier for TA encapsulation: PPS-PEG-RA@TA. A potent inhibitory effect on lipopolysaccharide (LPS) -induced proliferation of endothelial cells with significant intracellular uptake was observed with this system. This could have been the result of release of TA in a reactive oxygen species (ROS) environment and stronger antioxidant effect of RA. The synergistic effect was optimized when the combination was used in a molar ratio of 1:1. Mechanistic studies showed that, compared with PPS-PEG-RA and TA+RA, PPS-PEG-RA@TA micelles could more effectively regulate the nuclear factor-kappa B (NF-κB) pathway to reduce expression of vascular cell adhesion molecule-1 (VCAM-1), inhibit the inflammatory cascade and reduce endothelial-cell injury. One month after intravenous injection of PPS-PEG-RA@TA micelles, the plaque area in murine aortic vessels was reduced significantly, and serious toxic side-effects were not observed in vivo, along with excellent biocompatibility. In summary, PPS-PEG-RA@TA micelles could achieve synergistic treatment of atherosclerosis.

Trastuzumab- and Fab' fragment-modified curcumin PEG-PLGA nanoparticles: preparation and evaluation in vitro and in vivo.

INTRODUCTION: Nanoparticles (NPs) modified with bio-ligands represent a promising strategy for active targeted drug delivery to tumour. However, many targeted ligands, such as trastuzumab (TMAB), have high molecular weight, limiting their application for targeting. In this study, we prepared Fab' (antigen-binding fragments cut from TMAB)-modified NPs (Fab'-NPs) with curcumin (Cur) as a model drug for more effective targeting of human epidermal growth factor receptor 2 (HER2/ErbB2/Neu), which is overexpressed on breast cancer cells. MATERIAL AND METHODS: The release kinetics was conducted by dialysis bags. The ability to kill HER2-overexpressing BT-474 cells of Fab'-Cur-NPs compared with TMAB-Cur-NPs was conducted by cytotoxicity experiments. Qualitative and quantitative cell uptake studies using coumarin-6 (fluorescent probe)-loaded NPs were performed by fluorescence microscopy and flow cytometry. Pharmacokinetics and biodistribution experiments in vivo were assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: The release kinetics showed that both Fab'-Cur-NPs and TMAB-Cur-NPs provided continuous, slow release of curcumin for 72 h, with no significant difference. In vitro cytotoxicity experiments showed that Fab'-Cur-NPs manifested prominent ability to kill HER2-overexpressing BT-474 cells compared with TMAB-Cur-NPs. Qualitative and quantitative cell uptake studies indicated that the accumulation of Fab'-NPs was greater than that of TMAB-NPs in BT-474 (HER2+) cells; However, there was no significant difference in MDA-MB-231 (HER2-) cells. Pharmacokinetics and biodistribution experiments in vivo demonstrated that the half-life (t1/2) and area under the blood concentration-time curve (AUC0-t) of Fab'-Cur-NPs increased 5.30-fold and 1.76-fold relative to those of TMAB-Cur-NPs, respectively. Furthermore, the tumor accumulation of Fab'-Cur-NPs was higher than that of TMAB-Cur-NPs. CONCLUSION: Fab' fragment has greater capacity than the intact antibody to achieve tumor targeting through NP-based delivery.

Intranasal delivery of Huperzine A to the brain using lactoferrin-conjugated N-trimethylated chitosan surface-modified PLGA nanoparticles for treatment of Alzheimer's disease.

