Design and evaluation of mPEG-PLA micelles functionalized with drug-interactive domains as improved drug carriers for docetaxel delivery

Polymeric micelles are very attractive drug delivery systems for hydrophobic agents, owing to their readily tailorable chemical structure and ease for scale-up preparation. However, the intrinsic poor stability of drug-loaded micelles presents one of the major challenges for most micellar systems in the translation to clinical applications. In this study, a simple, well-defined, and easy-to-scale up 9-Fluorenylmethoxycarbonyl (Fmoc) and tert-butoxycarbonyl (Boc) containing lysine dendronized mPEG-PLA (mPEG-PLA-Lys(FB)₂) micellar formulation was designed and prepared for docetaxel (DTX) delivery, in an effort to improve the stability of the micelles, and its physicochemical properties, pharmacokinetics, and anti-tumor efficacy against SKOV-3 ovarian cancer were evaluated. MPEG-PLA-Lys(FB)₂ was synthesized via a three-step synthetic route, and it actively interacted with DTX in aqueous media to form stable micelles with small particle sizes (~17–19 nm) and narrow size distribution (PI < 0.1), which can be lyophilized and easily reconstituted in saline without significant change in particle size distribution. In vitro drug-release study demonstrated that mPEG-PLA-Lys(FB)₂ micelles achieved delayed and sustained release manner of DTX in comparison with mPEG-PLA micelles. Further in vivo xenograft tumor model in nude mice DTX/mPEG-PLA-Lys(FB)₂ micelles demonstrated significantly higher inhibitory effect on tumor growth than the marketed formulation Taxotere. Thus, our system may hold promise as a simple and effective delivery system for DTX with a potential for translation into clinical study.     

Synthesis,characterization, and in vitro evaluation of TRAIL-modified,cabazitaxel -loaded polymeric micelles for achieving synergistic anticancer therapy

Abstract

Combination therapy of two or more drugs has gradually become of outmost importance in cancer treatment. Cabazitaxel (CTX) is a taxoid drug and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of TNF superfamily. In this study, we prepared TRAIL-modified and CTX-loaded polymer micelle (TRAIL-M-CTX). This nanoparticle was self-assembled from biodegradable amphiphilic copolymers, monomethoxyl poly(ethylene glycol)–b-poly(DL-lactide) (mPEG-PLA) and COOH-PEG-PLA, via a nanoprecipitation method and were modified with the TRAIL protein, resulting in a particle size of 39.75 ± 0.17 nm in diameter and a drug encapsulation efficiency of 95.52 ± 1.69%. The successful coupling was confirmed by ¹H NMR, FTIR spectroscopy, and DLS article size measurement. Pharmacodynamic analysis in two human cancer cell lines with different TRAIL sensitivities showed that TRAIL-M-CTX has a significantly better anticancer efficacy than the individual CTX and TRAIL protein. Importantly, TRAIL-M-CTX showed synergistic effects against TRAIL-insensitive cells (MCF-7). A study of cellular uptake implied that the modified micelles were internalized into MCF-7 cells more effectively than unmodified micelles, owing to the coupled TRAIL protein. A cell cycle assay of MCF-7 cells revealed that TRAIL-M-CTX significantly increased the sub-G1 population compared with CTX or TRIAL, thus, facilitating cancer cell apoptosis. These results suggest that TRAIL-M-CTX micelles have potential as a cancer chemotherapy formulation.     

