Acidity-activatable dynamic hybrid nanoplatforms derived from extracellular vesicles of M1 macrophages enhance cancer immunotherapy through synergistic triple immunotherapy.

Immunotherapy exhibits considerable promise for sustained tumor reduction. However, current cancer immunotherapy methods elicit limited responses due to the inadequate immunogenicity exhibited by cancer cells. This obstacle may be addressed using nanoplatforms that can activate synergistic therapies (photodynamic therapy and ferroptosis) in response to the acidic pH of the tumor microenvironment. We previously developed an amphiphilic photosensitizer, SR780, which displays satisfactory photodynamic effects. This photosensitizer is inactivated when bound to Fe3+ (SR780Fe) but is activated upon release in mildly acidic conditions. In this study, M1 macrophage-derived extracellular vesicles (EVs) were fused with REV and SR780Fe-loaded liposomes (REV@SR780Fe@Lip) to form REV@SR780Fe@LEV hybrid nanovesicles. Further modification with the RS17 peptide for tumor targeting enabled a combination of photodynamic therapy, ferroptosis, and cGAS-STING pathway activation, resulting in enhanced antitumor efficacy through a synergistic effect. Upon laser irradiation, REV@SR780Fe@LEV-RS17 demonstrated antitumor effects in 4T1 breast cancer models, including the inhibition of lung and liver metastasis, as well as prevention of tumor recurrence.

Association between lipid metabolism and periodontitis in obese patients: a cross-sectional study.

BACKGROUND: To investigate the association between clinical periodontal parameters of periodontitis, serum lipid metabolism markers and adipokines' levels in patients with obesity and periodontitis. METHODS: A total of 112 patients admitted to Hospital of Xi'an Jiaotong University were included in this study. They were divided into normal body weight group (18.5 < body mass index, BMI < 25, n = 36), overweight group (25 ≤ BMI < 30, n = 38), and obesity group (BMI ≥ 30, n = 38) accordingly. The diagnosis of periodontitis was based on the newest international classification of periodontitis. Full-mouth clinical periodontal measurements included: plaque index, periodontal pocket depth, clinical attachment level, and bleeding on probing. Gingival crevicular fluid samples were analyzed for: Interleukin-1ß, tumor necrosis factor-α, Interleukin-6 and C-reactive protein. Serum triglycerides, total cholesterol, low density lipoprotein cholesterol, high density lipoprotein cholesterol and glycosylated hemoglobin levels were measured. Visfatin, leptin, resistin, and adiponectin levels in serum were also measured. RESULTS: The ratio of participants without periodontitis was significantly highest in normal weight group, and the proportion of severe periodontitis (stage III and IV) was highest in obesity group. The periodontal pocket depth, clinical attachment level, and the inflammatory cytokines in gingival crevicular fluid in obesity group and overweight group were higher than those in normal body weight group. The BMI and waist-to-hip ratio (WHR) were significantly positive correlated with periodontal pocket depth and clinical attachment level. Using a Multivariate logistic regression model, periodontitis correlates to BMI, WHR, serum levels of triglyceride, total cholesterol, low density lipoprotein, and adipokines such as visfatin, leptin, and resistin. CONCLUSIONS: Obesity is positively correlated with the aggravation of periodontitis. Obesity may aggravate the damage to periodontal tissue by regulating the secretion level of adipokines.

Establishment and validation of a sensitive LC-MS/MS method for the quantification of KRASG12C protein PROTAC molecule LC-2 in rat plasma and its application to in vivo pharmacokinetic studies of LC-2 PEGylated liposomes.

