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To concurrently suppress multidrug resistance (MDR) and metastasis of breast cancer cells, paclitaxel (PTX) and honokiol (HNK) were coencapsulated into pH-sensitive polymeric micelles based on poly(2-ethyl-2-oxazoline)-poly( d , l -lactide) (PEOz-PLA). The physicochemical properties of dual drug-loaded PEOz-PLA micelles were characterized in size, drug loading and in vitro release. The efficiency of MDR reversal for the micelles was testified by synergetic enhancement of cytotoxicity and uptake by MCF-7/ADR cells. The flow cytometry and fluorescence polarization measurement results reinforced the conclusion that down-regulation of P-gp expression and increase of plasma membrane fluidity appeared to be possible mechanisms of MDR reversal by dual drug-loaded PEOz-PLA micelles. Further, the efficient inhibition of tumor metastasis by dual drug-loaded PEOz-PLA micelles was demonstrated by in vitro anti-invasion and anti-migration assessment in MDA-MB-231 cells and in vivo bioluminescence imaging in nude mice. The suppression of MDR and metastasis by the micelles was assigned to synergistic effects of pH-triggered drug release and HNK/PEOz-PLA-aroused P-gp inhibition, and pH-triggered drug release and PTX/HNK-aroused MMPs inhibition, respectively. In conclusion, our findings strengthen the usefulness of co-delivery of PTX and HNK by pH-responsive polymeric micelles for suppression of tumor MDR and metastasis. Statement of Significance Multidrug resistance (MDR) and metastasis are considered to be two of the major barriers for successful chemotherapy. The combination of a chemotherapeutic drug with a modulator has emerged as a promising strategy for efficiently treating MDR cancer and preventing tumor metastasis. Herein, a dual drug (paclitaxel and honokiol)-loaded pH-sensitive polymeric micelle system based on PEOz-PLA was successfully fabricated to ensure that tumor MDR and metastasis could be concurrently suppressed, therefore achieving distinguishing endo/lysosomal pH from physiological pH by accelerating drug release and then enhancing the cytotoxicity of paclitaxel to drug-resistant tumor cells MCF-7/ADR by increasing cellular uptake of paclitaxel, preventing in vitro invasion and migration for MDA-MB-231 cells and in vivo metastasis in nude mice. Further, the mechanism of MDR reversal by dual drug-loaded PEOz-PLA micelles was elucidated to be down-regulation of P-gp expression and increase of plasma membrane fluidity of MCF-7/ADR cells. The present findings strengthen the usefulness of co-delivery of PTX and HNK by pH-responsive polymeric micelles for suppression of tumor MDR and metastasis.

To ensure that antitumor drugs can be effectively transported across intestinal barrier and then quickly released in tumor cells, mixed polymeric micelles (Mix-PMs) were designed and fabricated by combining poly(2-ethyl-2-oxazoline)-vitamin E succinate (PEOz-VES) with TPGS1000 for enhancing intestinal absorption of paclitaxel. PEOz-VES exhibited an extremely low critical micelle concentration and negligible cytotoxicity. The Mix-PMs were characterized to have about 20 nm in diameter, uniform spherical morphology, high drug-loading content and sustained drug release profile with a retained pH-sensitivity. The results of the transport through Caco-2 cell monolayers and intestinal absorption revealed that Mix-PMs displayed higher transcellular transport efficiency compared with PEOz-VES micelles and Taxol®. The possible mechanism of transcellular transport for Mix-PMs was elucidated to be mainly through clathrin- and caveolae/lipid rafts-mediated transcytosis. Confocal laser scanning micrographs revealed that late endosomes, lysosomes, endoplasmic reticulum, Golgi apparatus, and mitochondria were all involved in intracellular trafficking of Mix-PMs. The proteins involved in transcytosis of Mix-PMs and finally excreted were unraveled for the first time by the analysis of proteins in the basolateral media according to the proteomics method. Consequently, the fabricated mixed polymeric micelles may have great potential in enhancing intestinal absorption and accelerating drug release in tumor cells.

