Formulation conditions on the drug loading properties of polymeric micelles.

Methyl poly(ethylene glycol) grafted poly (lactide-co-(glycolic acid)-alt-(glutamic acid) amphiphilic copolymers (PLG-g-mPEG) were fabricated polymeric micelles to load anticancer drug doxorubicin (DOX). Both blank and drug loaded micelles were spherical nanoparticles with the mean sizes around 50 and 100nm, respectively. The effects of formulation conditions including compositions, concentrations, temperature, feeding doses and solvents on the size and drug loading content were investigated, the storage of the drug loaded micelles was explored. The results showed that the short graft mPEG chain length was favorable for the loading of DOX. The increase of temperature was preferable for receive micelles with higher drug loading content and smaller size. The encapsulation of polymeric micelles could protect the bioactivity of DOX. In vitro drug release profiles illustrated that the drug release from polymeric micelles with long mPEG chains was much faster than from micelles with short mPEG chains. The release kinetics of drug from micelles fitted to the Ritger-Peppas equation well and the release process followed diffusion mechanism.

[Enhancers on the transmembrane transport of chlorogenic acid].

To investigate the influence of the difference enhancers on the transport mechanism of chlorogenic acid (CGA) across Caco-2 cells model, a RP-HPLC method was adopted to detect the concentrations of CGA. At the concentrations of 20 to 80 microg x mL(-1), the difference of absorption rate constants (K(a)) was not statistically significant. At the concentrations of 40 and 20 microg x mL(-1), the ratios of apparent permeability coefficients (P(app)) of the apical to basolateral and the basolateral to apical were 1.14 and 1.18, respectively. With the effect of enhancers K(a) and P(app) increased, the absorption half-life (T1/2) decreased. CGA passed through the Caco-2 cell membrane mainly by passive transport. It showed that monocarboxylic acid transporter (MCT) could be involved in the across membrane transport process of CGA. Borneol had no effect on the cell membrane transport processes. The order of increasing absorption of CGA caused by the enhancers was sodium lauryl sulphate > sodium taurocholate > carbomer.

Pipette-tip micro-solid phase extraction based on melamine-foam@polydopamine followed by ultra-high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry for detection of psychotropic drugs in human serum.

In this work, pipette-tip micro-solid phase extraction (PT-µSPE) which packed by melamine-foam@polydopamine (MF@PDA) coupled with ultra-high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UHPLC-QTOF) was developed for extraction and determination of psychotropic drugs in serum samples. Considering the operation back pressure, the melamine-foam as carrier material with 3D cross-linked grid structure can provide high permeability and contact surface. MF@PDA was prepared by self-polymerization reaction of dopamine under weak alkaline conditions and characterized by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX) and X-ray photoelectron spectroscopy (XPS). The surface group of PDA containing catechol structure, quinone structure and amine group has multi-interaction with psychotropic drugs which can increase the adsorption capacity. Moreover, the parameters affecting extraction efficiencies such as extraction and desorption cycle, pH value, eluent type, ionic strength and amount of sorbent were investigated. Based on the high sensitivity and accuracy mass measurement by TOF/MS, under the optimized extraction condition, the limits of detection (LOD) of this method were obtained in the range of 0.002-0.1 ng ml-1. The linearity was ranged from 0.01 ng ml-1 to 600 ng ml-1, and all the correlation coefficients (R2) were above 0.993. The spiked recoveries were in the range of 80.04% to 109.18% in real sample test and RSD values obtained from 0.95% to 9.85%. The results demonstrate that MF@PDA-PT-µSPE-UHPLC-QTOF is a sample and reliable method for the detection of psychotropic drugs in serum sample.

Investigating the Fate of MP1000-LPX In Vivo by Adding Serum to Transfection Medium.