LC-2, is a molecule of proteolysis targeting chimeras (PROTACs), with a large molecular weight, poor water solubility and low system bioavailability, which was designed to degrade KRASG12C protein. In this study, LC-2 PEGylated liposomes were developed and characterized. Moreover, a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in rat plasma was established and effectively utilized for an in vivo pharmacokinetic investigation. LC-2 PEGylated liposomes with better properties were prepared by an improved ethanol injection method. The chromatographic separation was achieved on an Agilent Eclipse XDB-CN column (100 × 2.1 mm, 3.5 µm) with acetonitrile-ammonium deionized water (5 mm; 80:20, v/v) at a flow rate of 0.5 ml/min. The mass spectra of LC-2 and the IS (gefitinib) were obtained at m/z 1132.5 → 626.4 and 447.1 → 128.2, respectively. The pharmacokinetic study was carried out by analyzing plasma concentrations of LC-2 solution or produced LC-2 PEGylated liposomes in rats using the developed and validated method. The pharmacokinetic results indicate that PEGylated liposome-encapsulation protected LC-2 from the influence of endogenous protein binding, improved insolubility, prolonged half-life and increased system bioavailability. This study provides a feasible solution for future preclinical and clinical studies of LC-2 and/or other PROTACs.

Morphology/Interstitial Fluid Pressure-Tunable Nanopomegranate Designed by Alteration of Membrane Fluidity under Tumor Enzyme and PEGylation.

Up to now, insufficient drug accumulation in tumor remains a major challenge for nanochemotherapy. However, the spherical nanocarriers with large diameter, which are beneficial for blood circulation and tumor extravasation, cannot travel deep in a tumor. Additionally, high tumor interstitial fluid pressure (IFP) in the tumor microenvironment may promote the efflux of the penetrable nanodrugs. Therefore, the size and shape of nanocarriers as well as the tumoral IFP can be regulated synchronously for improved tumor penetration and combined chemotherapy. Herein, a novel dual-functional polymer-polypeptide (Biotin-PEG2000-GKGPRQITITK) for both verified tumor targeting and responsiveness was synthesized to construct the "peel" of nanopomegranate-like nanovectors (DI-MPL), in which docetaxel-loaded micelles was encapsulated as "seeds". Interestingly, DI-MPL was endowed multi-abilities of tunable size/shape switch and controlled release of IFP alleviator imatinib (IM), which were developed with one and the same strategy-alteration of membrane fluidity under the cleavage of polymer-polypeptide and PEGylation. As a result, the peel of DI-MPL could turn into small pieces with the seed scattered out in response to matrix metalloproteinase-9 (MMP-9), making nanopomegranate (180 nm) switch into spheres/disks (40 nm), during which IM is released to reduce IFP synchronously. With prominent tumor penetration ability in both multicellular tumor spheroids (MCTS) and tumor tissue, DI-MPL exhibited optimal inhibition of MCTS growth and the enhanced chemotherapy in comparison to other preparations. Meanwhile, the improved penetrability of DI-MPL in tumor tissue was found to be related to the reduced IFP, which is achieved via inhibiting expression of phosphorylated platelet-derived growth factor receptor-ß (p-PDGFR-ß) by IM. Altogether, the bilateral adjusting strategies from nanocarrier size/shape and tumoral IFP with a single enzyme-responsive material could provide a potential combined chemotherapy to improve tumor penetration.

Plasmonic tooth-multilayer structure with high enhancement field for surface enhanced Raman spectroscopy.

The significant enhancement seen in surface-enhanced Raman scattering (SERS) heavily relies on the ability of plasmonic structures to strongly confine light. Current techniques used to fabricate plasmonic nanostructures have been limited in their reproducibility for bottom-up techniques or their feature size for top-down techniques. Here, we propose a tooth multilayer structure that can be fabricated by using physical vapor deposition and selective wet etching, achieving extremely small feature sizes and high reproducibility. A multilayer structure composed of two alternating materials whose thicknesses can be controlled accurately in the nanometer range is deposited on a flat substrate using ion-beam sputtering. Subsequent selective wet etching is used to form nanogaps in one of the materials constituting the multilayer, with the depth of the nanogaps being controlled by the wet etching time. Combining both techniques can allow the nanogap dimensions to be controlled at sub 10 nm length scale, thus achieving a tooth multilayer structure with high enhancement and tunability of the resonance mode over a broad range, ideal for SERS applications.

Lipid Nanoparticular Codelivery System for Enhanced Antitumor Effects by Ferroptosis-Apoptosis Synergistic with Programmed Cell Death-Ligand 1 Downregulation.

