Manganese Sulfate Nanocomposites Fabricated by Hot-Melt Extrusion for Chemodynamic Therapy of Colorectal Cancer.

The development of metal salts-based nanocomposites is highly desired for the Fenton or Fenton-like reaction-based chemodynamic therapy of cancer. Manganese sulfate (MnSO4)-dispersed nanoparticles (NPs) were fabricated with a hot-melt extrusion (HME) system for the chemodynamic therapy of colorectal cancer in this study. MnSO4 was homogeneously distributed in polyethylene glycol (PEG) 6000 (as a hydrophilic polymer) with the aid of surfactants (Span 80 and Tween 80) by HME processing. Nano-size distribution was achieved after dispersing the pulverized extrudate of MnSO4-based composite in the aqueous media. The distribution of MnSO4 in HME extrudate and the interactions between MnSO4 and pharmaceutical additives were elucidated by Fourier-transform infrared, X-ray diffractometry, X-ray photoelectron spectroscopy, and scanning electron microscopy analyses. Hydroxyl radical generation efficiency by the Fenton-like chemistry capability of Mn2+ ion was also confirmed by catalytic assays. By using the intrinsic H2O2 in cancer cells, MnSO4 NPs provided an elevated cellular reactive oxygen species level, apoptosis induction capability, and antiproliferation efficiency. The designed HME-processed MnSO4 formulation can be efficiently used for the chemodynamic therapy of colorectal cancer.

Bio- Fortification of Angelica gigas Nakai Nano-Powder Using Bio-Polymer by Hot Melt Extrusion to Enhance the Bioaccessibility and Functionality of Nutraceutical Compounds.

Angelica gigas Nakai (AGN) is a popular traditional herbal medicine which has been used to alleviate various human diseases in Korea since ancient times. However, the low bioaccessibility of the nutraceutical compounds of AGN results in a poor water solubility, thereby limiting bioavailability. In this regard, a ternary AGN-biopolymer-plasticizer composite (AGNC) was developed to enhance the bioaccessibility of nutraceutical compounds from extrudate AGN formulations manufactured by hot melt extrusion (HME). The AGNC was prepared with extrudate AGN (EAGN) using different hydroxypropyl methylcellulose (HPMC) biopolymers (5% w/w) viz.: hypromellose phthalate (HP), hypromellose (AN), and hypromellose (CN) along with acetic acid (AA) (0.1 M, 20% w/v) as a plasticizer. The non-extrudate fresh AGN (FAGN) powder was used as a control. The physicochemical properties of the extrudate formulations and control were characterized by differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopy (FTIR). DSC analysis showed a lower enthalpy (ΔH) (12.22 J/g) and lower glass transition temperature (Tg) (41 °C) in HP-AA-EAGN compared to the control. FTIR confirmed the physical crosslinking between AGN and biopolymer in the extrudate composite and demonstrated that some functional groups formed viz., -OH and -CH2. The obtained result also shows that the particle size was reduced by 341 nm, and solubility was increased by 65.5% in HP-AA-EAGN compared to the control (1499 nm, 29.4%, respectively). The bioaccessibility of the total phenolic content and the total flavonoids-including decursin (D) and decursinol angelate (DA)-were significantly higher in HP-AA-EAGN compared to the control. The 2,2-diphenyl-1 picryl hydrazyl (DPPH) free radical scavenging capacity and ferric reducing antioxidant power assay (FRAP) indicated that the HP-AA-EAGN formulation preserves a greater antioxidant profile than the other formulations. Finally, it is summarized that the addition of acidified HP biopolymer increased the bioaccessibility, functionality, and improved the physicochemical properties of nutraceutical compounds in the extrudate AGN formulation.

Development of a Polymer-Mediated Soybean Nanocomposite by Hot Melt Extrusion to Improve Its Functionality and Antioxidant Properties.