BACKGROUND: Safe and effective delivery of therapeutic drugs to the brain is important for successful therapy of Alzheimer's disease (AD). PURPOSE: To develop Huperzine A (HupA)-loaded, mucoadhesive and targeted polylactide-co-glycoside (PLGA) nanoparticles (NPs) with surface modification by lactoferrin (Lf)-conjugated N-trimethylated chitosan (TMC) (HupA Lf-TMC NPs) for efficient intranasal delivery of HupA to the brain for AD treatment. METHODS: HupA Lf-TMC NPs were prepared using the emulsion-solvent evaporation method and optimized using the Box-Behnken design. The particle size, zeta potential, drug entrapment efficiency, adhesion and in vitro release behavior were investigated. The cellular uptake was investigated by fluorescence microscopy and flow cytometry. MTT assay was used to evaluate the cytotoxicity of the NPs. In vivo imaging system was used to investigate brain targeting effect of NPs after intranasal administration. The biodistribution of Hup-A NPs after intranasal administration was determined by liquid chromatography-tandem mass spectrometry. RESULTS: Optimized HupA Lf-TMC NPs had a particle size of 153.2±13.7 nm, polydispersity index of 0.229±0.078, zeta potential of +35.6±5.2 mV, drug entrapment efficiency of 73.8%±5.7%, and sustained release in vitro over a 48 h period. Adsorption of mucin onto Lf-TMC NPs was 86.9%±1.8%, which was significantly higher than that onto PLGA NPs (32.1%±2.5%). HupA Lf-TMC NPs showed lower toxicity in the 16HBE cell line compared with HupA solution. Qualitative and quantitative cellular uptake experiments indicated that accumulation of Lf-TMC NPs was higher than nontargeted analogs in 16HBE and SH-SY5Y cells. In vivo imaging results showed that Lf-TMC NPs exhibited a higher fluorescence intensity in the brain and a longer residence time than nontargeted NPs. After intranasal administration, Lf-TMC NPs facilitated the distribution of HupA in the brain, and the values of the drug targeting index in the mouse olfactory bulb, cerebrum (with hippocampus removal), cerebellum, and hippocampus were about 2.0, 1.6, 1.9, and 1.9, respectively. CONCLUSION: Lf-TMC NPs have good sustained-release effect, adhesion and targeting ability, and have a broad application prospect as a nasal drug delivery carrier.

IR-780-loaded polymeric micelles enhance the efficacy of photothermal therapy in treating breast cancer lymphatic metastasis in mice.

Cancer metastasis is responsible for over 90% of breast cancer-related deaths, and inhibiting lymph node metastasis is an option to treat metastatic disease. Herein, we report the use of IR-780-loaded polymeric micelles (IPMs) for effective photothermal therapy (PTT) of breast cancer lymphatic metastasis. The IPMs were nanometer-sized micelles with a mean diameter of 25.6 nm and had good stability in simulated physiological solutions. Under 808-nm laser irradiation, IPMs exhibited high heat-generating capability in both in vitro and in vivo experiments. After intravenous injection, IPMs specifically accumulated in the tumor and metastatic lymph nodes and penetrated into these tissues. Moreover, a single IPMs treatment plus laser irradiation significantly inhibited primary tumor growth and suppressed lymphatic metastasis by 88.2%. Therefore, IPMs are an encouraging platform for PTT applications in treatment of metastatic breast cancer.

Colon-Targeted Delivery of IgY Against Clostridium difficile Toxin A and B by Encapsulation in Chitosan-Ca Pectinate Microbeads.

This study investigated the use of a newly developed chitosan-Ca pectinate microbead formulation for the colon-targeted delivery of anti-A/B toxin immunoglobulin of egg yolk (IgY) to inhibit toxin binding to colon mucosa cells. The effect of the three components (pectinate, calcium chloride, and chitosan) used for the microbead production was examined with the aim of identifying the optimal levels to improve drug encapsulation efficiency, swelling ratio, and cumulative IgY release rate. The optimized IgY-loaded bead component was pectin 5% (w/v), CaCl2 3% (w/v), and chitosan 0.5% (w/v). Formulated beads were spherical with 1.2-mm diameter, and the drug loading was 45%. An in vitro release study revealed that chitosan-Ca pectinate microbeads inhibited IgY release in the upper gastrointestinal tract and significantly improved the site-specific release of IgY in the colon. An in vivo rat study demonstrated that 72.6% of biologically active IgY was released specifically in the colon. These results demonstrated that anti-A/B toxin IgY-loaded chitosan-Ca pectinate oral microbeads improved IgY release behavior in vivo, which could be used as an effective oral delivery platform for the biological treatment of Clostridium difficile infection (CDI).

[Study on preparation of ligustrazine ocular implant and correlation between in vivo and in vitro drug release].

OBJECTIVE: To prepare ligustrazine (TMPZ) ocular sustained-release implant, and investigate its in vitro drug release, pharmacokinetics in rabbit vitreum and in vitro correlation. METHOD: Ligustrazine ocular sustained-release implants were prepared by micro-twin conical screw mixers with hot-melting extrusion method, with polyactic-co-glycolic acid (PLGA) as the matrix. HPLC was adopted to determine the concentration in vitreum after ligustrazine was implanted in rabbit eyes, in order to examine its in vivo sustained-release behavior, and study the correlation between in vitro and in vivo. RESULT: Ligustrazine implants were prepared with a drug-loading rate between 10% and 30%, which was in conformity to the pharmacopoeia in terms of the content uniformity. Its in vitro release was in conformity to the zero-order release model. With PLGA 5050, 2. 5A as a vector, ligustrazine implants with a drug-loading rate of 30% could slowly release drug for more than 3 weeks, indicating a good correlation between in vitro and in vivo release. CONCLUSION: Ligustrazine ocular implants prepared with hot-melting extrusion method is practicable. Ligustrazine ocular implants release drug smoothly in rabbit vitreous vitreums, suggesting good sustained-release effect.