Intrinsic or acquired resistance to chemical drugs severely limits their therapeutic efficacy in cancer treatment. Various intracellular antioxidant molecules, particularly glutathione (GSH), play a crucial role in maintaining intracellular redox homeostasis by mitigating the overproduced reactive oxygen species (ROS) due to rapid cell proliferation. Notably, these antioxidants also eliminate chemical-drug-induced ROS, eventually diminishing their cytotoxicity and rendering them less effective. In this study, we combined erastin, a GSH biosynthesis inhibitor, with 2'-deoxy-5-fluorouridine 5'-monophosphate sodium salt (FdUMP), an ROS-based drug, to effectively disrupt intracellular redox homeostasis and reverse chemotherapy resistance. Therefore, efficient ferroptosis and apoptosis were simultaneously induced for enhanced antitumor effects. Additionally, we employed small interfering RNA targeting PD-L1 (siPD-L1) as a third agent to block immune-checkpoint recognition by CD8+ T cells. The highly immunogenic cell peroxidates or damage-associated molecular patterns (DAMPs) induced by erastin acted synergistically with downregulated PD-L1 to enhance the antitumor effects. To codeliver these three drugs simultaneously and efficiently, we designed GE11 peptide-modified lipid nanoparticles (LNPs) containing calcium phosphate cores to achieve high encapsulation efficiencies. In vitro studies verified its enhanced cytotoxicity, efficient intracellular ROS induction and GSH/GPX4 downregulation, substantial lipid peroxidation product accumulation, and mitochondrial depolarization. In vivo, this formulation effectively accumulated at tumor sites and achieved significant tumor inhibition in subcutaneous colon cancer (CRC) mouse models with a maximum tumor inhibition rate of 83.89% at a relatively low dose. Overall, a strategy to overcome clinical drug resistance was verified in this study by depleting GSH and activating adaptive immunity.

Complete genome sequencing and analysis of six enterovirus 71 strains with different clinical phenotypes.

BACKGROUND: Hand, foot and mouth diseases (HFMD) caused by enterovirus 71(EV71) presents a broad spectrum of clinical manifestations ranging from mild febrile disease to fatal neurolocal disease. However, the mechanism of virulence is unknown. METHODS: We isolated 6 strains of EV71 from HFMD patients with or without neurological symptoms, and sequenced the whole genomes of the viruses to reveal the virulence factors of EV71. RESULTS: Phylogenetic tree based on VP1 region showed that all six strains clustered into C4a of C4 sub-genotype. In the complete polypeptide, 298 positions were found to be variable in all strains, and three of these positions (Val(P814)/Ile(P814) in VP1, Val(P1148)/Ile(P1148) in 3A and Ala(P1728)/Cys)/Val(P1728) in 3C) were conserved among the strains with neurovirulence, but variable in strains without neurovirulence. In the 5'-UTR region, it showed that the first 10 nucleotides were mostly conserved, however from the 11th nucleotide, nucleotide insertions and deletions were quite common. The secondary structure prediction of 5'-UTR sequences showed that two of three strains without neurovirulence (SDLY11 and SDLY48) were almost the same, and all strains with neurovirulence (SDLY96, SDLY107 and SDLY153) were different from each other. SDLY107 (a fatal strain) was found different from other strains on four positions (C(P241)/T(P241), A(P571)/T(P571), C(P579)/T(P579) in 5'-UTR and T(P7335)/C(P7335) in 3'-UTR). CONCLUSIONS: The three positions (Val(P814)/Ile(P814) in VP1, Val(P1148)/Ile(P1148) in 3A and Ala(P1728)/Cys(P1728)/Val(P1728) in 3C), were different between two phenotypes. These suggested that the three positions might be potential virulent positions. And the three varied positions were also found to be conserved in strains with neurovirulence, and variable in strains without neurovirulence. These might reveal that the conservation of two of the three positions or the three together were specific for the strains with neurovirulence. Varation of secondary structure of 5'-UTR, might be correlated to the changes of viral virulence. SDLY107 (a fatal strain) was found different from other strains on four positions, these positions might be related with death.

Investigation on Flexural Fracture Behaviour of Bolted Spherical Joints with Crack Propagation in Screw Threads.