Enhanced solubility and targeted delivery of curcumin by lipopeptide micelles

A lipopeptide (LP)-containing KKGRGDS as the hydrophilic heads and lauric acid (C₁₂) as the hydrophobic tails has been designed and prepared by standard solid-phase peptide synthesis technique. LP can self-assemble into spherical micelles with the size of ~30 nm in PBS (phosphate buffer saline) (pH 7.4). Curcumin-loaded LP micelles were prepared in order to increase the water solubility, sustain the releasing rate, and improve the tumor targeted delivery of curcumin. Water solubility, cytotoxicity, in vitro release behavior, and intracellular uptake of curcumin-loaded LP micelles were investigated. The results showed that LP micelles can increase the water solubility of curcumin 1.1 × 10³ times and sustain the release of curcumin in a low rate. Curcumin-loaded LP micelles showed much higher cell inhibition than free curcumin on human cervix carcinoma (HeLa) and HepG2 cells. When incubating these curcumin-loaded micelles with HeLa and COS7 cells, due to the over-expression of integrins on cancer cells, the micelles can efficiently use the tumor-targeting function of RGD (functionalized peptide sequences: Arg-Gly-Asp) sequence to deliver the drug into HeLa cells, and better efficiency of the self-assembled LP micelles for curcumin delivery than crude curcumin was also confirmed by LCSM (laser confocal scanning microscope) assays. Combined with the enhanced solubility and higher cell inhibition, LP micelles reported in this study may be promising in clinical application for targeted curcumin delivery.     

Development of polyion complex micelles for encapsulating and delivering amphotericin B

A block copolymer poly(2-ethyl-2-oxazoline)-block-poly(aspartic acid) (PEOz-b-PAsp) was synthesized and investigated as the carrier of antifungal drug amphotericin B (AmB). Polyion complex (PIC) micelles with clear core–shell structures were identified by TEM, which revealed that the PAsp segment became hydrophobic after it interacted with AmB. PEOz-b-PAsp increased not only the solubility of AmB but also simultaneously the drug potency. The prolonged release of AmB from micelles effectively inhibited the growth of Candida albicans even after three days of administration. Moreover, the in vitro cytotoxicity of AmB-loaded micelles was less than that of Fungizone^®, which is a powerful antifungal antibiotic that is adopted to treat various fungal infections. The PEOz-b-PAsp PIC micelles with lower cytotoxicity and higher potency than Fungizone^® represent a potential means of encapsulating basic/amphoteric drugs.     

Glycyrrhetinic acid-modified poly(ethylene glycol)–b-poly(γ-benzyl l-glutamate) micelles for liver targeting therapy

Liver targeted micelles were successfully constructed via self-assembly of glycyrrhetinic acid (GA)-modified poly(ethylene glycol)–b-poly(γ-benzyl l-glutamate) (GA–PEG–PBLG) block co-polymers, which were fabricated via ring opening polymerization of γ-benzyl l-glutamate N-carboxyanhydride monomer initiated by GA-modified PEG. The in vivo biodistribution and the in vitro cellular uptake of these micelles were investigated. The results showed that the relative uptake of doxorubicin (DOX)-loaded micelles (DOX/GA–PEG–PBLG) in liver was much higher than in other tissues, and the resulting DOX concentration in liver was about 2.2-fold higher than that from the micelles without modification by GA. Moreover, the cellular uptake study demonstrated that the introduction of GA to the micelles could significantly increase the affinity for human hepatic carcinoma 7703 cells, which induced a 3.7-fold higher endocytosis than unmodified ones. The cytotoxicity of DOX/GA–PEG–PBLG micelles (IC₅₀ 47 ng ml^?1) was much higher than that of free DOX (IC₅₀ 90 ng ml^?1). These results indicate that GA-modified micelles have great potential in liver targeting therapy.     

Glutathione-dependent micelles based on carboxymethyl chitosan for delivery of doxorubicin