[Ocular pharmacokinetics of puerarin in anesthetic rabbits by microdialysis].

OBJECTIVE: To establish the model of microdialysis, and study the ocular pharmacokinetics of puerarin in anesthetic rabbits. METHOD: Implanted the probe into anterior chamber of anesthetic rabbit by surgery. After balanced for 2 h, 1% puerarin eye drop (100 microL) was applied into the cul-de-sac with micropipette. Immediately the dialysate was collected at different time and detected by HPLC with the detection wavelength of 249 nm. The mobile phase was methanol and 0.1% citric acid solution (30:70); the flow rate was 1.0 mL x min(-1). RESULT: After the administration, puerarin can be absorbed into aqueous humor quickly. The peak concentration of puerarin appeared at about 1 h and then reduced gradually. The peak concentration(C(max)) is (2.52 +/- 0.31) mg x L(-1). The other lower peak was shown at 3.5 h during the eliminate phase. This might be attributed to the inhibition of aqueous humor production by the puerarin and resulted in a high drug concentration. The area under concentration-time curve (AUC(0-t)) is (5.04 +/- 0.21) mg x h x L(-1) and the eliminate half life (t1/2) is (0.38 +/- 0.13) h. CONCLUSION: The microdialysis technique can be used to detect the ocular pharmacokinetics of puerarin, and support the valuable pharmacokinetics parameter for the clinical applications of puerarin eye drop.

[Corneal penetration of PAMAM dendrimers-coated puerarin liposomes].

OBJECTIVE: To study the corneal penetration of PAMAM dendrimers-coated puerarin liposomes in rabbits. METHOD: Evaluated PAMAM (G2, G3) dendrimers-coated puerarin liposomes were prepared and the in vitro transcorneal penetration were compared to puerarin drop solution and uncoated liposomes. The effect of different proportion of PAMAM to phospholipids in formulation on corneal penetration and the penetration parameters were investigated. RESULT: The steady state fluxes and permeability coefficients of puerarin by PAMAM G2 (1.0%) and PAMAM G3 (0.5%) coated puerarin liposomes were greater than that by puerarin drop solution and uncoated liposomess (P < 0.01), meanwhile the PAMAM G2 (1.0%) and PAMAM G3 (0.5%) coated liposomes were better than other ratios of coated liposomes for improvement of corneal penetration (P < 0.01). CONCLUSION: The PAMAM coated liposomes is able to enhance the corneal penetration of puerarin and promising as an ocular drug carriers.

[Preparation of sustained release multivesicular liposome for thymopentin and preliminary study on its pharmacokinetics in rats].

To optimize the formulation and preparation method of multivesicular liposome of thymopentin and to investigate its pharmacokinetics in rats, the multivesicular liposome of thymopentin was prepared by double emulsification method and the formulation was optimized by orthogonal design. The release characteristics of thymopentin from multivesicular liposome in PBS (pH 7.4) and in plasma were investigated. The multivesicular liposome of thymopentin labeled with fluorescein isothiocyanate was prepared by double emulsification method. Its pharmacokinetics was evaluated following intramuscular injection in rats. The optimal formulation of multivesicular liposome of thymopentin were formulated with 7.5% glucose in aqueous phase and 2.25 mol x L(-1) triolein, 2.68 mol x L(-1) DPPG and 16.96 mol x L(-1) DOPC in organic phase. The entrapment efficiency of the multivesicular liposome of thymopentin was above 85% and the mean particle size was about 22 microm. The in vitro release of thymopentin from multivesicular liposome in PBS (pH 7.4) and in plasma was found to be in a sustained manner. The release curves were fitted to Higuchi equation. The pharmacokinetics following intramuscular injection of the multivesicular liposome of thymopentin labeled with fluorescein isothiocyanate in rats showed that the peak concentration of thymopentin was lower and elimination of it was slower significantly than that of thymopentin labeled with fluorescein isothiocyanate solution in the same dose. The plasma concentration of thymopentin maintained above quantitative limitation at 120 h after administration of multivesicular liposome of thymopentin. The optimized formulation and preparation technology of multivesicular liposome of thymopentin with higher entrapment efficiency are feasible with good reproducibility. Multivesicular liposome of thymopentin showed significant sustained-release property following intramuscular injection in rats.