Quercetin and tanshinone prevent mitochondria from oxidation and autophagy to inhibit KGN cell apoptosis through the SIRT1/SIRT3-FOXO3a axis.

Granulosa cells are somatic cells located inside follicles that play a crucial role in the growth and development of follicles. Quercetin and tanshinone are two key monomers in traditional Chinese medicine that have antioxidant and anti-aging properties. The KGN cell apoptosis model caused by triptolide (TP) was employed in this work to investigate granulosa cell death and medication rescue. Quercetin and tanshinone therapy suppressed KGN cell death and oxidation while also regulating the expression of critical apoptosis and oxidation-related markers such as B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax). Further research revealed that the effects of Quercetin and Tanshinone were accomplished via deacetylation of FOXO3A in the cytoplasm and mitochondria via the SIRT1/SIRT3-FOXO3a axis. In summary, Quercetin and tanshinone protect KGN cells from apoptosis by reducing mitochondrial apoptosis and oxidation via the SIRT1/SIRT3-FOXO3a axis.

Target-Driven Annular DNA Walker Coupled with Electrochemiluminescent Silicon Quantum Dots for APE1 Bioanalysis.

Functional DNA walkers with substantial nanostructures have been extensively investigated; however, their stability still faces challenges when exposed to diverse nuclease in clinical biological samples, resulting in the unreliability of actual assessment. This work proposed a target-driven annular DNA walker with enhanced stability enabling the sensitive and reliable response to different concentrations of apurinic/apyrimidinic endonuclease 1 (APE1), by preparing silicon quantum dots (SiQDs) as electrochemiluminescence (ECL) emitters. Specifically, the SiQDs showed significant strong and stable ECL signals by purifying the microenvironment of SiQDs through the dialysis removal of the gel-like layers surrounding the SiQDs. The relative standard deviation (RSD) of their ECL signal had been improved 16.59 times under consecutive scanning compared to that of SiQDs without dialysis, demonstrating a significant improvement in ECL stability. Subsequently, in the presence of APE1, the designed annular DNA walker was activated to move along the numerous quenching probes within the continuous cross-based DNA orbits, which were immobilized to the SiQD-modified electrode, providing ECL readout signals. The linear range of this ECL biosensor was 1.0 × 10-13 U·µL-1 to 1.0 × 10-7 U·µL-1, and the limit of detection (LOD) was as low as 1.766 × 10-14 U·µL-1. This work provides a novel structure of a DNA walker with nuclease resistance for clinical sample detection and designs a new strategy for synthesizing SiQDs with favorable ECL performance, tremendously expanding the ECL application of SiQDs.

Lipid rafts-mediated endocytosis and physiology-based cell membrane traffic models of doxorubicin liposomes.

The clathrin-mediated endocytosis is likely a major mechanism of liposomes' internalization. A kinetic approach was used to assess the internalization mechanism of doxorubicin (Dox) loaded cationic liposomes and to establish physiology-based cell membrane traffic mathematic models. Lipid rafts-mediated endocytosis, including dynamin-dependent or -independent endocytosis of noncaveolar structure, was a dominant process. The mathematic models divided Dox loaded liposomes binding lipid rafts (B) into saturable binding (SB) and nonsaturable binding (NSB) followed by energy-driven endocytosis. The intracellular trafficking demonstrated early endosome-late endosome-lysosome or early/late endosome-cytoplasm-nucleus pathways. The three properties of liposome structures, i.e., cationic lipid, fusogenic lipid, and pegylation, were investigated to compare their contributions to cell membrane and intracellular traffic. The results revealed great contribution of cationic lipid DOTAP and fusogenic lipid DOPE to cell membrane binding and internalization. The valid Dox in the nuclei of HepG2 and A375 cells treated with cationic liposomes containing 40mol% of DOPE were 1.2-fold and 1.5-fold higher than that in the nuclei of HepG2 and A375 cells treated with liposomes containing 20mol% of DOPE, respectively, suggesting the dependence of cell type. This tendency was proportional to the increase of cell-associated total liposomal Dox. The mathematic models would be useful to predict intracellular trafficking of liposomal Dox.