Novel glutathione (GSH)-dependent micelles based on carboxymethyl chitosan (CMCS) were developed for triggered intracellular release of doxorubicin (DOX). DOX-33′-Dithiobis (N-hydroxysuccinimidyl propionate)-CMCS (DOX-DSP-CMCS) prodrugs were synthesized. DOX was attached to the amino group on CMCS via disulfide bonds and drug-loaded micelles were formed by self-assembly. The micelles formed core–shell structure with CMCS and DOX as the shell and core, respectively, in aqueous media. The structure of the prodrugs was confirmed by IR and proton nuclear magnetic resonance (¹H NMR) spectroscopy. The drug-loading capacity determined by UV spectrophotometry was 4.96% and the critical micelle concentration of polymer prodrugs determined by pyrene fluorescence was 0.089 mg/mL. Micelles were spherical and the mean size of the nanoparticles was 174 nm, with a narrow polydispersity index of 0.106. Moreover, in vitro drug release experiments showed that the micelles were highly GSH-sensitive owing to the reductively degradable disulfide bonds. Cell counting kit (CCK-8) assays revealed that DOX-DSP-CMCS micelles exhibited effective cytotoxicity against HeLa cells. Moreover, confocal laser scanning microscopy (CLSM) demonstrated that DOX-DSP-CMCS micelles could efficiently deliver and release DOX in the cancer cells. In conclusion, the DOX-DSP-CMCS nanosystem is a promising drug delivery vehicle for cancer therapy.     

Thermosensitive AB4 four-armed star PNIPAM-b-HTPB multiblock copolymer micelles for camptothecin drug release

Thermo-sensitive poly(N-isoproplacrylamide)_m-block-hydroxyl-terminated polybutadiene-block-poly(N-isoproplacrylamide)_m (PNIPAM_m-b-HTPB-b-PNIPAM_m, m = 1 or 2) block copolymers, AB₄ four-armed star multiblock and linear triblock copolymers, were synthesized by ATRP with HTPB as central blocks, and characterization was performed by ¹H NMR, Fourier transform infrared, and size exclusion chromatography. The multiblock copolymers could spontaneously assemble into more regular spherical core–shell nanoscale micelles than the linear triblock copolymer. The physicochemical properties were detected by a surface tension, nanoparticle analyzer, transmission electron microscope (TEM), dynamic light scattering, and UV–vis measurements. The multiblock copolymer micelles had lower critical micelle concentration than the linear counterpart, TEM size from 100 to 120 nm, and the hydrodynamic diameters below 150 nm. The micelles exhibited thermo-dependent size change, with low critical solution temperature of about 33–35 °C. The characteristic parameters were affected by the composition ratios, length of PNIPAM blocks, and molecular architectures. The camptothecin release demonstrated that the drug release was thermo-responsive, accompanied by the temperature-induced structural changes of the micelles. MTT assays were performed to evaluate the biocompatibility or cytotoxicity of the prepared copolymer micelles.     

Mutant prevention concentration of colistin alone and in combination with rifampicin for multidrug-resistant <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis>

Colistin-susceptible isolates of Acinetobacter baumannii often contain subpopulations that are resistant to colistin. Monotherapy with colistin can lead to selective growth of these subpopulations and emergence of colistin-resistant strains. Our objectives were to explore the susceptibility pattern of colistin-resistant subpopulations and investigate if combining colistin with a second antibiotic could prevent their selective growth. Four colistin-susceptible clinical isolates of A. baumannii and one reference isolate were used. The mutant prevention concentration (MPC) of colistin, i.e. the concentration required to block growth of all single-step-mutant subpopulations, was determined by plating an inoculum of 10⁹ CFU on Mueller Hinton agar (MHA)-plates containing 2-fold dilutions of colistin (0.125–128 mg/L). Susceptibility testing of colistin-resistant subpopulations, obtained in the MPC assay, was performed with Etest. The MPC of colistin, in combination with rifampicin, was determined by plating an inoculum of 10⁹ CFU on MHA-plates containing colistin (0.125–128 mg/L) and fixed concentrations of rifampicin (1.1 mg/L or 4.4 mg/L). The colistin-resistant subpopulations demonstrated increased susceptibility to a number of agents compared to their main populations. These subpopulations were even susceptible to agents that normally are inactive against gram-negative bacteria and all had rifampicin MICs of?<?0.002 mg/L. The combination of colistin and rifampicin completely inhibited the growth of all colistin-resistant subpopulations and significantly lowered the MPC of colistin for A. baumannii. Combining colistin with rifampicin could be a way to prevent selective growth of colistin-resistant subpopulations of A. baumannii and possibly the emergence of colistin-resistant strains.     