Long-circulation (PEGLip), pH-sensitive (PEOzLip), and active targeted liposomes (PEG-TATLip)-loading doxorubicin (DOX) and harmine (HM) were prepared. Their physicochemical properties and antitumor effect were investigated.The aims of the present study were to evaluate synergistic antitumor efficacy.Liposomes were prepared by using thin-film dispersion, active drug-loading and target post-insertion method. Subsequently physiochemical properties including particle size distribution, zeta potential, entrapment efficiency (EE), drug-loading content and in-vitro release were determined. Besides, the in vitro cytotoxicity of free drugs and drug-loaded liposomes was explored by using a Sulforhodamine-B Staining assay and the combination index values (CI Value) were calculated. Finally, the cellular uptake experiments by MCF-7cells were carried out via flow cytometry.All liposomes enhanced the antitumor effect significantly compared to free drugs. Among liposomes, PEG-TATLip enhanced the antitumor effect significantly compared to others. DOX and HM had moderate synergism with CI Value 0.85 for free drugs, 0.81 for PEGLip, 0.72 for PEOzLip, and 0.84 for PEG-TATLip respectively when the weight ratio of two drugs was 1:2. Moreover, the similarity between DOX and HM such as physicochemical properties, in vitro release modes and in vitro uptake kinetics characteristics when they were in the same formulations proved it possible for them to be delivered together.Active targeting liposomes were the most effective delivery system as compared with pH-sensitive and long circulation liposomes. Additionally, DOX and HM could be co-delivered in liposomes and they could play moderate synergism effect in antitumor efficacy.

Sirolimus is recognized as a P-glycoprotein (P-gp) substrate with poor water-solubility. To improve its solubility and bioabsorption, self-microemulsifying drug delivery systems (SMEDDS) containing a novel P-gp inhibitor, honokiol, were prepared. The aim of this work was to evaluate the enhanced transport of sirolimus SMEDDS as well as the roles of honokiol. In situ single-pass intestinal perfusion and in vitro human colon adenocarcinoma (Caco-2) cell models were applied to study the effects of honokiol within SMEDDS on the transport of sirolimus. The results indicated that a combination of honokiol with sirolimus in SMEDDS did not significantly alter the particle size, polydispersity index and release of drugs. In addition, the absorption rate constant (Ka) as well as the effective permeability coefficients (Peff) of sirolimus in situ intestinal absorption, and the apparent permeability coefficients (Papp) of sirolimus in caco-2 cells were significantly enhanced by cremophor EL-based SMEDDS with honokiol as compared with those of SMEDDS without honokiol. Rhodamine123 uptake rate in caco-2 cells and in vitro cytotoxicity of sirolimus were enhanced by honokiol in SMEDDS indicating a substantial P-gp inhibition of honokiol. In conclusion, coadministration of honokiol with poor soluble P-gp substrate in SMEDDS, could serve as a favorable approach for oral delivery.

The aim of the present study was to develop an oil-free o/w microemulsion, composed of pluronic F68, propylene glycol and saline, which solubilized poorly soluble anesthetic drug propofol for intravenous administration. The ternary diagram was constructed to identify the regions of microemulsions, and the optimal composition of microemulsion was determined by in vitro evaluation such as globule size upon dilution and rheology. The droplet size of the diluent emulsion corresponding to oil-in-water type ranged from 200 to 300nm in diameter. Stability analysis of the microemulsions indicated that they were stable upon storage for at least 6 months. Hemolysis percent of propofol microemulsions was lower than that of commercial lipid emulsion (CLE) at 4h. Acute toxicity test showed that median lethal dose of propofol microemulsion was the same as that of CLE. No significant difference in time for unconsciousness and recovery of righting reflex was observed between the prepared microemulsions and CLE. In conclusion, microemulsion would be a promising intravenous delivery system for propofol.

To evaluate novel mixed polymeric micelles based on monomethoxy poly(ethylene glycol)-poly(D,L-lactic acid) (mPEG-PLA) and Pluronic L61 for delivery of paclitaxel (PTX) to circumvent unfavorable effects resulting from Cremophore EL in Cremophore EL-based PTX formulation and overcoming multidrug resistance (MDR) in tumor cells at the same time.PTX-loaded plain micelles and mixed micelles were prepared and characterized by determining PTX release in vitro, MDR reversal effect in human breast cancer MDR MCF-7/ADR cell sublines and pharmacokinetics in vivo.Both PTX-loaded plain micelles and mixed micelles had similar in vitro release profile. Mixed micellar PTX significantly reduced IC(50) of PTX in MCF-7/ADR cells compared to free PTX and plain micellar PTX, and mixed micelles substantially enhanced cellular accumulation of R 123 in MCF-7/ADR cells compared to free R123 and plain micelles. PTX-loaded mixed micelles with lower content of L61 exhibited comparable cytotoxicity to that observed with Cremophore EL-based PTX formulation in inhibiting the growth of MCF-7/ADR cells. Moreover, plain micelles and mixed micelles retained the pharmacokinetic characteristics of PTX in rats compared with Cremophore EL-based PTX formulation.This study suggested that the mixed micelles could enhance delivery of PTX and cell-killing effect for MDR MCF-7/ADR cells.