The poor bioaccessibility of the phenolic compounds of soybeans is a key challenge to developing functional food products. Therefore, a novel hydrophilic food-grade hydroxypropyl methylcellulose (HPMC) polymer was added to soybean to prepare a soybean food composite (SFC), in order to improve the soybean's functionality. The SFC was prepared with soybean (95%) plus HPMC (5%) (w/w) mixes (HSE), as well as 100% soybean extrudate (SE), at 80 °C and 130 °C by a hot melt extrusion (HME) process. A non-extrudate 100% soybean material was considered as a control. It is observed that water solubility was significantly increased (35.18%), and particle size reached to nano-size (171.5 nm) in HSE at 130 °C compared to the control (7.14% and 1166 nm, respectively). The total phenolic, flavonoid, and single isoflavones content, including daidzin, daidzein, glycitein, genistein, and genistin was significantly increased in HSE at 130 °C compared to the control. The antioxidant properties were also significantly increased in HSE at 130 °C compared to the control, measured by 2,2-diphenyl-1 picryl hydrazyl (DPPH), a ferric reducing antioxidant power assay (FRAP), and the phosphomolybdenum method (PPMD). Finally, it is concluded that the HPMC polymer could be used as a novel excipient to develop nanocomposite via HME, in order to improve the functionality of soybean food products.

Development of iron(II) sulfate nanoparticles produced by hot-melt extrusion and their therapeutic potentials for colon cancer.

Particle size reduction of FeSO4 (iron(II) sulfate, IS) from micron to nano size was achieved by a combination of hot-melt extrusion (HME) processing and the input of Span 80, Tween 80, and poly(ethylene glycol) (PEG) 6000. Conveying, kneading, and extruding steps of the HME process and a decrease in the surface tension by surfactants were introduced to produce FeSO4 nanoparticles (NPs) in an aqueous environment. The FeSO4-based NPs (ISNPs) in the dispersion were composed of FeSO4, Span 80, Tween 80, and PEG 6000 and displayed a hydrodynamic size of 350-400 nm (5-50 mg/mL ISNPs concentration range) and a spherical shape. Considering the feeding ratio of FeSO4 (20%, w/w) used for preparing the ISNPs, FeSO4 appears to be wrapped by Span 80, Tween 80, and PEG 6000 according to the results of X-ray photoelectron spectroscopy (XPS) analysis. ISNPs exhibited different thermodynamic properties from those of FeSO4 itself. In colon adenocarcinoma (Caco-2) cells, the ISNPs group exhibited enhanced antiproliferation and apoptosis potentials compared to the FeSO4 group (p < 0.05). Histological staining data of a dissected intestine after oral administration of ISNPs suggest the absence of severe intestinal toxicities compared to the control (no treatment) group. All of these results imply the feasibility of the use of the developed ISNPs for the treatment of colon cancers with oral administration.

Preparation of cupric sulfate-based self-emulsifiable nanocomposites and their application to the photothermal therapy of colon adenocarcinoma.

Nanocomposites (NCs) of cupric sulfate monohydrate (CuSO4) were fabricated by hot-melt extrusion (HME) system equipped with twin screws. Micron-sized bulk powder of CuSO4 was dispersed in the mixture of surfactants (Span 80 and Tween 80) and hydrophilic polymer (polyethylene glycol (PEG) 6000) by HME process. Reduction of surface tension by surfactants and homogeneous dispersion in hydrophilic polymer along with HME technique were introduced to prepare CuSO4 NCs. Dispersion of CuSO4 NCs exhibited approximately 204 nm hydrodynamic size, unimodal size distribution, and positive zeta potential values. Encapsulation of CuSO4 in CuSO4 NCs and the physicochemical interactions between CuSO4 and pharmaceutical excipients were investigated by solid-state studies. Of note, CuSO4 NCs group exhibited higher antiproliferation efficacies, compared with bulk CuSO4, in Caco-2 (human adenocarcinoma) cells at 75 and 100 µg/mL CuSO4 concentrations (p < 0.05). Also, near-infrared laser irradiation to CuSO4 NCs group elevated the antiproliferation efficacies, compared with non-irradiation group, in Caco-2 cells. After intravenous injection in mice, CuSO4 NCs did not show severe in vivo toxicities. Developed CuSO4 NCs can be one of promising candidates of photothermal therapeutic agents for colon cancers.