In vitro and in vivo evaluation of redox-responsive sorafenib carrier nanomicelles synthesized from poly (acryic acid) -cystamine hydrochloride-D-α-tocopherol succinate

In this study, a controlled drug release system based on redox-responsive nanomicelles for drug delivery was described. The system was constructed by linking poly (acryic acid) (PAA) with D-α-tocopherol succinate (VES) via a disulfide bond linker (ss). This amphiphilic polymer (PAAssVES) was synthesized by coupling reaction and its chemical structure was confirmed by FT-IR and ¹HNMR analyses. PAAssVES was found to self-assemble into nanomicelles with diameter of about 130 nm, and a critical micelle concentration of about 6.3 μg/mL. The Sorafenib-loaded nanomicelles (SFN-NM) were almost spherical as observed by transmission electron microscopy. Differential scanning calorimetry analysis showed that Sorafenib (SFN) was entrapped in the micelles in an amorphous or molecular state. The safety of SFN-NM was confirmed by hemolysis study. The release of SFN from the nanomicelles was dependent on the concentration of glutathione (GSH), with 85% of the drug being released under the maximum concentration (40 mM) of GSH used. SFN-NM exhibited stronger cytotoxicity than free SFN against BGC-823 cells under the same SFN concentration. Furthermore, pharmacokinetics study showed that the bioavailability of SFN in rat obtained by injecting the animal with SFN-NM was about 2.8-fold the bioavailability of SFN obtained by injecting the animal with free SFN. Thus, the redox-responsive SFN delivery system described in the current study could be considered as a carrier for delivering SFN.     

Cytokine response to acute running in recreationally-active and endurance-trained men

To compare the cytokine response to exhaustive running in recreationally-active (RA) and endurance-trained (ET) men. Eleven RA men (VO_2max 55 ± 7 mL·min^?1·kg^?1) and 10 ET men (VO_2max 68 ± 7 mL·min^?1·kg^?1) followed a controlled diet and refrained from volitional exercise for 8 days. On the fourth day, participants completed 60 min of treadmill running (65 % VO_2max), followed by intermittent running to exhaustion (70 % VO_2max). Fasting blood was obtained at baseline, after 20, 40 and 60 min of exercise, at the end of intermittent exercise, during 2 h of recovery and on four follow-up days (FU1–FU4). Tumour necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-1 receptor antagonist (IL-1ra) and creatine kinase (CK) were measured. Exercise increased the concentrations of all cytokines and CK, but there were no significant differences between groups. IL-1β increased (2.2–2.5-fold, P < 0.001) during exercise, while TNF-α was increased (1.6–2.0-fold, P < 0.001) during exercise and for 2 h post-exercise. IL-6 (71–84-fold, P < 0.001) and IL-1ra (52–64-fold, P < 0.001) were increased throughout exercise and up to FU1, peaking immediately after exercise and at 1.5–2 h post-exercise, respectively. CK concentrations were increased (P < 0.001) throughout exercise and up to FU4, peaking at FU1, but were not associated with changes in any cytokines. Exhaustive running resulted in modest and transient increases in TNF-α and IL-1β, and more marked and prolonged increases in IL-6 and IL-1ra, but improved training status did not affect this response. Increased CK might indicate either exercise-induced muscle cell disruption or increased cell permeability, although neither appears to have contributed to the increased cytokine concentrations.     