The purpose of this work was to investigate feasibility of a promising topical drug delivery system (TDDS) for sinomenine hydrochloride (SMH), extracted from the Chinese medicinal plant sinomenine acutum and currently used for the treatment of rheumatoid arthritis. It was found that SMH was a weak base (p K a , 7.98 ± 0.04) with pH‐dependent solubility and partition coefficient. The result of in vitro permeation studies demonstrated that the permeation enhancer azone was the most effective. In contrast, spray had higher accumulative permeated amounts of SMH than patch, but permeated duration of spray was shorter than that of patch. The efficacy on Freund's complete adjuvant‐induced arthritis suggested that there was near arthritis index for SMH spray with medium dose (i.e., 15 mg/rat) and oral solution at a dose of 12 mg/rat, indicating that topical SMH delivery system could achieve the similar anti‐inflammatory efficacy with oral administration. Pharmacokinetic parameters including C max and AUC for both topical preparations were lower than those for oral preparation, which hinted that systemic side effect could be ignored. Therefore, the spray and patch were promising formulations for successful topical delivery of SMH through the skin instead of oral administration with side effects. © 2009 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 1790–1799, 2010

The purpose of the present study was to develop a new nanoparticulate formulation for delivery of tacrolimus to reduce its kidney distribution and functional nephrotoxicity. Tacrolimus (TAC)-loaded bovine serum albumin (BSA) nanoparticles (TAC-BSA-NPs) were prepared by emulsification-dispersion technique. The obtained TAC-BSA-NPs, with 189.50±7.15 nm of diameter and -20.86±0.45 mV of Zeta potential determined by DLS, were spherical in shape observed by TEM. The drug loading content and encapsulation efficiency were (1.7±0.13)% and (85±3.0)%, respectively. The in vitro release of TAC-BSA-NPs exhibited biphasic drug release pattern with an initial burst release and subsequently sustained release. Pharmacokinetic analysis displayed that TAC-BSA-NPs could enhance the drug blood level and prolong the circulation time in comparison to Prograf(®). Meanwhile, compared with Prograf(®), TAC-BSA-NPs could deliver less TAC to kidney and simultaneously reduce the functional nephrotoxicity of TAC to kidney. In conclusion, BSA nanoparticles might be a more safe carrier for delivery of hydrophobic drug TAC.

The purpose of the present study was to design and fabricate endosomal pH-sensitive dual-ligand-modified micellar nanoparticles to achieve enhanced drug delivery to tumor cells and facilitated intracellular drug release.End-group-carboxylated poly(2-ethyl-2-oxazoline)-poly(D,L-lactide) and cyclic Arg-Gly-Asp-Tyr-Lys- and anti-prostate specific membrane antigen antibody-modified diblock copolymer poly(2-ethyl-2-oxazoline)-poly(D,L-lactide) were synthesized and characterized by (1)H NMR and gel permeation chromatography, and self-assembled into micelles. Paclitaxel-loaded dual-ligand-modified micelles were prepared by thin-film hydration method, and characterized by dynamic light scattering, transmission electron microscope, pH-dependent in vitro release and stability. Intracellular paclitaxel delivery was measured by flow cytometry and imaged by confocal microscopy. In vitro cytotoxicity was studied in the 22Rv1 xenograft prostate tumor cell lines.The prepared dual-ligand-modified micelles with about 30 nm in diameter and rapid intracellular drug release behavior at endo/lysosomal pH were very effective in enhancing the cytotoxicity of paclitaxel against 22Rv1 cells by increasing the cellular uptake, which was verified the correlation with the expression of integrin αvβ3 and prostate specific membrane antigen in tumor cells by flow cytometric analysis and confocal microscopy, compared with single ligand-modified micelles.These findings provided valuable information that the application of combining of dual-ligand modifications with pH-sensitivity to polymeric micelles may be a promising approach in the efficient delivery of anticancer drugs for treatment of integrin αvβ3 and prostate specific membrane antigen expressing prostate cancers.