Development of Resveratrol-Loaded Herbal Extract-Based Nanocomposites and Their Application to the Therapy of Ovarian Cancer.

Resveratrol (RSV) and the ethanol extract of Angelica gigas Nakai (AGN Ex)-based nanoparticles (NPs) were prepared using the nanocrystal concept. AGN/RSV NPs with 224 nm hydrodynamic size, unimodal size distribution, and negative zeta potential values were developed with the emulsification and solvent evaporation techniques. The crystalline properties of AGN Ex and RSV were transformed during the emulsification and solvent evaporation processes, thus, AGN NPs and AGN/RSV NPs exhibited amorphous states. AGN/RSV NPs held up their initial hydrodynamic size after 24 h of incubation in serum-included media. Sustained release profiles (for 5 days) of decursin (D) and decursinol angelate (DA) (the representative markers of AGN Ex) and RSV were observed at normal physiological pH (pH 7.4). In ovarian cancer (SKOV-3) cells, although AGN/RSV NPs showed a lower cellular entry rate rather than AGN NPs, the cellular accumulated amount of AGN/RSV NPs was similar with that of AGN NPs after 4 h of incubation. The antiproliferation efficiency of AGN/RSV NPs group was significantly higher than the AGN Ex, AGN NPs, and AGN NPs + RSV groups in SKOV-3 cells. AGN/RSV NPs can be one of the promising candidates for therapeutic nanoplatforms against ovarian cancers.

Polydopamine-coated nanocomposites of Angelica gigas Nakai extract and their therapeutic potential for triple-negative breast cancer cells.

Polydopamine (PD)-coated nanocomposites (NCs) based on the ethanol extract of Angelica gigas Nakai (AGN EtOH ext) were fabricated and evaluated for breast cancer therapy. AGN NCs were prepared using a modified emulsification-solvent evaporation method and were further incubated in dopamine solution (at pH 8.6) to be covered with the PD layer. PD-AGN NCs with a 213-nm mean diameter, narrow size distribution, and negative zeta potential values were fabricated in this study. Less negative (close to zero) zeta potential value of PD-AGN NCs than that of AGN NCs implied the existence of the PD layer in the outer surface of NCs. The PD layer in PD-AGN NCs was also identified by X-ray photoelectron spectroscopy (XPS) and ultraviolet (UV)/visible absorption analyses. The sustained release of decursin (D) and decursinol angelate (DA), as major active pharmacological components of AGN, was observed in both AGN NCs and PD-AGN NCs. Enhanced cellular binding property of PD-AGN NCs, compared to AGN NCs, in MDA-MB-231 (human breast adenocarcinoma; triple-negative breast cancer) cells was observed. Improved anticancer activities of PD-AGN NCs compared with those of AGN EtOH ext and AGN NCs were also shown in MDA-MB-231 cells. The developed PD-AGN NCs may be used as remarkable platform nanocarriers for efficient breast cancer therapy.

Fabrication of polymer matrix-free nanocomposites based on Angelica gigas Nakai extract and their application to breast cancer therapy.