In vitro drug release of natamycin from β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin-functionalized contact lens materials

Purpose: The antifungal agent natamycin can effectively form inclusion complexes with beta-cyclodextrin (β-CD) and 2-hydroxypropyl-β-cyclodextrin (HP-βCD) to improve the water solubility of natamycin by 16-fold and 152-fold, respectively (Koontz, J. Agric. Food. Chem. 2003). The purpose of this study was to develop contact lens materials functionalized with methacrylated β-CD (MβCD) and methacrylated HP-βCD (MHP-βCD), and to evaluate their ability to deliver natamycin in vitro. Methods: Model conventional hydrogel (CH) materials were synthesized by adding varying amounts of MβCD and MHP-βCD (0, 0.22, 0.44, 0.65, 0.87, 1.08% of total monomer weight) to a monomer solution containing 2-hydroxyethyl methacrylate (HEMA). Model silicone hydrogel (SH) materials were synthesized by adding similar concentrations of MβCD and MHP-βCD to N,N-dimethylacrylamide (DMAA)/10% 3-methacryloxypropyltris(trimethylsiloxy)silane (TRIS). The gels were cured with UV light, washed with ethanol and then, hydrated for 24 h (h). The model materials were then incubated with 2 mL of 100 μg/mL of natamycin in phosphate buffered saline (PBS) pH 7.4 for 48 h at room temperature. The release of natamycin from these materials in 2 mL of PBS, pH 7.4 at 32 ± 2 °C was monitored using UV–vis spectrophotometry at 304 nm over 24 h. Results: For both CH and SH materials, functionalization with MβCD and MHP-βCD improved the total amount of drugs released up to a threshold loading concentration, after which further addition of methacrylated CDs decreased the amount of drugs released (p < 0.05). The addition of CDs did not extend the drug release duration; the release of natamycin by all model materials reached a plateau after 12 h (p < 0.05). Overall, DMAA/10% TRIS materials released significantly more drug than HEMA materials (p < 0.05). The addition of MHP-βCD had a higher improvement in drug release than MβCD for both HEMA and DMAA/10% TRIS gels (p < 0.05). Conclusions: A high loading concentration of methacrylated CDs decreases overall drug delivery efficiency, which likely results from an unfavorable arrangement of the CDs within the polymer network leading to reduced binding of natamycin to the CDs. HEMA and DMAA/10% TRIS materials functionalized with MHP-βCD are more effective than those functionalized with MβCD to deliver natamycin.     

Synthetic polymannose as a drug carrier: synthesis,toxicity and anti-fungal activity of polymannose-amphotericin B conjugates

Polymannose (PM) having a weight-average molar mass (M_w) of 30–53 kDa was synthesized by the polycondensation of mannose using phosphorous acid as the catalyst and characterized by various techniques such as NMR, IR, GPC and polarimetry. 2D NMR results confirmed the presence of (1 → 6)-linked α-D-mannose residues as backbone with O-3 and O-2 substituted linear or branched chains in PM. Amphotericin B (AmB) was conjugated to periodate-oxidized PM through Schiff’s linkages at 20 wt% concentration. The AmB-PM conjugates were highly soluble in phosphate buffered saline (180–250 mg/mL), exhibited negligible hemolytic potential to human erythrocytes even at a concentration of 200 μg/mL (equivalent to ~40 μg/mL AmB) and were non-toxic to human embryonic kidney (HEK293T) cells even at a concentration of 250 μg/mL (equivalent to ~50 μg/mL AmB). The minimum inhibitory concentration of the AmB-PM conjugates against C. albicans, C. parapsilosis and C. neoformans was in the range of 0.5–1.0 μg/mL. Mannose receptors are widely expressed on myeloid cells such as macrophages, neutrophils, and dendritic cells. Therefore, apart from treating fungal infections, AmB-PM conjugates also may have therapeutic potential for the treatment of macrophage-associated diseases such as leishmaniasis where mannose receptors are overexpressed.     