Bolted spherical joints, due to their prominent merits in installation, have been widely used in modern spatial structures. Despite significant research, there is a lack of understanding of their flexural fracture behaviour, which is important for the catastrophe prevention of the whole structure. Given the recent development to fill this knowledge gap, it is the objective of this paper to experimentally investigate the flexural bending capacity of the overall fracture section featured by a heightened neutral axis and fracture behaviour related to variable crack depth in screw threads. Accordingly, two full-scale bolted spherical joints with different bolt diameters were evaluated under three-point bending. The fracture behaviour of bolted spherical joints is first revealed with respect to typical stress distribution and fracture mode. A new theoretical flexural bending capacity expression for the fracture section with a heightened neutral axis is proposed and validated. A numerical model is then developed to estimate the stress amplification and stress intensity factors related to the crack opening (mode-I) fracture for the screw threads of these joints. The model is validated against the theoretical solutions of the thread-tooth-root model. The maximum stress of the screw thread is shown to take place at the same location as the test bolted sphere, while its magnitude can be greatly reduced with an increased thread root radius and flank angle. Finally, different design variants related to threads that have influences on the SIFs are compared, and the moderate steepness of the flank thread has been found to be efficient in reducing the joint fracture. The research findings could thus be beneficial for further improving the fracture resistance of bolted spherical joints.

Novel oxoisoaporphine-based inhibitors of acetyl- and butyrylcholinesterase and acetylcholinesterase-induced beta-amyloid aggregation.

A series of novel oxoisoaporphine-based inhibitors (10-aminoalkylamino-1-azabenzanthrone Ar-NH(CH(2))(n)NR(1)R(2)) of acetylcholinesterase (AChE) has been designed, synthesized, and tested for their ability to inhibit AChE, butyrylcholinesterase (BChE) and AChE-induced ß-amyloid (Aß) aggregation. The synthetic compounds exhibited high AChE inhibitory activity with IC(50) values in the submicromolar range in most cases. Non-competitive binding mode was found for these derivatives by the graphical analysis of steady-state inhibition data. Moreover, all compounds exhibit significant inhibitory activity on AChE-induced Aß aggregation with inhibitory potency from 54.5% to 93.5%. Finally, six out of twelve synthetic compounds were predicted to be able to cross the blood-brain barrier (BBB) to reach their targets in the central nervous system (CNS) according to a parallel artificial membrane permeation assay for BBB. The result encourages us to study this class of compounds thoroughly and systematically.

Porous metal membranes for solid-phase extraction of polycyclic aromatic hydrocarbons.

Solid-phase extraction (SPE) is one of the most important techniques for sample preparation, purification, concentration and cleanup. Membranes made from synthetic organic polymers, cellulose, or glass fibers are used for sample pretreatment. In this work, we report that a porous metal membrane, the metal filter in HPLC, was used as a novel kind of solid-phase extraction adsorbent material. To evaluate the performance of the porous metal membrane for the SPE, naphthalene, fluorene, anthracene, phenanthrene, fluoranthene, pyrene, chrysene, perylene and benzo(a)pyrene were selected as analytes. Several parameters that affected the extraction efficiency such as the extraction time, the concentration of NaCl, the extraction temperature and the agitation speed were optimized. The experimental result indicates that the porous metal membrane possesses high adsorption ability to the tested polycyclic aromatic hydrocarbons (PAHs). Under the optimum conditions, the detection limits of the developed method were in the range of 0.03-0.082 µg L(-1) (S/N = 5), and excellent linear correlations between peak area and concentration of PAHs were found over the range of 0.1-60 µg L(-1). The precisions (RSD) for five replicate extractions of the PAHs from sample solutions were in the range of 2.6-5.0%. The recoveries of the PAHs from tap water and river water samples spiked with 9 PAHs (20 µg L(-1) of each individual PAH) ranged from 83.0% to 112.5%. The porous metal membrane is durable, simple, inexpensive, reproducible and has a high adsorption ability for use in SPE of PAHs.

Hyaluronic Acid Capped, Irinotecan and Gene Co-Loaded Lipid-Polymer Hybrid Nanocarrier-Based Combination Therapy Platform for Colorectal Cancer.