BACKGROUND: Cationic liposomes (CLs) based messenger RNA (mRNA) vaccine has been a promising approach for cancer treatment. However, rapid lung accumulation after intraveous injection and significantly decreased transfection efficacy (TE) in serum substantially hamper its application. OBJECTIVE: In this study, we attempt to investigate the fate of Mannose-PEG1000-lipoplex (MP1000-LPX) in vivo, a previous reported mRNA vaccine, and potential mechanism in it. METHODS: MP1000-CLs and different type of MP1000-LPX were produced by previous method and characterized by dynamic light scattering (DLS). Organ distribution and Luc-mRNA expression of DiD loaded luciferase (Luc-mRNA)-MP1000-LPX were evaluated by IVIS Spectrum imaging system. Cellular transfection and uptake under serum-free and serum-containing conditions were analysed by flow cytometry and counted by FlowJo software. RESULTS: MP1000-CLs had an average size of 45.3 ± 0.9 nm, a positive charge of 39.9 ± 0.9 mV. When MP1000-LPX formed, the particle size increased to about 130 nm, and zeta potential decreased to about 30 mV. All formulations were in narrow size distribution with PDI < 0.3. 6 h after intraveous injection, Luc-MP1000-LPX mostly distributed to liver, lung and spleen, while only successfully expressed Luc in lung. DC2.4 cellular transfection assay indicated serum substantially lowered TE of MP1000-LPX. However, the cellular uptake on DC2.4 cells was enhanced in the presence of serum. CONCLUSION: MP1000-LPX distributed to spleen but failed to transfect. Because serum dramatically decreased TE of MP1000-LPX on DC2.4 cells, but not by impeding its interaction to cell membrane. Serum resistance and avoidance of lung accumulation might be prerequisites for CLs based intravenous mRNA vaccines. Lay Summary: mRNA vaccine has been promising immunotherapy to treat cancer by delivering mRNA encoding tumor antigens to APCs and activating immune system against tumor cells. We are investigating the in vivo fate of MP1000-LPX, a CLs based mRNA vaccine. To see if serum causes the fate, we'll be looking at the influence of serum on transfection and uptake efficacy of MP1000-LPX by DC2.4 cells experiments in vitro. Our findings will imply that serum inhibits transfection but not by decreasing uptake. Thus, we can ultilize serum to enhance transfection if we make intracellular process of MP1000-LPX successful.

Pharmacokinetics and tolerability of multiple-dose rosuvastatin: An open-label, randomized-sequence, three-way crossover trial in healthy Chinese volunteers.

BACKGROUND: Rosuvastatin has been reported to be beneficial in the treatment of dyslipidemia. The Cmax and AUC0-t of rosuvastatin were reported to be ~2 to 4 times higher in Chinese subjects compared with white subjects after administration of a single 1-mg/kg dose. OBJECTIVES: The aims of this study were to assess the pharmacokinetics and tolerability of multiple doses of rosuvastatin in healthy Chinese volunteers. METHODS: This open-label, randomized-sequence, 3-way crossover trial consisted of three 7-day treatment periods and two 10-day washout periods. Healthy volunteers were randomly allocated to 1 of 3 daily treatment regimens: rosuvastatin 5, 10, or 20 mg. To assess the pharmacokinetics and tolerability of rosuvastatin, blood samples were drawn before dosing (hour 0) on days 5, 6, and 7 and 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 12, 15, 24, 36, 48, 72, and 96 hours after the final dose was administered on day 7. A validated HPLC-MS/MS method was used to determine rosuvastatin levels. A 2-compartment pharmacokinetic model was fitted to the plasma concentration-time profiles obtained for each volunteer. Adverse events (AEs) were monitored throughout the study via subject interview, vital signs, and blood sampling. Serious AEs were those requiring hospitalization, treatment discontinuation, or resulting in death. RESULTS: Twelve healthy Chinese volunteers (6 men: mean [SD] age, 21.8 [1.7] years; weight, 62.3 [5.8] kg; height, 174.3 [7.2] cm; 6 women: age, 20.8 [1.2] years; weight, 53.2 [4.7] kg; height, 161.3 [4.3] cm) participated in and completed the trial. The mean (SD) steady-state Cmax was significantly greater after ro-suvastatin administration in the 20-mg group compared with the 5-mg group (37.69 [29.83] vs 6.17 [6.03] ng/mL; P = 0.04). The t1/2 was significantly greater in the 20-mg group (15.51 [6.43] hours) compared with the 5-mg group (5.65 [5.08] hours; P = 0.001) and the 10-mg group (8.58 [5.17] hours; P = 0.002). The mean AUC0-t was significantly greater in the 20-mg group compared with the 5-mg group (349.16 [257.20] vs 40.63 [39.31] ng/mL/h; P = 0.02). All AEs were considered by the investigators to be mild in intensity, with the exception of 2 cases of abdominal discomfort (1 man and 1 woman, both in the 5-mg dose group). Two women in the 20-mg group experienced dizziness and cold sweats simultaneously. In the 10-mg group, 1 woman had abdominal discomfort and nausea and 1 woman had jaw pain. All reported AEs were considered possibly related to study drug administration. CONCLUSIONS: In this small study in healthy Chinese volunteers, rosuvastatin systemic exposure appeared to be dose-proportional over the dosing range of 5 to 20 mg with multiple-dose administration. There was no accumulation of rosuvastatin in the body with the 5- and 10-mg doses. However, the results suggest that rosuva-statin might accumulate when the dose is increased to 20 mg. No serious AEs occurred in any of the 3 dosing groups.

