Separable and Inseparable Silk Fibroin Microneedles for the Transdermal Delivery of Colchicine: Development, Characterization, and Comparisons.

Colchicine is the first-line option for both the treatment and prophylaxis of gout flares. However, due to potentially severe side effects, the clinical use of colchicine is limited. A well-tolerated and safe delivery system for colchicine is widely desired. For this purpose, colchicine-loaded inseparable microneedles were fabricated using silk fibroin. Additionally, separable microneedles made of silk fibroin as the needle tips and PVP K30 as the base material were developed. Both types of microneedles were evaluated for their mechanical strength, swelling and dissolution characteristics, insertion abilities, degradation properties, in vitro penetration, skin irritation, and in vivo anti-gout effects. The results demonstrated that separable microneedles had greater mechanical strength and insertion ability. Moreover, the separable microneedles separated quickly and caused little skin irritation. In the pharmacodynamic test, mice with acute gouty arthritis responded significantly to treatment with separable microneedles. In conclusion, the separable silk fibroin-based microneedles provide a promising route for colchicine delivery.

A Novel Oral Drugs Delivery System for Borneol Based on HiCap®100 and Maltodextrin: Preparation, Characterization, and the Investigation as an Intestinal Absorption Enhancer.

The objective of this study was to create a new method for delivering oral borneol (BN) drug that would improve stability. This was accomplished through microencapsulation using HiCap®100 and maltodextrin (MD), resulting in HiCap®100/MD/BN microcapsules (MCs). The HiCap®100/MD/BN MCs were evaluated in terms of encapsulation efficiency (EE%), drug loading (DL%), morphological observations, particle size distribution, Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), thermal analysis, drug degradation rate studies, and in vitro release behavior. The effect of MCs on intestinal permeability in a rat model was assessed using the model drug "florfenicol" (FF) in single-pass intestinal perfusion (SPIP) study. The relationship between MCs and P-glycoprotein (P-gp) was further investigated in comparison with verapamil (Ver). The irritation of MCs was assessed by histological analysis. The MCs in a spherical structure with micron-scale dimensions were obtained. The EE% and DL% were (86.71 ± 0.96)% and (6.03 ± 0.32)%, respectively. MCs played a significantly protective role in drug degradation rate studies. In vitro release studies indicated that the release behavior of MCs was significantly better than BN at the three-release media, and the cumulative release rate exceeded 90% in 15 min. The SPIP studies showed that MCs significantly enhanced the absorption of FF in rats. Compared with Ver, MCs were not promoted by a single inhibition of P-gp. Hematoxylin-eosin (HE)-stained images showed that MCs had no obvious irritation and toxic effects on the intestines of rats. Thus, the preparation of HiCap®100/MD/BN MCs improves the stability of BN, which has certain scientific value for the development and application of BN, and provides unique perspectives for future BN-related researches.

Determination of the number of observers needed to evaluate a subjective test and its application in two PD-L1 studies.

In pathological studies, subjective assays, especially companion diagnostic tests, can dramatically affect treatment of cancer. Binary diagnostic test results (ie, positive vs negative) may vary between pathologists or observers who read the tumor slides. Some tests have clearly defined criteria resulting in highly concordant outcomes, even with minimal training. Other tests are more challenging. Observers may achieve poor concordance even with training. While there are many statistically rigorous methods for measuring concordance between observers, we are unaware of a method that can identify how many observers are needed to determine whether a test can reach an acceptable concordance, if at all. Here we introduce a statistical approach to the assessment of test performance when the test is read by multiple observers, as would occur in the real world. By plotting the number of observers against the estimated overall agreement proportion, we can obtain a curve that plateaus to the average observer concordance. Diagnostic tests that are well-defined and easily judged show high concordance and plateau with few interobserver comparisons. More challenging tests do not plateau until many interobserver comparisons are made, and typically reach a lower plateau or even 0. We further propose a statistical test of whether the overall agreement proportion will drop to 0 with a large number of pathologists. The proposed analytical framework can be used to evaluate the difficulty in the interpretation of pathological test criteria and platforms, and to determine how pathology-based subjective tests will perform in the real world. The method could also be used outside of pathology, where concordance of a diagnosis or decision point relies on the subjective application of multiple criteria. We apply this method in two recent PD-L1 studies to test whether the curve of overall agreement proportion will converge to 0 and determine the minimal sufficient number of observers required to estimate the concordance plateau of their reads.