BACKGROUND: The current approach for treating colorectal cancer favors the use of drug and gene combination therapy, and targeted nano-systems are gaining considerable attention for minimizing toxicity and improving the efficacy of anticancer treatment. The aim of this study was to develop ligand-modified, irinotecan and gene co-loaded lipid-polymer hybrid nanocarriers for targeted colorectal cancer combination therapy. METHODS: Hyaluronic acid modified, irinotecan and gene co-loaded LPNs (HA-I/D-LPNs) were prepared using a solvent-evaporation method. Their average size, zeta potential, drug and gene loading capacity were characterized. The in vitro and in vivo gene transfection and anti-tumor ability of this nano-system were evaluated on colorectal cancer cells and mice bearing colorectal cancer model. RESULTS: HA-I/D-LPNs had a size of 182.3 ± 5.1, over 80% drug encapsulation efficiency and over 90% of gene loading capacity. The peak plasma concentration (Cmax) and half-life (T1/2) achieved from HA-I/D-LPNs were 41.31 ± 1.58 µg/mL and 12.56 ± 0.67 h. HA-I/D-LPNs achieved the highest tumor growth inhibition efficacy and the most prominent transfection efficiency in vivo. CONCLUSION: HA-I/D-LPNs exhibited the most remarkable tumor inhibition efficacy and best gene transfection efficiency in the tumor, which could prove the effects of the drug and gene combination therapy.

Anti-angiogenesis effect of generation 4 polyamidoamine/vascular endothelial growth factor antisense oligodeoxynucleotide on breast cancer in vitro.

OBJECTIVE: To study the effects of the generation 4 polyamidoamine/vascular endothelial growth factor antisense oligodeoxynucleotide (G4PAMAM/VEGFASODN) compound on the expressions of vascular endothelial growth factor (VEGF) and its mRNA of breast cancer cells and on the inhibition of vascular endothelial cells. METHODS: We examined the morphology of G4PAMAM/VEGFASODN compound and its pH stability, in vitro transfection efficiency and toxicity, and the expressions of VEGF and its mRNA. Methyl thiazolyl tetrazolium assay was used to detect the inhibitory function of the compound on vascular endothelial cells. RESULTS: The compound was about 10 nm in diameter and was homogeneously netlike. From pH 5 to 10, it showed quite a buffered ability. The 48-h transfection rate in the charge ratio of 1:40 was 98.76%, significantly higher than that of the liposome group (P<0.05). None of the transfection products showed obvious toxicity on the cells. The expressions of both VEGF protein and its mRNA after G4PAMAM/VEGFASODN transfection decreased markedly. CONCLUSION: With a low toxicity, high safety, and high transfection rate, G4PAMAM/VEGFASODN could be a promising gene vector. Specifically, it inhibits VEGF gene expression efficiently, laying a basis for further in vivo animal studies.

The untranslated regions of EV-A71 contribute to its pathogenicity and virulence.

Enterovirus A71 (EV-A71) is known for its manifestation as hand foot and mouth disease (HFMD), which has caused countless large-scale epidemic outbreaks throughout the world. However, the molecular pathogenesis of EV-A71 infection is still elusive. Previous studies found that the biological characteristics of a mild EV-A71 strain (SDLY1) and a severe EV-A71 strain (SDLY107) are significantly different, and sequence analysis showed that there are several differences in nucleotide sites of UTRs (88 nt, 123 nt, 143 nt, 154 nt, 187 nt, 241 nt, 243 nt, 253 nt, 291 nt, 438 nt, 440 nt, 571 nt, 579 nt, 602 nt, 658 nt, 664 nt, 690 nt, 696 nt, 7328 nt, 7335 nt, 7367 nt, and 7395 nt). The aim of this study was to determine whether these amino sites in UTRs are associated with the pathogenesis of EV-A71 and are responsible for different clinical manifestations. Based on the reverse genetics technology, we rescued two chimeric viruses SDLY107(1-5'UTR) and SDLY107(1-3'UTR) by replacing 5'UTR/3'UTR gene fragments of an infectious cDNA clone. Replication kinetics and cytotoxicity assays showed that the virulence of the two chimeric strains significantly changed in vitro. The viral loads of the two chimeric strains in infected ICR mice were reduced and pathological damage in the brains, lungs, intestinal tissues, and muscles were lightened. Our findings suggest that some nucleotide sites in UTRs may have a function in the pathogenicity and virulence of EV-A71.