Preparation and evaluation of poly-butylcyanoacrylate nanoparticles for oral delivery of thymopentin.

Thymopentin (Tp5) was loaded in poly-butylcyanoacrylate (PBCA) nanoparticles (NP) in order to enhance the oral bioavailability of Tp5. PBCA-Tp5-NP was prepared by nanoprecipitation methods. Dialyzing membrane method was employed to examine the in vitro release of PBCA-Tp5-NP in PBS, and Tp5 samples in the release medium were detected by HPLC. The cell proliferation test ((3)H-thymidine) was conducted to verify the PBCA-Tp5-NP bioactivity in vitro. The pharmacodynamical studies were performed on preimmunoinhibited rats and in flow cytometer. The size and the entrapment efficiency of PBCA-Tp5-NP were 178 +/- 39 nm and 92.21 +/- 1.08%, respectively. In vitro release data show that less than 60% Tp5 was released from lyophilized PBCA-Tp5-NP while 80% Tp5 was released from the colloidal PBCA-Tp5-NPs in 48 h. The proliferation test showed that PBCA-Tp5-NP had the similar effect as Tp5. The in vivo data showed that oral PBCA-Tp5-NPs had similar function as what intravenous Tp5 did. The oral bioavailability of Tp5 could be enhanced by PBCA nanoparticles. PBCA-Tp5-NP had the property of sustained-release and the efficacy of Tp5 was not changed after formulation.

Enhanced anticancer efficacy of paclitaxel through multistage tumor-targeting liposomes modified with RGD and KLA peptides.

Mitochondria serve as both "energy factories" and "suicide weapon stores" of cells. Targeted delivery of cytotoxic drugs to the mitochondria of tumor cells and tumor vascular cells is a promising strategy to improve the efficacy of chemotherapy. Here, multistage tumor-targeting liposomes containing two targeted peptide-modified lipids, cRGD-PEG2000-DSPE and KLA-PEG2000-DSPE, were developed for encapsulation of the anticancer drug paclitaxel (PTX, RGD-KLA/PTX-Lips). Compared with Taxol (free PTX), RGD/PTX-Lips and KLA/PTX-Lips, the half-maximal inhibitory concentration (IC50) value of RGD-KLA/PTX-Lips in vitro was 1.9-, 36.7- and 22.7-fold lower with 4T1 cells, respectively, because of higher levels of cellular uptake. Similar results were also observed with human umbilical vascular endothelial cells (HUVECs). An apoptosis assay showed that the total apoptotic ratio of RGD-KLA/PTX-Lips was the highest because of the mitochondria-targeted drug delivery and the activation of mitochondrial apoptosis pathways, as evidenced by visible mitochondrial localization, decreased mitochondrial membrane potential, release of cytochrome c and increased activities of caspase-9 and caspase-3. The strongest tumor growth inhibition (TGI; 80.6%) and antiangiogenesis effects without systemic toxicity were also observed in RGD-KLA/PTX-Lip-treated 4T1 tumor xenograft BALB/c mice. In conclusion, these multistage tumor-targeting liposomes represent a promising anticancer drug delivery system (DDS) capable of maximizing anticancer therapeutic efficacy and minimizing systemic toxicity.