The combination of a chemotherapeutic drug with a P-glycoprotein (P-gp) inhibitor has emerged as a promising strategy for treating multidrug resistance (MDR) cancer. To ensure that two drugs can be co-delivered to the tumor region and quickly released in tumor cells, tumor-targeted and pH-sensitive polymeric micelles were designed and prepared by combining cationic ring-opening polymerization of 2-ethyl-2-oxazoline (EOz) with anionic ring-opening polymerization of D,L-lactide (LA), and then encapsulating doxorubicin (DOX) and D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS1000) into the micelles self-assembled by poly(2-ethyl-2-oxazoline)-poly(D,L-lactide) (PEOz-PLA) and DSPE-PEG-folate. PEOz-PLA exhibited a low critical micelle concentration and negligible cytotoxicity. The micelles enabled the rapid release of DOX when pH decreased from 7.4 to 5.0. The targeting ability of the micelles was demonstrated by in vitro flow cytometry in KBv cells and in vivo real time near-infrared fluorescence imaging in KBv tumor-bearing nude mice. The efficiency of MDR reversion for the micelles was testified by enhancement of intracellular DOX accumulation and cytotoxicity. The efficient drug delivery by the micelles was attributed to synergistic effects of folate-mediated targeting, pH-triggered drug release and TPGS1000-aroused P-gp inhibition. Therefore, the designed multifunctional polymeric micelles may have significant promise for therapeutic application of MDR cancer.

The purpose of this work was to explore the feasibility of ethosomes for improving the antiarthritic efficacy of tetrandrine by topical application. It was found that tetrandrine was a weak base (pKa=7.06) with pH-dependent partition coefficient. The spherical-shaped ethosomes were prepared by pH gradient loading method.Ex vivopermeation and deposition behavior demonstrated that the drug flux across rat skin and deposition of the drug in rat skin for ethosomes was 2.1- and 1.7-fold higher than that of liposomes, respectively. Confocal laser scanning microscopy confirmed that ethosomes could enhance the topical delivery of the drug in terms of depth and quantity compared with liposomes. The ethosomes were shown to generate substantial enhancement of therapeutic efficacy of tetrandrine on Freund’s complete adjuvant-induced arthritis with regard to liposomes. These results indicated that ethosomes would be a promising carrier for topical delivery of tetrandrine into and across the skin.

The purpose of this work was to prepare and characterize a novel ethosomal carrier for tacrolimus, an immunosuppressant treating atopic dermatitis (AD), and to investigate inhibition action upon allergic reactions of mice aiming at improving pharmacological effect for tacrolimus in that commercial tacrolimus ointment (Protopic®) with poor penetration capability exhibited weak impact on AD compared with common glucocorticoid. Results indicated that the ethosomes showed lower vesicle size and higher encapsulation efficiency (EE) as compared with traditional liposomes with cholesterol. In addition, the quantity of tacrolimus remaining in the epidermis at the end of the 24-h experiment was statistically significantly greater from the ethosomal delivery system than from commercial ointment (Protopic®) (p

Polymeric micelles, based on lactobionic acid (LA)-conjugated Pluronic P105 (P105), were prepared to achieve liver-targeted delivery of silybin. In the triblock copolymer structure of PEO-PPO-PEO, LA was successfully conjugated with the terminal end of PEO to produce LA-P105. The success of synthesis was confirmed using FTIR and 1H NMR. The triblock copolymers with functional moiety were physically mixed with silybin to form micelles. Silybin-loaded LA-P105 micelles characterized by dynamic light scattering and transmission electron microscopy (TEM) were uniform spherical particles. There was a remarkable increase in the dissolubility for silybin in LA-P105 micelle solution (627 microg/mL) when compared with that for water (4.6 microg/mL). The pharmacokinetic experiments showed that the area under the curve of silybin plasma concentration-time profile in rats for LA-P105 micelles was lower than that for P105 micelles. Biodistribution studies indicated that a significantly increased amount of silybin was accumulated in liver, suggesting that LA locating on the surface of the micelles played an important role in transporting an increased amount of silybin into liver. This polymeric vehicle is, therefore, expected to be widely used as target-specific delivery vehicles for diverse water-insoluble therapeutic and diagnostic agents.