Improving intestinal absorption and antibacterial effect of florfenicol via nanocrystallisation technology.

To study the effects of nanocrystallisation technology on the intestinal absorption properties and antibacterial activity of florfenicol (FF). The florfenicol nanocrystals (FF-NC) were prepared by wet grinding and spray drying. Additionally, changes in particle size, charge, morphology, and dissolution of FF-NC in the long-term stability were monitored by laser particle sizer, TEM, SEM, paddle method, and the structure of FF-NC powder was characterised by nuclear magnetic resonance (NMR) test. The antibacterial activity, intestinal absorption and intestinal histocompatibility of FF-NC were investigated by the stiletto, mini broth dilution susceptibility test, in situ single-pass intestinal perfusion (SPIP) and haematoxylin-eosin (H-E) staining. After 12 months of storage, the particle size and zeta potential of FF-NC were 280.43 ± 8.21 nm and -19.64 ± 3.45 mV, and the electron microscopy results showed that FF-NC were nearly circular with no adhesion between particles. In addition, the drug loading, encapsulation efficiency, and dissolution of FF-NC did not change significantly during storage. The inhibition zone of FF-NC against Escherichia coli and Staphylococcus aureus was 21.37 ± 1.70 mm and 25.17 ± 2.47 mm, respectively. Compared with the FF, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of FF-NC are reduced, and the absorption rate constant (Ka) and efficient permeability coefficient (Peff) of FF-NC in the three intestinal segments were increased by 1.28, 0.25, and 9.10 times and 0.59, 0.17, and 6.0 times, respectively. The results of tissue sections showed that FF-NC had little damage to the small intestinal. Nanocrystallisation technology is an effective method to increase the intestinal absorption and antibacterial activity of FF.

Increased bioaffinity and anti-inflammatory activity of florfenicol nanocrystals by wet grinding method.

Context: The main objective of the current study is to improve the water solubility of florfenicol (FF) and evaluate changes in its pharmacokinetics and anti-inflammatory activity.Materials and methods: Florfenicol nanocrystals (FF-NC) were prepared by wet grinding combined with spray drying. The characterisations, pharmacokinetics, and anti-inflammatory activity of FF-NC were evaluated.Results: The particle size, polydispersity index (PDI), and zeta potential of FF-NC were 276.4 ± 19.4 nm, 0.166 ± 0.011, and -18.66 ± 5.25 mV, respectively. Compared with FF, FF-NC showed a better dissolution rate in media at different pH. Pharmacokinetic experiments showed the area under the curve (AUC0-t), maximum concentration (Cmax), and mean residence time (MRT) of FF-NC were about 4.62-fold, 2.86-fold, and 1.68-fold higher compared with FF, respectively. In vitro anti-inflammatory experiments showed that FF inhibited the secretion of tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), and synthesis of NO in a dose-dependent manner, while FF-NC showed a stronger anti-inflammatory effect than FF under the same dose.Conclusion: FF-NC are an effective way to improve the bioaffinity and anti-inflammatory effects of FF.

Design and Self-Assembly of Peptide-Copolymer Conjugates into Nanoparticle Hydrogel for Wound Healing in Diabetes.