Synthesis, characterization and in situ intestinal absorption of different molecular weight scutellarin-PEG conjugates.

Highly water soluble esters of scutellarin with different molecular weight polyethylene glycol (PEG) were synthesized. The physicochemical properties, the stabilities under different conditions and the in situ intestinal absorption of the conjugates in rats were investigated. By PEG modification, greatly increased water solubility and a desirable partition coefficient were obtained. These compounds act as prodrugs i.e. breakdown occurrs in a predictable fashion: in vitro, the t1/2 of them in PBS buffer at pH 7.4 was above 12 h (37 degrees C), while in plasma a more rapid breakdown was observed (t1/2 1.5-3 h). PEGylation could enhance the absorption of scutellarin in rat intestine, and scutellarin, its PEG conjugates are absorbed through intestine mainly via passive transport. When the molecular weight of PEG increased from 200 to 1000 Da, the absorption of the conjugates decreased accordingly. The range of PEG molecular weight used for the PEGylation of scutellarin was about 400-1000 Da based on considerations of the yield, the stability and the absorption.

[The pharmacokinetic study on cytarabine nanoparticle lyophilization injection in rabbits].

OBJECTIVE: The pharmacokinetics of Cytarabine Polybutylcyanoacrylate Nanoparticles (Ara-C-PBCA-NP) lyophilization injection in rabbits was studied. METHODS: Ara-C water solution was taken as reference, the concentration of Ara-C was determined by HPLC, and the pharmacokinetic parameters were calculated. RESULTS: Pharmacokinetic properties of Ara-C water solution and Ara-C-PBCA-NP lyophilization injection complied with two-department model. The parameters t1/2 beta and MRT of Ara-C-PBCA-NP lyophilization injection were prolonged, and CL was reduced dramatically (P < 0.05). CONCLUSION: Ara-C-PBCA-NP lyophilization injection possesses prolonged retention time in vivo and significant sustained releasing character.

[The synthesis, characterization and in situ intestinal absorption of different molecular weight scutellarin-PEG conjugates].

In this report, highly water soluble esters of scutellarin with different molecular weight polyethylene glycol (PEG) were synthesized in order to improve the bioavailability of scutellarin. The physicochemical properties, the stabilities under different conditions and the in situ intestinal absorption in rats of the conjugates were investigated. By PEG modification, the greatly increased water solubility and desirable partition coefficient of scutellarin were obtained. These compounds function as prodrugs i. e. breakdown occurred in a predictable fashion: the t1/2 of them in PBS buffer at pH7.4 was above 12 h (37 degrees C) in vitro, while in plasma a rapid breakdown was observed, with a t1/2 of about 1. 5-3 h. The stabilities of the prodrugs were improved according to the increase of the molecular weight of PEG, thus, PEGylated prodrugs with desirable rates of hydrolysis could be obtained by the use of variable molecular weight PEGs. The PEGylation could enhance the absorption of scutellarin in rat intestine, and the absorption of scutellarin and its PEG conjugates by intestine was mainly via passive transport, for when the concentration was raised, the uptake did not appear saturable, and the permeability coefficient kept at an equilibrium level. When the molecular weight of PEG increased from 200 to 1000 Da, the absorption of the conjugates decreased. In conclusion, by overall consideration of the yield, stability and absorption, the present authors estimate that the PEG molecular weight used for the PEGylation of scutellarin should be within the range of 400-1 000 Da.

Poly(ethylene glycol)-block-poly(ε-caprolactone)-and phospholipid-based stealth nanoparticles with enhanced therapeutic efficacy on murine breast cancer by improved intracellular drug delivery.