Enterovirus 71 VP1 Protein Regulates Viral Replication in SH-SY5Y Cells via the mTOR Autophagy Signaling Pathway.

BACKGROUND: Enterovirus 71 (EV71) is the main pathogen that causes severe hand, foot, and mouth disease with fatal neurological complications. However, its neurovirulence mechanism is still unclear. Candidate virulence sites were screened out at structural protein VP1, but the function of these candidate virulence sites remains unclear. Several studies have shown that autophagy is associated with viral replication. However, the relationship between VP1 and autophagy in human neurons has not been studied. METHODS: A recombinant virus-SDLY107-VP1, obtained by replacing the VP1 full-length gene of the SDLY107 strain with the VP1 full-length gene of the attenuated strain SDJN2015-01-was constructed and tested for replication and virulence. We then tested the effect of the recombinant virus on autophagy in nerve cells. The effect of autophagy on virus replication was detected by western blot and plaque test. Finally, the changes of mTOR signaling molecules during EV71 infection and the effect of mTOR on virus replication at the RNA level were detected. RESULTS: Viral recombination triggered virulence attenuation. The replication ability of recombinant virus SDLY107-VP1 was significantly weaker than that of the parent strain SDLY107. The SDLY107 strain could inhibit autophagic flux and led to accumulation of autophagosomes, while the SDLY107-VP1 strain could not cause autophagosome accumulation. The synthesis of EV71 RNA was inhibited by inhibiting mTOR. CONCLUSIONS: Replacement of VP1 weakened the replication ability of virulent strains and reduced the level of autophagy in nerve cells. This autophagy facilitates the replication of virulent strains in nerve cells. VP1 is an important neurovirulence determinant of EV71, which affects virus replication by regulating cell autophagy. mTOR is a key molecule in this type of autophagy.

[Anti-angiogenesis effect of G4PAMAM/VEGFASODN on breast cancer in vitro].

OBJECTIVE: To investigate the effect of G4PAMAM/VEGFASODN compound on expression of VEGF and VEGF mRNA in MDA-MB-231 breast cancer cells and the growth inhibition of vascular endothelial cells. METHODS: The diameter of G4PAMAM/VEGFASODN granule was measured by transmission electron microscopy, and its stability under different pH was also observed. The efficiency of transfection in vitro was detected by flow cytometer and the positively transfected cells were detected by laser scanning confocal microscope. The survival rate and the inhibitory rate of vascular endothelial cells were determined by MTT assay. The expression of protein VEGF was detected by immunohistochemical method and the expression of VEGF mRNA was detected by RT-PCR. RESULT: The diameter of G4PAMAM/VEGFASODN granules was about 10 nm and it arranged as homogeneous netlike. Under pH 5-10 G4PAMAM/VEGFASODN presented highly stable and no dissociation was observed with different charge ratios. The 48-hour transfection rate of G4PAMAM/VEGFASODN in charge ratio of 1:40 was significantly higher than that of the lipofectamine group. None of the transfection products in each group showed cell toxicity. The staining of VEGF protein in the cytoplasm of MDA-MB-231 cells after G4PAMAM/ASODN transfection weakened markedly, and the positive expression rate decreased. Meanwhile, the VEGF mRNA expression was also decreased. CONCLUSION: With good stability and transfection rate, compound G4PAMAM/VEGFASODN can be a promising new nanometer vector of gene transfer system. The G4PAMAM/VEGFASODN can inhibit the expression of VEGF gene specifically and efficiently, which may be used for in vivo animal experiment.

Nonstructural protein 2A modulates replication and virulence of enterovirus 71.