Nanocomposites (NCs) based on the ethanol extract of Angelica gigas Nakai (AGN EtOH ext) were developed for breast cancer therapy. Polymer matrix-free nano-sized particles based on the extract of natural product were fabricated using a modified emulsification-solvent evaporation method. Without the use of polymer matrix, toxicity can be minimized and the clinical application may be assured. AGN NCs with approximately 200nm mean diameter, narrow size distribution, and negative zeta potential were prepared in this study. Sustained release of decursin (D) and decursinol angelate (DA) (as major components of AGN) from AGN NCs was observed at pH 7.4. Cellular accumulation efficiency and intracellular distribution of AGN NCs were evaluated in MCF-7 (human breast adenocarcinoma) cells. According to the results of antiproliferation assay in MCF-7 cells, IC50 value of AGN NCs group (27.4±4.0µg/mL) was lower than that of AGN EtOH ext group (75.3±13.7µg/mL) (p<0.05). Also, the percentage of apoptotic events of AGN NCs group was significantly higher than that of AGN EtOH ext group (p<0.05). All these findings suggest that developed AGN NCs can be used as one of promising nanosystems for the therapy of breast cancers.

Angelica gigas Nakai extract-loaded fast-dissolving nanofiber based on poly(vinyl alcohol) and Soluplus for oral cancer therapy.

A poly(vinyl alcohol) (PVA) and Soluplus (SP)-based nanofiber (NF) mat was fabricated using an electrospinning method for the delivery of Angelica gigas Nakai (AGN) extract (ext) to oral cancers. AGN/SP NF (mean diameter: 75±26nm; entrapment efficiency: 84.6±18.6%) and AGN/PVA/SP NF (mean diameter: 170±35nm; entrapment efficiency: 81.0±10.1%) were fabricated using an electrospinning method. Amorphization of AGN EtOH ext was verified by X-ray diffractometry (XRD) analysis during the electrospinning process for the fabrication of NF structures. The AGN/PVA/SP NF group exhibited instant wetting (within 2s) and rapid disintegration (within 3min) properties compared with those in the AGN/PVA NF group, assuring the successful and conventional application of AGN/PVA/SP NF film in the oral cavity without the intake of beverages. After the spontaneous dispersion of NF in the aqueous media, it was converted to nanoparticles with a narrow size distribution. In YD-9 cells (oral squamous cell carcinoma from buccal cheek), the anti-proliferation activity was ordered as follows: AGN EtOH ext suspension < AGN/PVA NF < AGN/PVA/SP NF. All of these findings indicated that AGN/PVA/SP NF can be used as a fast-dissolving mat formulation for the therapy of oral cancers.

Anticancer effect of joboksansam, Korean wild ginseng germinated from bird feces.

BACKGROUND: Joboksansam, Korean bird wild ginseng, is an artificially cultivated wild ginseng germinated from bird feces. Although numerous pharmacologic activities of wild ginsengs have been reported, the beneficial effect of joboksansam in cancer has not been elucidated. In this study, we investigated the in vivo and in vitro anticancer activities of joboksansam powder. METHODS: To evaluate the in vivo anticancer activity of joboksansam, we established a xenograft mouse model bearing RMA cell-derived cancer. Direct cytotoxicity induced by joboksansam powder was also investigated in vitro using (3-4-5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT) assay. The inhibitory activity of this powder on the activation of cell survival signaling involving Akt and Src was examined with immunoblot analysis. RESULTS: Joboksansam powder displayed strong inhibitory activity against the increased tumor size, increased weight of total body and cancer tissues, and mortality of tumor-bearing mice. Joboksansam powder also suppressed the activation of survival regulatory enzymes Akt and Src, as assessed by phosphorylation levels in the immunoblot analysis of tumor tissues. Interestingly, the viability of RMA cells in vitro was directly decreased by joboksansam treatment. CONCLUSION: Overall, our results strongly suggest that joboksansam powder has the potential to protect against cancer generation by direct cytotoxic effects on cancer cells resulting from suppression of cell survival signaling.

Nanocomposites based on Soluplus and Angelica gigas Nakai extract fabricated by an electrohydrodynamic method for oral administration.