BACKGROUND: Effective anticancer drug delivery to the tumor site without rapid body clearance is a prerequisite for successful chemotherapy. 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-N-(methoxy[polyethyleneglycol]-2000) (DSPE-PEG2000) has been widely used in the preparation of stealth liposomes. Although PEG chains can efficiently preserve liposomes from rapid clearance by the reticuloendothelial system (RES), its application has been hindered by poor cellular uptake and unsatisfactory therapeutic effect. METHODS: To address the dilemma, we presented a facile approach to fabricate novel stealth nanoparticles generated by poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-b-PCL), soybean phosphatidylcholine (SPC), and cholesterol, namely LPPs (L represented lipid and PP represented PEG-b-PCL), for the delivery of anticancer drug paclitaxel (PTX). LPPs were prepared using the thin film hydration method. Two PEG-b-PCL polymers with different molecular weights (MW; PEG2000-b-PCL2000, MW: 4,000 Da and PEG5000-b-PCL5000, MW: 10,000 Da) were used to fabricate stealth nanoparticles. Conventional PEGylated liposome (LDP2000, L represented lipid and DP2000 represented DSPE-PEG2000) composed of SPC, cholesterol, and DSPE-PEG2000 was used as the control. The physical properties, cellular uptake, endocytosis pathway, cytotoxicity, pharmacokinetics, tumor accumulation, and anticancer efficacy of free PTX, PTX-loaded LPPs, and LDP2000 were systemically investigated after injection into 4T1 breast tumor-bearing mice. RESULTS: LPPs were vesicles around 100 nm in size with negative zeta potential. With enhanced stability, LPPs achieved sustainable release of cancer therapeutics. The cellular uptake level was closely related to the PEG chain length of PEG-b-PCL; a shorter PEG chain resulted in higher cellular uptake. Moreover, the cellular internalization of LPP2000 modified by PEG2000-b-PCL2000 on 4T1 cells was 2.1-fold higher than LDP2000 due to the improved stability of LPP2000. The cytotoxicity of PTX-loaded LPP2000 was also higher than that of LDP2000 and LPP5000 as observed using a WST-8 assay, while blank LPPs showed negligible toxicity. Consistent with the results of the in vitro study, in vivo experiments showed that LPPs allowed significantly improved bioavailability and prolonged T1/2ß as compared to free PTX injection. More importantly, LPPs mainly accumulated at the tumor site, probably due to the enhanced permeation and retention effect (EPR effect). As a nanomedicine, LPP2000 (tumor inhibition rate of 75.1%) significantly enhanced the therapeutic effect of PTX in 4T1 breast tumor-bearing mice by inhibiting tumor growth compared to LDP2000 and LPP5000 (tumor inhibition rates of 56.3% and 49.5%, respectively). CONCLUSION: Modification of liposomes with PEG2000-b-PCL2000 can simultaneously improve drug accumulation at the target tumor site and tumor cells, showing great promise for utilization as a PEG modification tool in the fabrication of stealth nanoparticles for cancer chemotherapy.

Effects of Labrasol and other pharmaceutical excipients on the intestinal transport and absorption of rhodamine123, a P-glycoprotein substrate, in rats.

Effects of Labrasol and other pharmaceutical excipients on the intestinal transport and absorption of rhodamine123, a P-glycoprotein substrate (P-gp) were examined. Intestinal transport and absorption studies were examined by an in vitro diffusion chamber method and an in situ closed loop method. We evaluated the intestinal membrane damage produced by Labrasol by measuring the release of protein and alkaline phosphatase (ALP). Labrasol (0.075-0.1% (v/v)) increased the absorptive transport of rhodamine123 and decreased its secretory transport in the in vitro transport studies. However, Labrasol did not change the transport of Lucifer yellow, a non-P-gp substrate, suggesting that the effect of Labrasol on the transport of drugs was specific for rhodamine123. We observed almost no intestinal membrane damage in the presence of Labrasol. These findings suggest that the increase in the absorptive transport of rhodamine123 in the presence of Labrasol may not be due to its intestinal membrane damage. In the in situ absorption studies, we found that Labrasol (0.1% (v/v)) significantly enhanced the intestinal absorption of rhodamine123 in rats, although the absorption enhancing effect of Labrasol was much less than that of verapamil. These findings suggest that low concentrations of Labrasol might inhibit the function of P-gp in the intestine, thereby increasing intestinal absorption and bioavailability of P-gp substrates including rhodamine123. However, we may also consider the contribution to the enhanced intestinal absorption of rhodamine123 via a passive transport in addition to the inhibitory action of Labrasol for the function of P-gp in the intestine.