Enterovirus 71 (EV71) can cause hand, foot, and mouth disease in children, and severe infections can induce neurological complications and even death. However, the pathogenesis of EV71 remains unknown. The 2A proteinase (2Apro) of EV71 plays an important role in segmenting the precursor polyprotein during viral replication, inhibiting host protein synthesis, and evading innate immunity. This study was to determine the function of EV71 2Apro in replication and virulence. A chimeric strain (SDLY 107-2A-1) was recombined by replacing 2Apro of a severe strain (SDLY107) with that of a mild strain (SDLY1) based on an infectious cDNA clone. The replication kinetics of the chimeric strain in vitro and in vivo were determined by qRT-PCR, which showed that the chimeric strain replicated slower and generated less viral RNA than the severe strain. The pathological change and viral load of chimeric strain infected mice were intermediate between severe strain infected mice and mild strain infected mice. Cellular cytotoxicity assays revealed that 2Apro was associated with the neurotoxicity of EV71. Histopathological and immunohistochemical assays detected tissue pathological damage in the lungs, muscles, brain, and intestinal tissues. Together, these results suggest that 2Apro modulates replication and virulence of EV71. This provides a theoretical basis for virulence determination of EV71.

The effect of DSPE-PEG2000, cholesterol and drug incorporated in bilayer on the formation of discoidal micelles.

In cholesterol/DSPE-PEG2000/DPPC systems, nano-disks could evolve between liposomes and spherical micelles in a certain range of PEG-lipids. How cholesterol or drug influences this evolution and what about the properties of discoidal micelles as drug carrier are still not clear. Aiming at this, nanocarriers with different contents of cholesterol and DSPE-PEG2000 were prepared by thin-film-hydration method. Firstly, the bilayer fluidity of nanocarriers was investigated and proved to decrease with the increase of cholesterol, and the cooperative degree between phospholipids was also related to cholesterol content in an order of 30 > 40 > 15 > 0 mol%. Then three different levels of cholesterol were chosen to study its effect on DSPE-PEG2000 limit used to form discoidal micelles. For transition from liposomes to disks, the limit of DSPE-PEG2000 employed is above 0.5 mol% at 0 mol% cholesterol, above 15 mol% at 30 mol% cholesterol and above 5 mol% at 40 mol% cholesterol observed from TEM images, indicating that nanocarriers with 30 mol% cholesterol formed discoidal micelles most difficultly due to the strongest interaction between phospholipids. However, membrane fluidity seems to have little responsibility for the different morphologies. And imatinib (IM) could promote the formation of discoidal micelles, resulting from the interaction between IM and polar headgroups of phospholipid demonstrated by DSC. The relative reduction of entrapment efficiency for three agents with different dissolving properties showed that lipophilic drugs were the most suitable drug that could be loaded into discoidal micelles. In addition, the discoidal micelle formulations tested could readily change to other morphologies after 80 days' storage. In conclusion, both the cholesterol content and IM could affect the formation of discoidal micelles by probably influencing the interaction between phospholipids, also the discoidal structure was not stable enough and only suitable for lipophilic drug loading. We hope this study could help to design the formula and choose the proper drugs that may retain the discoidal morphology.

Colorectal cancer combination therapy using drug and gene co-delivered, targeted poly(ethylene glycol)-ε-poly(caprolactone) nanocarriers.

PURPOSE: Combination therapy is a promising strategy to treat cancer due to the synergistic effects. The drug and gene co-delivered systems attract more attention in the field of combination therapy. MATERIALS AND METHODS: In the present research, poly(ethylene glycol)-ε-poly(caprolactone) block copolymer was used for the co-loading of 5-fluorouracil (5-FU) and gene. The physicochemical characteristics, in vitro and in vivo anticancer, and gene transfection efficiency were tested on colon cancer cells and tumor-bearing mice. RESULTS: 5-FU and gene co-loaded nanocarriers had a size of 145 nm. In vivo gene delivery results showed about 60% of gene-positive cells. Tumor volume of nanocarrier groups at day 21 was around 320 mm3, which is significantly smaller compared with free 5-FU group (852 mm3) and control group (1,059 mm3). The maximum 5-FU plasma concentration in nanocarrier groups (49 µg/mL) was significantly greater than free 5-FU (13 µg/mL). At 24 hours, drug level of nanocarrier groups was about 2.8 µg/mL compared with 0.02 µg/mL of free 5-FU. CONCLUSION: The resulting nanocarriers co-loaded with the anticancer drugs and genes could be considered as a promising nanomedicine for colorectal cancer therapy.