RGD Peptide-Pegylated PLLA Nanoparticles Containing Epirubicin Hydrochloride Exhibit Receptor-Dependent Tumor Trafficking In Vitro and In Vivo.

PURPOSE: A novel hydrophilic conjugate of arginine-glycine-aspartic acid (RGD) and polyethylene glycol (PEG), i.e., RGD-PEG Mw (M W = 300, 600, 1000 or 4000), was synthesized and employed in epirubicin (EPI) loaded poly L-lactic acid (PLLA) nanoparticles (NPs) to improve its tumor targeting effect. METHODS: In vitro studies were performed to assess EPI release from NPs in tumor-mimic acidic medium, cytotoxicity and cell cycle assay in HepG2 cells, and cellular uptake kinetics in four types of tumor cells including A375 cells (high integrin receptor expression), HeLa cells (low integrin receptor expression), and metabolic HepG2/SMMC7721 cells. In vivo pharmacodynamics (PD) and pharmacokinetic (PK) studies were determined in a murine ascites tumor model. RESULTS: Cellular uptake kinetics showed integrin receptor-dependent binding and internalization. In vitro release results showed that PLLA and PEG groups retarded EPI release from NPs and promoted drug release amount in acidic medium, which benefited in vivo trafficking to the acidic tumors. In vivo PD and PK studies revealed that RGD-PEG Mw (M W = 600 ~ 1000) improved tumor targeting capacity of NPs by ~2.4-fold, compared to conventional EPI NPs. CONCLUSIONS: RGD-PEG Mw (M W = 600 ~ 1000) modified PLLA NPs provide a promising strategy to improve tumor selectivity in cancer treatment.

Bioinspired zwitterionic polyphosphoester modified porous silicon nanoparticles for efficient oral insulin delivery.

The intestinal epithelial and mucus barriers on the gastrointestinal tract limit the bioavailability of oral protein or peptide drugs. Therefore, efficient mucus permeability and cellular internalization are required properties for oral delivery systems. To overcome these two obstacles, porous silicon nanoparticles were modified with poly (pyridyl disulfide ethylene phosphate/sulfobetaine) polymers to make P(PyEP-g-SBm)n-AmPSiNPs (m = 0.1, 0.2, 0.3 and n = 10, 20, 30) nanoparticles (NPs). The insulin-loaded P(PyEP-g-SB)-AmPSiNPs showed favorable stability and good biocompatibility in vitro. The zwitterionic dodecyl sulfobetaine (SB) coated nanoparticles improved the mucus permeability. P(PyEP-g-SBm)20 with the optimal conjugated ratio (m = 0.3) of SB units was determined by evaluating the mucus diffusion rate of NPs. The cellular uptake of P(PyEP-g-SB0.3)n-AmPSiNPs (n = 10, 20, 30) was much higher than AmPSiNPs in the presence of inhibitors (N-acetylcysteine solution and sodium chlorate) (p < 0.01) due to the enhanced charge shielding effect of P(PyEP-g-SB) modification. The P(PyEP-g-SB0.3)20-AmPSiNPs showed about 1.4-1.7 fold increase in the apparent permeability of insulin across Caco-2/HT-29-MTX cell monolayers, compared to AmPSiNPs (p < 0.01). Finally, the in vivo study showed that insulin-loaded P(PyEP-g-SB0.3)20-AmPSiNPs generated 20% reduction of the blood glucose level with an 2-fold increase in oral bioavailability. These suggested that zwitterionic polyphosphoester modified porous silicon nanoparticles, which were of enhanced mucus permeability and cellular internalization, represent a promising carrier for oral delivery of peptide and protein.

Enhanced anticancer effect of doxorubicin by TPGS-coated liposomes with Bcl-2 siRNA-corona for dual suppression of drug resistance.