Single-channel biophysical and pharmacological characterizations of native human large-conductance calcium-activated potassium channels in freshly isolated detrusor smooth muscle cells

Recent studies have demonstrated the importance of large-conductance Ca²⁺-activated K⁺ (BK) channels in detrusor smooth muscle (DSM) function in vitro and in vivo. However, in-depth characterization of human native DSM single BK channels has not yet been provided. Here, we conducted single-channel recordings from excised patches from native human DSM cells. Inside-out and outside-out recordings in high K⁺ symmetrical solution (containing 140 mM KCl and ~300 nM free Ca²⁺) showed single-channel conductance of 215–220 pS, half-maximum constant for activation of ~+75 to +80 mV, and low probability of opening (P _o) at +20 mV that increased ~10-fold at +40 mV and ~60-fold at +60 mV. Using the inside-out configuration at +30 mV, reduction of intracellular [Ca²⁺] from ~300 nM to Ca²⁺-free decreased the P _o by ~85 %, whereas elevation to ~800 nM increased P _o by ~50-fold. The BK channel activator NS1619 (10 μM) enhanced the P _o by ~10-fold at +30 mV; subsequent application of the selective BK channel inhibitor paxilline (500 nM) blocked the activity. Changes in intracellular [Ca²⁺] or the addition of NS1619 did not significantly alter the current amplitude or single-channel conductance. This is the first report to provide biophysical and pharmacological profiles of native human DSM single BK channels highlighting their importance in regulating human DSM excitability.     

Methoxy poly (ethylene glycol)-b-poly (δ-valerolactone) copolymeric micelles for improved skin delivery of ketoconazole

Ketoconazole is a broad spectrum imidazole antifungal drug. For the treatment of superficial fungal infections with ketoconazole, it needs to be permeated to deep skin layers. In order to develop topical formulation of ketoconazole for improving its skin deposition and water-solubility, ketoconazole-loaded methoxy poly (ethylene glycol)-b-poly (δ-valerolactone) micelles were developed through thin-film hydration method. Particle size, drug loading capacity, infrared spectrum and X-ray diffraction of drug-loaded micelles were characterized. The optimal drug formulation was selected for skin delivery and deposition investigation performed by use of mice skin, and its in vitro release and antifungal activity were also investigated. Penetration and distribution in the skin were also visualized using fluorescein-loaded micelles and fluorescence microscopy. The drug-loaded micelles were obtained with encapsulation efficiency of 86.39% and particle diameter of about 12 nm. The micelles made ketoconazole aqueous solubility increase to 86-fold higher than crude one. Ketoconazole-loaded micelles showed no skin permeation of ketoconazole, obviously enhance skin deposition and demonstrated similar antifungal activity as compared with marketed ketoconazole cream. Fluorescein-loaded micelles displayed higher skin deposition than fluorescein water solution. These results demonstrate that the MPEG-PVL micelle is a potential delivery system for ketoconazole in the field of skin delivery.     

pH-sensitive micelles self-assembled from multi-arm star triblock co-polymers poly(ε-caprolactone)-b-poly(2-(diethylamino)ethyl methacrylate)-b-poly(poly(ethylene glycol) methyl ether methacrylate) for controlled anticancer drug delivery

A series of amphiphilic 4- and 6-armed star triblock co-polymers poly(ε-caprolactone)-b-poly(2-(diethylamino)ethyl methacrylate)-b-poly(poly(ethylene glycol) methyl ether methacrylate) (4/6AS-PCL-b-PDEAEMA-b-PPEGMA) were developed by a combination of ring opening polymerization and continuous activators regenerated by electron transfer atom transfer radical polymerization. The critical micelle concentration values of the star co-polymers in aqueous solution were extremely low (2.2–4.0 mg l^–1), depending on the architecture of the co-polymers. The self-assembled blank and doxorubicin (DOX)-loaded three layer micelles were spherical in shape with an average size of 60–220 nm determined by scanning electron microscopy and dynamic light scattering. The in vitro release behavior of DOX from the three layer micelles exhibited pH-dependent properties. The DOX release rate was significantly accelerated by decreasing the pH from 7.4 to 5.0, due to swelling of the micelles at lower pH values caused by the protonation of tertiary amine groups in DEAEMA in the middle layer of the micelles. The in vitro cytotoxicity of DOX-loaded micelles to HepG2 cells suggested that the 4/6AS-PCL-b-PDEAEMA-b-PPEGMA micelles could provide equivalent or even enhanced anticancer activity and bioavailability of DOX and thus a lower dosage is sufficient for the same therapeutic efficacy. The results demonstrate that the pH-sensitive multilayer micelles could have great potential application in delivering hydrophobic anticancer drugs for improved cancer therapy.     