Background: Delayed wound healing in skin injuries has become a significant problem in clinics, seriously affecting and even threatening life and health. Recently, research interest has increased in developing wound dressings containing bioactive compounds capable of improving outcomes for complex healing needs. Methods: In this study, Puerarin-loaded nanoparticles (Pue-NPs) were prepared using the cell-penetrating peptide-poly (lactic-co-glycolic acid) (CPP-PLGA) as a drug carrier by the emulsified solvent evaporation method. Then, they were added into poly (acrylic acid) to obtain a self-assembled nanocomposite hydrogels (SANHs) drug delivery system using the co-polymerization method. The particle size, zeta potential, and micromorphology of Pue-NPs were measured; the appearance, mechanical properties, adhesive strength, and biological activity of SANHs were performed. Finally, the potential of SANHs for wound healing was further evaluated in streptozotocin-induced diabetic mice. Results: Pue-NPs were regularly spherical, with an average particle size of 134.57 ± 1.42 nm and a zeta potential of 2.14 ± 0.78 mV. SANHs was colorless and transparent with a honeycomb-like porous structure and had an excellent swelling ratio (917%), water vapor transmission rate (3077 g·m-2·day-1), mechanical properties (Young's modulus of 18 kPa, elongation at break of 307%), and adhesive strength (15.5 kPa). SANHs exhibited sustained release of Pue over 48h, with a cumulative release of 55.60 ± 6.01%. In vitro tests revealed that the SANHs presented a 92.22% antibacterial rate against Escherichia coli after 4h, and a 61.91% scavenging rate of 1.1-diphenyl-2-trinitrophenylhydrazine (DPPH) radical. In vivo experiments showed that SANHs accelerated wound repair by reducing the inflammatory response at the wound site, promoting angiogenesis, and facilitating epidermal regeneration and collagen deposition. Conclusion: In conclusion, we successfully prepared SANHs. Our results show that SANHs have excellent performance and improves wound healing in diabetic mice model, indicating that it can be used to develop an effective strategy for the treatment of diabetic wounds.

Dissolution enhancement of cefdinir with hydroxypropyl-ß-cyclodextrin.

The solid state properties and dissolution behavior of binary systems of cefdinir (CEF) with hydroxypropyl-ß-cyclodextrin (HP-ß-CD) were investigated. CEF-HP-ß-CD interaction in the solution state was studied by phase-solubility analysis and demonstrates the ability of HP-ß-CD to complex with CEF giving A(L) type profile with 65.28 ± 1.3 M⁻¹ stability constant. The freeze drying technique was adopted to prepare binary systems of CEF with HP-ß-CD in 1:1 molar ratio. The solid inclusion was characterized by fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), X-ray powder diffractometry (XRD), and scanning electron microscopy (SEM). Aqueous solubility of CEF-HP-ß-CD inclusion complex was 2.36-fold of pure CEF. The dissolution profiles of inclusion complexes were determined and compared with those of CEF alone and their physical mixtures. The dissolution rate of inclusion complex was superior than the CEF alone. These approaches indicated that CEF was able to form an inclusion complex with HP-ß-CD, and the inclusion compounds exhibited different spectroscopic features and properties.

High resveratrol-loaded microcapsules with trehalose and OSA starch as the wall materials: Fabrication, characterization, and evaluation.

To improve the solubility and stability of resveratrol (Res), Res nanocrystals (Res-ncs) as the capsule core were prepared by wet milling using hydroxypropyl methyl cellulose (HPMCE5), sodium dodecyl sulfate (SDS), and polyvinylpyrrolidone (PVPK30) as stabilizers, along with trehalose and octenyl succinic anhydride (OSA) modified starch were used as the wall material to produce Res microcapsules (Res-mcs) via spray drying. The fresh-prepared Res-ncs and rehydrated Res-mcs had mean particle sizes of 190.30 ± 3.43 and 204.70 ± 3.60 nm, zeta potentials of -13.90 ± 0.28 and - 11.20 ± 0.34 mV, and the loading capacities (LC) were as high as 73.03 % and 28.83 %. Particle morphology showed that Res-mcs had more regular and smooth spherical structures. FTIR indicated that Res may have hydrogen bonding with the walls. XRD and DSC exhibited that Res in nanocrystals and microcapsules existed mostly as amorphous structures. The solubility of Res-mcs and Res-ncs was increased, with excellent redispersibility and rapid dissolution of Res in vitro. The antioxidant properties of Res-mcs were protected and improved. With the walls acting as a physical barrier, Res-mcs have better photothermal stability than raw Res. Res-mcs have a relative bioavailability of 171.25 %, which is higher than that of raw Res.

HPLC assay and pharmacokinetics and tissue distribution study of glycyrrhetinic acid liposomes modified with galactosylated lipid.