Multiple drug resistance (MDR) is a tough problem in developing hepatocellular carcinoma (HCC) therapy. Here, we developed TPGS-coated cationic liposomes with Bcl-2 siRNA corona to load doxorubicin (Dox) i.e., Bcl-2 siRNA/Dox-TPGS-LPs, to enhance anticancer effect of Dox in HCC-MDR. TPGS i.e., d-α-tocopheryl polyethylene glycol 1000 succinate, inhibited P-glycoprotein (P-gp) efflux pump and Bcl-2 siRNA suppressed anti-apoptotic Bcl-2 protein. The Bcl-2 siRNA loaded in the liposomal corona was observed under transmission electron microscopy. The stability and hemolysis evaluation demonstrated Bcl-2 siRNA/Dox-TPGS-LPs had good biocompatibility and siRNA-corona could protect the liposomal core to avoid the attachment of fetal bovine serum. In drug-resistant cells, TPGS effectively prolonged intracellular Dox retention time and siRNA-corona did improve the internalization of Dox from liposomes. In vitro and in vivo anticancer effect of this dual-functional nanostructure was examined in HCC-MDR Bel7402/5-FU tumor model. MTT assay confirmed the IC50 value of Dox was 20-50 fold higher in Bel7402/5-FU MDR cells than that in sensitive Bel7402 cells. Bcl-2 siRNA corona successfully entered the cytosol of Bel7402/5-FU MDR cells to downregulate Bcl-2 protein levels in vitro and in vivo. Bcl-2 siRNA/Dox-TPGS-LPs showed superior to TPGS- (or siRNA-) linked Dox liposomes in cell apoptosis and cytotoxicity assay in Bel7402/5-FU MDR cells, and 7-fold greater effect than free Dox in tumor growth inhibition of Bel7402/5-FU xenograft nude mice. In conclusion, TPGS-coated cationic liposomes with Bcl-2 siRNA corona had the capacity to inhibit MDR dual-pathways and subsequently improved the anti-tumor activity of the chemotherapeutic agent co-delivered to a level that cannot be achieved by inhibiting a MDR single way.

D-α-tocopherol polyethylene glycol 1000 succinate-modified liposomes with an siRNA corona confer enhanced cellular uptake and targeted delivery of doxorubicin via tumor priming.

BACKGROUND: Combination therapy employing siRNAs and antitumor drugs is a promising method for the treatment of solid tumors. However, regarding combined treatments involving siRNAs and chemotherapeutic reagents, most prior research has focused on the enhanced cytotoxicity against tumor cells conferred by downregulation of the targeted protein. PURPOSE: We developed D-α-tocopherol polyethylene glycol 1000 succinate (TPGS)-modified cationic liposomes (LPs) to simultaneously deliver doxorubicin (Dox) and the Bcl-2 siRNA (siBcl-2) for synergistic chemotherapy. The co-loading of siBcl-2 onto the Dox-loaded cationic LPs (siBcl-2/Dox-TPGS-LPs) could promote cellular uptake, cytotoxicity against 3D H22 tumor spheroids, circulation in the blood, drug accumulation at tumor sites, and synergistic chemotherapy in vivo. METHODS: The siBcl-2/Dox-TPGS-LPs were constructed by co-loading siBcl-2 onto the Dox-loaded TPGS-modified cationic LPs (Dox-TPGS-LPs), and Dox entrapment into the LPs was achieved using an ammonium sulfate gradient method. The antitumor effects of siBcl-2/Dox-TPGS-LPs were studied in murine hepatic carcinoma H22 cells, 3D H22 tumor spheroids, and H22 tumor-bearing mice. RESULTS: Dynamic light scattering technique and transmission electron microscopy images revealed that siBcl-2 loaded onto the Dox-TPGS-LPs formed a prominent corona at an nitrogen to phosphorus (N/P) ratio of 4:1, resulting in particle size increase from 155 to 210 nm and a weak positive zeta potential (+12.5 mV). The siBcl-2/Dox-TPGS-LPs enhanced the cellular uptake of Dox, promoted toxicity against 3D H22 tumor spheroids via tumor priming, prolonged Dox circulation in the blood, and increased accumulation of Dox at tumor sites, thereby enhancing the cytotoxicity of Dox in vitro and its chemotherapeutic efficacy in vivo. CONCLUSION: The siBcl-2/Dox-TPGS-LPs demonstrated a strong potential for application in synergistic chemotherapy. The co-loading of siRNAs both sensitized cells toward antitumor drugs by downregulating the expression level of a specific protein and influenced the pharmacokinetic behavior of the co-delivery system in vitro and in vivo.