Simulation of Intrathrombus Fluid and Solute Transport Using In Vivo Clot Structures with Single Platelet Resolution

The mouse laser injury thrombosis model provides up to 0.22 μm-resolved voxel information about the pore architecture of the dense inner core and loose outer shell regions of an in vivo arterial thrombus. Computational studies were conducted on this 3D structure to quantify transport within and around the clot: Lattice Boltzmann method defined vessel hemodynamics, while passive Lagrangian Scalar Tracking with Brownian motion contribution simulated diffusive-convective transport of various inert solutes (released from lumen or the injured wall). For an input average lumen blood velocity of 0.478 cm/s (measured by Doppler velocimetry), a 0.2 mm/s mean flow rate was obtained within the thrombus structure, most of which occurred in the 100-fold more permeable outer shell region (calculated permeability of the inner core was 10^?11 cm²). Average wall shear stresses were 80–100 dyne/cm² (peak values >200 dyne/cm²) on the outer rough surface of the thrombus. Within the thrombus, small molecule tracers (0.1 kDa) experienced ~70,000 collisions/s and penetrated/exited it in about 1 s, whereas proteins (~50 kDa) had ~9000 collisions/s and required about 10 s (tortuosity ~2–2.5). These simulations help define physical processes during thrombosis and constraints for drug delivery to the thrombus.     

Improving the anti-ovarian cancer activity of docetaxel with biodegradable self-assembly micelles through various evaluations

Docetaxel (DOC) produces anti-tumor effects by inducing apoptosis and inhibiting cell growth. However, its clinical application is limited by its hydrophobicity and low biocompatibility. Therefore, improving DOC's water solubility, biocompatibility, and anti-tumor effects are important goals that will improve its clinical utility. In this work, DOC and methoxy poly(ethylene glycol) (MPEG)/polycaprolactone (PCL) (MPEG-PCL) showed good compatibility through computer simulations. We prepared DOC-loaded polymeric micelles (DOC/MPEG-PCL micelles) with drug loading of 6.82% and encapsulation efficiency of 98.36%; these were monodispersed and approximately 30 nm in diameter, and released DOC over an extended period in vitro and in vivo. In addition, DOC/MPEG-PCL micelles inhibited cell growth and induced apoptosis more effectively than free DOC in vitro. Furthermore, DOC/MPEG-PCL micelles inhibited ovarian tumor growth more significantly than free DOC. Immunohistochemical analysis indicated that DOC/MPEG-PCL micelles improved DOC's anti-tumor effect by enhancing tumor cell apoptosis and suppressing tumor cell proliferation. Moreover, in bio-imaging analysis, DOC/MPEG-PCL micelles showed a higher concentration and a longer retention time in ovarian tumor tissue than did free DOC, indicating that the DOC/MPEG-PCL micelles delivered more anti-tumor drug to the tumor. Our data suggest that DOC/MPEG-PCL micelles have the potential to be applied clinically in ovarian cancer therapy.     

Preparation and characterization of magnetic thermosensitive fluorouracil micelles