The aim of this study was to evaluate the pharmacokinetics and tissue distribution of the glycyrrhetinic acid (GA) liposome modified with galactosylated lipid (NOH-GA-LP), compared with GA conventional liposome (GA-LP) and GA solution in mice. The pharmacokinetics and biodistribution of liposomal and solution formulation of GA in mice were studied after intravenous administration. Plasma and tissues were treated using liquid-liquid extraction and determined using reversed-phase high-performance liquid chromatography. Results showed that the mean residence times of NOH-GA-LP (2.99-fold) and GA-LP (2.94-fold) were higher than that of the GA solution in plasma. NOH-GA-LP produced a drug concentration in the liver that was markedly higher than that in other tissues and was approximately 2.0- and 4.8-fold of that of GA-LP and GA solution, respectively. In conclusion, the NOH-GA-LP prepared in this study is a promising sustained-release and drug-targeting system for antitumor drugs.

Age-Associated Dysregulation of Integrin Function in Vascular Smooth Muscle.

Arterial aging results in a progressive reduction in elasticity of the vessel wall and an impaired ability of aged blood vessels to control local blood flow and pressure. Recently, a new concept has emerged that the stiffness and decreased contractility of vascular smooth muscle (VSM) cells are important contributors to age-induced arterial dysfunction. This study investigated the hypothesis that aging alters integrin function in a matrix stiffness-dependent manner, which contributes to decreased VSM contractility in aged soleus muscle feed arteries (SFA). The effect of RGD-binding integrins on contractile function of cannulated SFA isolated from young (4 months) and old (24 months) Fischer 344 rats was assessed by measuring constrictor responses to norepinephrine, phenylephrine, and angiotensin II. Results indicated that constrictor responses in presence of RGD were impaired in old compared to young SFA. VSM cells isolated from young and old SFA were used for functional experiments using atomic force microscopy and high-resolution imaging. Aging was associated with a modulation of integrin ß1 recruitment at cell-matrix adhesions that was matrix and substrate stiffness dependent. Our data showed that substrate stiffening drives altered integrin ß1 expression in aging, while soft substrates abolish age-induced differences in overall integrin ß1 expression. In addition, substrate stiffness and matrix composition contribute to the modulation of SMα-actin cytoskeleton architecture with soft substrates reducing age effects. Our results provide new insights into age-induced structural changes at VSM cell level that translates to decreased functionality of aged resistance soleus feed arteries.

Preparation and characterization of galactose-modified liposomes by a nonaqueous enzymatic reaction.

In this study, NOH (NOH = N-octadecyl-4-[(D-galactopyranosyl)oxy]-2,3,5,6-tetrahydroxy hexanamide) was enzymatically synthesized as a targeting molecule and incorporated into liposomes to prepare a liposome surface modified with galactose. Glycyrrhetinic-acid-loaded liposome (GA-LP) and glycyrrhetinic-acid-loaded liposome surface modified with galactose (NOH-GA-LP) were prepared by the ethanol-injection method. NOH-GA-LP was characterized by morphology, particle size, zeta potential, encapsulation efficiency, release in vitro, and stability. The size of spherical particles was in the range of 179-211 nm. Spherical particles exhibit a positive electrical charge (38.7 mV) and possess high encapsulation efficiency (91.3%) and show sustained release (72% over 48 hours) in vitro. This novel approach for the liposome surface modified with galactose by enzymatic synthesis is expected to provide potential application as a drug carrier for active targeted delivery to hepatocytes.

Diagnostic accuracy of photodynamic diagnosis for upper urinary tract urothelial carcinoma: A systematic review and meta-analysis.