Follicular dynamics of glycerophospholipid and sphingolipid metabolisms in polycystic ovary syndrome patients.

Polycystic ovary syndrome (PCOS) is a common heterogeneous disease, affecting up to 5-10% women at reproductive age. Although PCOS patients could produce morphologically normal metaphase II oocytes undergoing assisted reproductive techniques (ART), oocyte developmental competence and embryo development have been impaired in following in-vitro fertilization (IVF) steps. Follicular fluid (FF) provides a variety of information in oocyte environment when oocytes grow. In the present work, based on ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS), the metabolic signatures of PCOS FF have been compared with healthy women using untargeted metabolomics approach. Significant abundance differences of a series of glycerolipid, glycerophospholipids, sphingolipids, and carboxylic acids have been discovered. Among them, reduced levels of phosphatidylglycerolphosphate (PGP) and a triglyceride (TG) were highly related to the lower fertilization rate in PCOS; increased abundance of lysoPE and decreased amount of PC were significantly correlated with LH/FSH (ratio of luteinizing hormone to follicle stimulating hormone). Some metabolites, including decreased sphingolipids, glycerophospholipids, and fluctuated fatty acyls, also performed close relationship with other ART and clinical results. We concluded that dysfunctions in the metabolism of glycerolipid, glycerophospholipid, sphingolipid, and glycosphingolipid biosynthesis in PCOS patients' follicles play a non-ignorable role in declining the 2 pronuclei (PN) fertilization rate during IVF procedure.

Multifunctional hybrid micelles with tunable active targeting and acid/phosphatase-stimulated drug release for enhanced tumor suppression.

Therapeutic efficacy of conventional single PEGylated polymeric micelles is significantly reduced by limited endocytosis and intracellular drug release. To improve drug delivery efficiency, poly (ethylene glycol)-block-poly (l-lactic acid)/(Arg-Gly-Asp-Phe)-poly (aminoethyl ethylene phosphate)-block-poly (l-lactic acid) (PEG-PLLA/RGDF-PAEEP-PLLA) hybrid micelles with tunable active targeting and acid/phosphatase-stimulated drug release are developed. The optimized hybrid micelles with 6 wt % of RGDF have favorable in vitro and in vivo activities. The hybrid micelles could temporarily shield the targeting efficacy of RGDF at pH 7.4 due to the steric effect exerted by concealment of RGDF peptides in the PEG corona, which strongly decreases the clearance by mononuclear phagocyte system and consequently improves the tumor accumulation. Inside the solid tumor with a lower acidic pH, the hybrid micelles restore the active tumor targeting property with exposed RGDF on the surface of the micelles because of the increased protonation and stretching degree of PAEEP blocks. RGDF-mediated endocytosis improves the tumor cell uptake. The hybrid micelles would also enhance intracellular drug release because of the hydrolysis of the acid/phosphatase-sensitivity of PAEEP blocks in endo/lysosome. Systemic administration of the hybrid micelles significantly inhibits tumor growth by 96% due to the integration of enhanced circulation time, tumor accumulation, cell uptake and intracellular drug release.

Self-Assembled PAEEP-PLLA Micelles with Varied Hydrophilic Block Lengths for Tumor Cell Targeting.