In this study, we synthesized P(NIPAM-co-DMAM)-b-PLA polymers with free radical polymerization and ring-opening addition polymerization, and immediately assembled ‘dextran magnetic layered double hydroxide fluorouracil’ (DMF) magnetic particles into the core of the amphiphilic polymer micelles with synchronous hydration and dialysis, to generate a magnetic thermosensitive fluorouracil drug delivery system. The basic properties of the micelle particles, such as the core–shell-type structure, size, and zeta potential, were studied with ¹H-NMR, FTIR, TEM, TGA, laser nanoparticle size analysis, and other characterization techniques. The thermosensitivity of the micelles was investigated by measuring parameters such as the lower critical solution temperature (LCST) and the relationship between the particle size variation and temperature. The drug release curves for the micelles at different temperatures were constructed with a dialysis method. The LCST of the triblock polymers was 42 °C. The particle sizes of the blank micelles and DMF-loaded micelles were 493.6 ± 1.8 nm and 464.9 ± 4.1 nm, respectively, at 25 °C. When the temperature was higher than LSCT, a contraction phase change in the micelle structure occurred, a significant characteristic of the core–shell-type structure, and reversible phase transition phenomena. The release behavior of the drug-loaded micelles showed obvious variations with temperature. Therefore, the magnetic thermosensitive fluorouracil drug delivery system has a good magnetic response and excellent temperature controlled release characteristics, so it can be used as a drug delivery system in magnetically and thermally targeted chemotherapy for tumors.     

The detection of endotoxin by in vitro production of endogenous pyrogen: Comparison with limulus amebocyte lysate gelation

The sensitivities of leukocyte endogenous pyrogen (EP) production and limulus amebocyte lysate (LAL) gelation to endotoxin from E. coli (minimum i.v. pyrogenic dose 4 ng/kg in rabbits) were determined. Concentrations of 0.5–1.0 ng/ml could be detected by LAL. The minimum endotoxin concentration which generated detectable EP from 2 × 10⁶ monocytes was 10-fold lower (0.05–0.1 ng/ml). At an endotoxin concentration of 0.4 ng/ml the minimum number of monocytes required for detectable EP production was 5 × 10⁵. It is concluded that the LAL gelation test cannot safely be used to exclude significant endotoxin contamination in a cellular system where EP production is being measured. The same conclusion applies even more forcibly to the in vitro production of lymphocyte activating factor (LAF, interleukin-1), since it appears that LAF and EP are identical and sub-pyrogenic amounts of EP are easily detectable in the LAF assay.     

Bone formation is suppressed with multi-stressor military training

Purpose

To determine the effects of US Army Ranger Training, an 8-week, physically demanding program (energy expenditure of 2,500–4,500 kcal/day) with energy restriction (deficit of 1,000–4,000 kcal/day) and sleep deprivation (<4 h sleep/night) on bone metabolism.

Methods

Blood was collected from 22 men (age 24 ± 4 years) before and after training. Follow-up measurements were made in a subset of 8 subjects between 2 and 6 weeks after training. Serum was analyzed for bone formation biomarkers [bone alkaline phosphatase (BAP) and osteocalcin (OCN)], bone resorption biomarkers [C-telopeptide cross-links of type I collagen (CTX) and tartrate-resistant acid phosphatase (TRAP5b)], calcium, parathyroid hormone (PTH), and vitamin D (25(OH)D). Data were analyzed using a paired t test to compare baseline to immediate post-training measures. A repeated-measures ANOVA with time as the only factor was used to analyze data on the subset of 8 subjects who completed follow-up data collection.

Results

BAP and OCN significantly decreased by 22.8 ± 15.5 % (pre 41.9 ± 10.1; post 31.7 ± 7.8 ng/ml) and 21.0 ± 23.3 % (pre 15.0 ± 3.5; post 11.3 ± 2.1 ng/ml), respectively, with training, suggesting suppressed bone formation. OCN returned to baseline, while BAP remained suppressed 2–6 weeks post-training. TRAP5b significantly increased by 57.5 ± 51.6 % (pre 3.0 ± 0.9; post 4.6 ± 1.4 ng/ml) from pre- to post-training, suggesting increased bone resorption, and returned to baseline 2–6 weeks post-training. PTH Increased significantly by 37.3 ± 45.2 % with training. No changes in CTX, calcium, or PTH were detected.

Conclusions

These data indicate that multi-stressor military training results in increased bone resorption and suppressed bone formation, with recovery of bone metabolism 2–6 weeks after completion of training.