BACKGROUND: This study aimed to evaluate the effectiveness of photodynamic diagnosis (PDD) for upper urinary tract urothelial carcinoma (UTUC) by performing a meta-analysis. METHOD: Relevant articles were retrieved from the Cochrane Library, PubMed, and Embase databases. Studies evaluating the accuracy of PDD for the diagnosis of upper UTUC were included. The pooled sensitivity, specificity, and area under the curve (AUC) were calculated by STATA 16.0 at the per-lesion level. RESULTS: Six studies with 289 lesions were included in this systematic review and meta-analysis. The pooled results showed that PDD can differentiate upper UTUC from benign lesions with a sensitivity of 0.96 (95 % confidence interval: 0.85-0.99) and a specificity of 0.86 (95 % confidence interval: [0.64-0.95]; AUC, 0.97). Compared with white-light ureterorenoscopy, PDD can significantly improve the additional detection rate of UTUC (RR 0.16, 95 % CI 0.07-0.34 P = 0.000). CONCLUSIONS: PDD is a valid technique that improves the diagnostic accuracy of UTUC compared with standard white-light ureterorenoscopy at the per-lesion level. PDD is a promising endoscopic technique for upper UTUC.

Enhancing Betulinic Acid Dissolution Rate and Improving Antitumor Activity via Nanosuspension Constructed by Anti-Solvent Technique.

PURPOSE: The aim of this study was to prepare and evaluate betulinic acid nanosuspension (BA-NS) for new drug delivery to enhance its solubility and in vitro anti-tumor activity. METHODS: BA-NS was formulated by an anti-solvent precipitation method using the Box-Behnken design (BBD). Particle size (PS) and Zeta potential were measured by laser particle size analysis. The drug solid state after freeze drying was characterized by scanning electron microscope (SEM), transmission electron microscope (TEM), differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD) and Fourier transform infrared spectroscopy (FTIR) after freeze drying. The saturation solubility and dissolution rate were determined by solubility assay and in vitro dissolution studies, respectively. The in vitro cytotoxicity assay was performed using 3-(4,5-dimethylthiazole)-2,5-diphenltetraazolium bromide (MTT) method. RESULTS: The PS was 129.7±12.2 nm having a Zeta potential of -28.1±4.5 mV and the polydispersity index (PDI) was 0.231±0.013, which confirmed that the nanosuspension was in the stable amorphous state. A series of characterization experiments demonstrated that nanoparticles retained original effective structure and existed as spherical or near-spherical nanoparticles in the nanosuspension, but the drug transferred from the crystal state to the amorphous state. The form of lyophilized BA-NS was very successful in enhancing the dissolution rate in PH-dependent way. The cytotoxicity assay revealed that BA-NS could significantly enhance the in vitro anti-proliferation against tumor cells compared to the BA suspension (BA-S). CONCLUSION: The BA-NS can remarkably improve solubility and in vitro antitumor activity, which seems very promising for the treatment of cancers in practical application.

Preliminary study on conjugation of formononetin with multiwalled carbon nanotubes for inducing apoptosis via ROS production in HeLa cells.

BACKGROUND: The present work was conducted to prepare and evaluate multiwalled carbon nanotube-formononetin (MWCNT-FMN) composite for sustained delivery and inducing apop-tosis via reactive oxygen species (ROS) production in HeLa cells. METHODS: The composite was prepared by solution mixing with short carboxylic group-functionalized multiwalled carbon nanotubes (MWCNT-COOH). Then the composite was characterized by laser particle size analysis, Fourier transform infrared spectrometry, X-ray diffractometry, differential scanning calorimetry, and scanning electron microscopy. Drug release rates in vitro were determined by dialysis method. The in vitro cytotoxicity study was performed using water soluble tetrazolium assay. The cellular apoptosis assay, ROS, and mitochondrial membrane potential (MMP) of HeLa cells were investigated by acridine orange and ethidium bromide double dye, 2',7'-dichlorodihydrofluorescein diacetate, and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide probe, respectively. RESULTS: The entrapment efficiency was 28.77%±0.15%, and the loading capacity was 12.05%±0.20%. The release of MWCNT-FMN was sustained, and the cumulative release rate of formononetin (FMN) from MWCNT-COOH was higher at pH 7.4 than at pH 5.3. The in vitro cytotoxicity assay demonstrated that FMN, MWCNT-COOH, and MWCNT-FMN had no significant effects on the proliferation and viability of mouse fibroblast 3T3 cells over 48 hours, while the cell growth inhibition of the three samples showed concentration-dependent for HeLa cells. Biological assay suggested FMN and MWCNT-FMN could induce apoptosis in HeLa cells, meanwhile the cells exhibited stronger ROS signal and more depolarized MMP than that of the control group. CONCLUSION: These results preliminarily demonstrated that MWCNT-FMN exerted anticancer efficacy through cellular apoptosis induced by ROS-mediated mitochondrial dysfunctions in HeLa cells.