The properties of hydrophilic shell in micelles significantly affect the interaction between micelles and cells. Compared with frequently used polyethylene glycol (PEG) as the hydrophilic block, polyphosphoesters (PPEs) are superior in functionality, biocompatibility, and degradability. A series of amphiphilic poly(aminoethyl ethylene phosphate)/poly(l-lactide acid) (PAEEP-PLLA) copolymers were synthesized with hydrophilic PAEEP with different chain lengths. The corresponding self-assembled micelles were used for doxorubicin (Dox) entrapment. The length of hydrophilic PAEEP block on the shell affected the structure of micelles. PAEEPm-PLLA168 (m = 130 or 37) polymers formed vesicles, while PAEEPm-PLLA168 (m = 15 or 9) formed large compound micelles (LCMs), suggesting a difference in tumor cell uptake and intracellular trafficking. PAEEP15-PLLA168 polymer showed superiority on cellular uptake amount, intracellular drug release, and cell apoptosis. Lipid rafts and macropinocytosis are the leading endocytic pathways of PAEEP-PLLA micelles. The shape coupling between micelles and cell membrane facilitated cell surface features such as flattened protrusions (membrane protein) and inward-pointing hollows as well as efficient endocytosis. These results suggested that PAEEP-PLLA self-assembled block copolymer micelles may be an excellent drug delivery system for tumor treatment and that the hydrophilic chain length could regulate drug targeting to tumor cells.

Modulation of combined-release behaviors from a novel "tablets-in-capsule system".

A multifunctional and multiple unit system, which contains versatile mini-tablets in a hard gelation capsule, is developed by preparing Rapid-release Mini-Tablets (RMTs), Sustained-release Mini-Tablets (SMTs), Pulsatile Mini-Tablets (PMTs), and Delayed-onset Sustained-release Mini-Tablets (DSMTs), each with various lag times of release. Based on the combinations of mini-tablets, multiplied pulsatile drug delivery system (DDS), site-specific DDS, slow/quick DDS, quick/slow DDS, and zero-order DDS could be obtained. Velocity-time curve, instead of the cumulative percentage drug release profile, is plotted. The nonlinear least square model fit program is applied to process the velocity data of dissolution. The test curves coincided with the theoretical curves from simple summation of v-t equations of individual mini-tablets. Therefore, the programmed DDS can be predicted by adding the v-t equations of various mini-tablets to calculate the theoretical equations and be implemented.

[Studies on combined release behaviors of versatile mini-tablets in capsule systems and fittings of their mathematical model].

AIM: To prepare nifedipine (NP) rapid release mini-tablet, sustained release mini-tablets, pulsed release mini-tablets and delayed-onset sustained release mini-tablets and develop multiplied pulsed drug delivery system (DDS), site-specific DDS, zero-order DDS and quick/slow DDS by various ways. METHODS: Velocity-time (v-t) equation of each mini-tablet was deduced by non-linear least square model fit. The difference of combinations in v-t profiles between theoretical value and test value was compared. RESULTS: According to the v-t equations, the combined release behaviors were observed directly from v-t profiles and the test values coincided with the theoretical profiles. CONCLUSION: The programmed DDS, which consist of a variety of mini-tablets with different dosages and combinations in capsules, could be predicted by summing up the v-t equation of each tablet.

RGD-fatty alcohol-modified docetaxel liposomes improve tumor selectivity in vivo.

The tripeptide arginine-glycine-aspartate (RGD) was conjugated with various fatty alcohols to obtain RGDOCnH2n+1 (n=8, 10, 12, 14, 16, 18), which were incorporated into the bilayer of docetaxel liposomes to improve their tumor specificity. The fatty alcohols were accepted as linking groups to insert the tetrapeptide RGDX (X=amino acid) into the bilayer of liposomes. RGDX was previously shown to be a potent ligand to target tumor cell-surface integrin receptors, whereas RGD was not shown to have this ability. We hypothesized that RGD-fatty alcohol conjugates lacking the fourth amine X can guide liposomes to tumors without reducing their binding affinity to integrins. Antitumor activity, pharmacokinetics and biodistribution studies were evaluated in mice inoculated with S180 sarcoma. Compared with unmodified liposomes, RGD-fatty alcohol-modified liposomes successfully delivered significantly more docetaxel to tumors, which led to significant tumor weight loss and increased tumor docetaxel concentrations accompanied by reduced liver accumulation. Improved affinity of RGD-fatty alcohols to integrins was also confirmed on A375 cell model. Further comparisons among the tumor-targeting capacities of liposomes containing RGD-fatty alcohols, RGDF-fatty alcohols and RGDV-fatty acids demonstrated that RGD-fatty alcohols were as effective as the other two tetrapeptide derivatives. Therefore, a simplified tumor-targeting delivery system using RGD-fatty alcohols was developed.