Multiseed liposomal drug delivery system using micelle gradient as driving force to improve amphiphilic drug retention and its anti-tumor efficacy.

To improve drug retention in carriers for amphiphilic asulacrine (ASL), a novel active loading method using micelle gradient was developed to fabricate the ASL-loaded multiseed liposomes (ASL-ML). The empty ML were prepared by hydrating a thin film with empty micelles. Then the micelles in liposomal compartment acting as 'micelle pool' drove the drug to be loaded after the outer micelles were removed. Some reasoning studies including critical micelle concentration (CMC) determination, influencing factors tests on entrapment efficiency (EE), structure visualization, and drug release were carried out to explore the mechanism of active loading, ASL location, and the structure of ASL-ML. Comparisons were made between pre-loading and active loading method. Finally, the extended drug retention capacity of ML was evaluated through pharmacokinetic, drug tissue irritancy, and in vivo anti-tumor activity studies. Comprehensive results from fluorescent and transmission electron microscope (TEM) observation, encapsulation efficiency (EE) comparison, and release studies demonstrated the formation of ML-shell structure for ASL-ML without inter-carrier fusion. The location of drug mainly in inner micelles as well as the superiority of post-loading to the pre-loading method , in which drug in micelles shifted onto the bilayer membrane was an additional positive of this delivery system. It was observed that the drug amphiphilicity and interaction of micelles with drug were the two prerequisites for this active loading method. The extended retention capacity of ML has been verified through the prolonged half-life, reduced paw-lick responses in rats, and enhanced tumor inhibition in model mice. In conclusion, ASL-ML prepared by active loading method can effectively load drug into micelles with expected structure and improve drug retention.

Plaque-hyaluronidase-responsive high-density-lipoprotein-mimetic nanoparticles for multistage intimal-macrophage-targeted drug delivery and enhanced anti-atherosclerotic therapy.

Increasing evidence has highlighted the pivotal role that intimal macrophage (iMΦ) plays in the pathophysiology of atherosclerotic plaques, which represents an attractive target for atherosclerosis treatment. In this work, to address the insufficient specificity of conventional reconstituted high-density lipoprotein (rHDL) for iMΦ and its limited cholesterol efflux ability, we designed a hyaluronan (HA)-anchored core-shell rHDL. This nanoparticle achieved efficient iMΦ-targeted drug delivery via a multistage-targeting approach, and excellent cellular cholesterol removal. It contained a biodegradable poly (lactic-co-glycolic acid) (PLGA) core within a lipid bilayer, and apolipoprotein A-I (apoA-I) absorbing on the lipid bilayer was covalently decorated with HA. The covalent HA coating with superior stability and greater shielding was favorable for not only minimizing the liver uptake but also facilitating the accumulation of nanoparticles at leaky endothelium overexpressing CD44 receptors in atherosclerotic plaques. The ultimate iMΦ homing was achieved via apoA-I after HA coating degraded by hyaluronidase (HAase) (abundant in atherosclerotic plaque). The multistage-targeting mechanism was revealed on the established injured endothelium-macrophage co-culture dynamic system. Upon treatment with HAase in vitro, the nanoparticle HA-(C)-PLGA-rHDL exhibited a greater cholesterol efflux capacity compared with conventional rHDL (2.43-fold). Better targeting efficiency toward iMΦ and attenuated liver accumulation were further proved by results from ex vivo imaging and iMΦ-specific fluorescence localization. Ultimately, HA-(C)-PLGA-rHDL loaded with simvastatin realized the most potent anti-atherogenic efficacies in model animals over other preparations. Thus, the HAase-responsive HDL-mimetic nanoparticle was shown in this study to be a promising nanocarrier for anti-atherogenic therapy, in the light of efficient iMΦ-targeted drug delivery and excellent function of mediating cellular cholesterol efflux.