Ruthenium complex delivery using liposomes to improve bioactivity against HeLa cells via the mitochondrial pathway.

AIM: The aim of this study was to encapsulate a ruthenium complex [Ru(ttbpy)2PIP](ClO4)2 (Ru) in liposomes to enhance their antitumor effect on human cervical cancer. METHODS: The Ru-loaded PEGylated liposomes (Ru-Lip) were prepared using thin-film hydration method. The mechanism of action was studied. RESULTS: A novel Ru was successfully synthesized. Ru-Lip showed stronger cytotoxic activity against HeLa cells than Ru. Ru-Lip demonstrated a more significant increase in apoptosis, reactive oxygen species production and apoptosis-associated processes (intracellular calcium concentration, cytochrome c release and activation of Bax and caspase-3) than Ru. Ru-Lip exhibited greater blockade efficacy in the cell cycle G1 phase and greater DNA damage than Ru. CONCLUSION: Ru-Lip significantly elevates the anticancer effect via reactive oxygen species-mediated mitochondrial dysfunctional pathway.

Iridium (III) complex-loaded liposomes as a drug delivery system for lung cancer through mitochondrial dysfunction.

BACKGROUND AND AIM: Iridium (Ir)-based complex is a potential antitumor ingredient, but its poor physicochemical properties such as hydrophobicity and low biocompatibility hamper further application. Liposome provides a potential delivery approach for improving the poor physicochemical property and reducing the side effects of antitumor drug. In this study, we aimed at incorporating Ir ([Ir(ppy)2(BTCP)]PF6) into liposomes to enhance the biocompatibility and sustained release of Ir for intravenous administration and to elucidate the mechanism in A549 cells. MATERIALS AND METHODS: Ir-loaded PEGylated liposomes (Lipo-Ir) were formulated by thin-film dispersion and ultrasonic method. Morphology, size distribution, and zeta potential of Lipo-Ir were examined by transmission electron microscopy (TEM) and Zetasizer. The released profile and biocompatibility were investigated by dialysis method and hemolysis test, respectively. Additionally, the cytotoxic activity and mechanism of Lipo-Ir and Ir inducing apoptosis in A549 cells were evaluated. RESULTS: Lipo-Ir can keep sustained release, excellent biocompatibility, and physical stability. The average particle size, polydispersity index, zeta potential, encapsulation efficiency, and drug loading are 112.57±1.15 nm, 0.19±0.02, -10.66±0.61 mV, 94.71%±3.21%, and 4.71%±0.41%, respectively. 3-(4,5-dimethylthiazole)-2,5-diphenltetraazolium bromide (MTT) assay show that Lipo-Ir and Ir display high cytotoxicity against selected cancer cells. Furthermore, the apoptotic features of morphology, depolarization of mitochondrial membrane potential, increase in the reactive oxygen species (ROS) levels, and disorder of Ca2+ homeostasis are observed after treating A549 cells with Ir and Lipo-Ir. Besides, Lipo-Ir can arrest the cell growth in G0/G1 phase. CONCLUSION: The studies demonstrate that Lipo-Ir can trigger apoptosis in A549 cells via ROS-mediated mitochondrial dysfunctions, and the biocompatible and sustained Lipo-Ir will be a promising drug delivery system.

An iridium (III) complex as potent anticancer agent induces apoptosis and autophagy in B16 cells through inhibition of the AKT/mTOR pathway.