Delivery of nanomedicines to extracellular and intracellular compartments of a solid tumor.

Advances in molecular medicines have led to identification of promising targets on cellular and molecular levels. These targets are located in extracellular and intracellular compartments. The latter include cytosol, nucleus, mitochondrion, Golgi apparatus and endoplasmic reticulum. This report gives an overview on the barriers to delivering nanomedicines to various target sites within a solid tumor, the experimental approaches to overcome such barriers, and the potential utility of nanotechnology.

Relationships between liposome properties, cell membrane binding, intracellular processing, and intracellular bioavailability.

Positive surface charge enhances liposome uptake into cells. Pegylation, used to confer stealth properties to enable in vivo applications of cationic liposomes, compromises internalization. The goal of this study was to determine the quantitative relationships between these two liposome properties (separately and jointly), liposomes binding to cell membrane, and the subsequent internalization and residence in intracellular space (referred to as intracellular bioavailability). The results, obtained in pancreatic Hs-766T cancer cells, revealed nonlinear and inter-dependent relationships, as well as substantial qualitative and quantitative differences. The proportionality constant K of intracellular and membrane-bound liposomes at equilibrium (i.e., I(eq) and B(eq)) showed a positive triphasic relationship with surface charge and a negative biphasic relationship with pegylation. Near-neutral liposomes showed little internalization of the membrane-bound moiety, increasing to a constant K value for medium charge liposomes (+15 to +35 mV zeta potential), followed by a further increase for highly charged liposomes (greater than or equal to +46 mV). The decline of pegylation with K value showed a breakpoint at 2%. The negative consequences of pegylation (%PEG) were partially offset by increasing charge (ZP). The best-fitting regression equations are: B(eq) = -1.36 × %PEG + 0.33 × ZP; I(eq) = -1.52 × %PEG + 0.34 × ZP. It suggested that 1% pegylation increase can be offset with 4 mV ZP. The differences are such that it may be possible to balance these parameters to simultaneously maximize the stealth property and intracellular bioavailability of cationic liposomes.

Preparation and characterization of solid dispersions of dipyridamole with a carrier "copolyvidonum Plasdone S-630".

Solid dispersions (SDs) of dipyridamole (DIP) with a novel carrier copolyvidonum Plasdone S-630 (CoPVP) were developed by solvent evaporation method. The solid state of SDs of DIP with CoPVP (SDs CoPVP) was characterized by fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), X-ray diffraction (XRD) and polarizing microscopy, compared with that of SDs of DIP with polyvinylpyrrolidone Plasdone K-29/32 (SDs PVP). FT-IR analysis demonstrated the presence of intermolecular hydrogen bonding between DIP and CoPVP or PVP in SDs. DSC and XRD studies indicated that DIP presented in amorphous state in both SDs CoPVP and SDs PVP at higher weight ratios. The dissolution property of SDs CoPVP was significantly improved in comparison of pure DIP and physical mixtures with CoPVP (PM CoPVP). Both SDs CoPVP and SDs PVP powder showed the favorable flowability. However, SDs CoPVP showed better compressibility than SDs PVP. The lower hydroscopicity of SDs CoPVP could be advantageous to the stability to SDs. This study proves the potential of CoPVP as a carrier in the formulations of SDs for poorly soluble drugs.