A new ligand THPDP (THPDP = 11-(6,7,8,9-tetrahydrophenazin-2-yl)dipyrido[3,2-a:2',3'-c]phenazine) and its iridium(III) complex [Ir(ppy)2(THPDP)]PF6 (Ir-1) was synthesized and characterized by elemental analysis, IR, ESI-MS, 1H NMR and 13C NMR. The cytotoxicity in vitro of the complex against cancer cells B16, A549, Eca-109, SGC-7901, BEL-7402 and normal NIH 3T3 cell lines was evaluated using MTT method. The IC50 values of the complex toward B16, A549 and Eca-109 cells are 1.0 ±â€¯0.02, 1.4 ±â€¯0.03 and 1.6 ±â€¯0.06 µM, respectively. The apoptosis was investigated with AO/EB and DAPI staining methods. The complex shows strong ability to inhibit the cell growth in B16, A549 and Eca-109 cells. Ir-1 can induce apoptosis, increase the intracellular ROS level, and cause a decrease in the mitochondrial membrane potential. The intracellular Ca2+ level and the release of cytochrome c were studied under a fluorescent microscope. The cell invasion and autophagy were also performed, and the cell cycle arrest was assayed by flow cytometry. The expression of Bcl-2 family proteins, PI3K, AKT, mTOR, P-mTOR was investigated by western blot. The results show that the complex induces apoptosis through ROS-mediated mitochondria dysfunction and inhibition of AKT/mTOR pathways. These findings are helpful for design and synthesis of iridium(III) complexes as potent anticancer drugs.

Enzymatic synthesis and in vitro evaluation of folate-functionalized liposomes.

In this study, folate-poly(ethylene glycol)3400-cholesterol conjugates (FA-PEG-Chol) were enzymatically synthesized in one step and incorporated into liposomes to prepare folate (FA)-functionalized liposomes for targeted drug delivery. The FA-functionalized liposomes loaded with betulinic acid (BA) (FA-L-BA) were prepared by thin lipid film method. The FA-L-BA was characterized by their morphology, particle size, zeta potential, encapsulation efficiency (EE), stability, cell cytotoxicity and cellular uptake. The average size of FA-L-BA was 222±8 nm. The spherical particles exhibited a negative electrical charge of -20.12±1.45 mV and high EE of 91.61%±1.16%. The liposomes were taken up selectively by HepG2 cells. FA-L-BA showed enhanced cytotoxicity (50% inhibitory concentration [IC50] =63.07±2.22 µg/mL) compared to nontargeted control normal liposomes loaded with BA (L-BA; IC50 =93.14±2.19 µg/mL) in HepG2 cells in vitro. In addition, FA-functionalized liposomes loaded with Ir-1 (FA-L-Ir-1) showed significantly higher cellular uptake in HepG2 cells compared to nontargeted control normal liposomes loaded with Ir-1 (L-Ir-1). This novel approach for the liposomes surface modified with FA by a one-step enzymatic amidation was expected to provide potential application as a drug carrier for active targeted delivery to tumor cells.

Synthesis and anticancer properties of ruthenium (II) complexes as potent apoptosis inducers through mitochondrial disruption.

A new ligand MHPIP (MHPIP = 2-(1-methyl-1H-pyrazol-4-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) and its three ruthenium (II) complexes [Ru(N-N)2(MHPIP)](ClO4)2 (N-N = phen: 1,10-phenanthroline 1; dmp = 2,9-dimethyl-1,10-phenanthroline 2; ttbpy = 4,4'-ditertiarybutyl-2,2'-bipyridine 3) were synthesized and characterized. The cytotoxic activity in vitro was studied by MTT method. The complexes 1-3 show moderate cytotoxic effects on the cell growth in HepG2 cells with an IC50 value of 25.5 ± 3.5, 35.6 ± 1.9 and 27.4 ± 2.3 µM, respectively. The apoptosis was investigated with AO/EB and Annex V/PI staining methods and comet assay. The reactive oxygen species, mitochondrial membrane potential were investigated under a fluorescent microscope. Autophagy assay shows that the complexes can cause autophagy and up-regulate the expression of Beclin-1 protein. Additionally, the complexes inhibit the cell growth in HepG2 cells at G0/G1 phase, and the complexes can regulate the expression of caspase 3 and Bcl-2 family proteins. The studies demonstrate that the complexes induce apoptosis in HepG2 cells through DNA damage and ROS-mediated mitochondrial dysfunction pathways.