Nanocomposites (NCs) based on Soluplus (SP) were fabricated by an electrohydrodynamic (EHD) method for the oral delivery of Angelica gigas Nakai (AGN). Nano-sized particles were obtained after dispersing the resultant, produced by the EHD technique, in the aqueous environment. AGN/SP2 (AGN:SP=1:2, w/w) NC dispersion in aqueous media exhibited a 130nm mean diameter, narrow size distribution, and robust stability in the tested concentration range of the ethanol extract of AGN (AGN EtOH ext) and at pH 1.2 and 6.8. Amorphization of the components of AGN and their interactions with SP in the AGN/SP2 NC formulation were demonstrated by X-ray diffractometry (XRD) analysis. The released amounts of decursin (D) and decursinol angelate (DA), major components of AGN, from NCs were improved compared with those from the AGN EtOH ext group at both pH 1.2 and 6.8. As D and DA can be metabolized into decursinol (DOH) in the liver after oral administration, the DOH concentrations in plasma were quantitatively determined to evaluate the oral absorption of AGN. In a pharmacokinetic study in rats, higher oral absorption and the maximum concentration in plasma (Cmax) were presented in the AGN/SP2 NC group compared with the AGN EtOH ext and AGN NC groups. These findings indicate the successful application of developed SP-based NCs for the oral delivery of AGN.

Omega-3 fatty acids incorporated colloidal systems for the delivery of Angelica gigas Nakai extract.

Omega-3 (ω-3) fish oil-enriched colloidal systems were developed for the oral delivery of Angelica gigas Nakai (AGN) extract (ext). By constructing a pseudo-ternary phase diagram, the composition of oil-in-water (o/w) microemulsion (ME) systems based on ω-3 (oil), Labrasol (surfactant), and water was determined. AGN ext was dissolved into the ME system and d-α-tocopherol polyethylene glycol 1000 succinate (TPGS) was added to the ME formulation in order to enhance the mucosal absorption of the pharmacologically active ingredients in the AGN ext. The droplet size of AGN-loaded MEs was 205-277 nm and their morphology was spherical. The release of major components of AGN, decursin (D) and decursinol angelate (DA), from ME formulations in pH 1.2 and 6.8 buffers was significantly greater (P<0.05) than that from the AGN suspension group. The pharmacokinetic properties of AGN-loaded MEs in rats were evaluated by measuring decursinol (DOH) concentrations in plasma after oral administration. TPGS-included ME (F2) resulted in significantly greater (P<0.05) systemic exposure of DOH than that with ME without TPGS (F1), AGN ext+TPGS, and AGN in suspension. Severe toxicity of F1 and F2 on the intestinal epithelium was not observed by histological staining. The colloidal carriers described herein are promising delivery systems for oral administration of AGN ext.

Improvement in antiproliferative activity of Angelica gigas Nakai by solid dispersion formation via hot-melt extrusion and induction of cell cycle arrest and apoptosis in HeLa cells.

Angelica gigas Nakai (AGN) is one of the most popular herbal medicines and widely used as a functional food product. In this study, AGN was firstly processed by a low-temperature turbo mill and a hot melting extruder to reduce particle size and form solid dispersion (SD). Anticancer activity against HeLa cells was then examined. AGN-SD based on Soluplus was formed via hot-melt extrusion (HME) and showed the strongest cytotoxic effect on HeLa cells. In addition, the possible mechanism of cell death induced by AGN-SD on HeLa cells was also investigated. AGN-SD decreased cell viability, induced apoptosis, increased the production of reactive oxygen species, regulated the expression of Bcl-2 and Bax, and induced G2/M phase arrest in HeLa cells. This study suggested that AGN-SD based on Soluplus and the method to improve antiproliferative effect by SD formation via HME may be suitable for application in the pharmaceutical industry.

Soluplus®/TPGS-based solid dispersions prepared by hot-melt extrusion equipped with twin-screw systems for enhancing oral bioavailability of valsartan.

BACKGROUND: Soluplus(®) (SP) and D-alpha-tocopherol polyethylene glycol 1000 succinate (TPGS)-based solid dispersion (SD) formulations were developed by hot-melt extrusion (HME) to improve oral bioavailability of valsartan (VST). METHODS: HME process with twin-screw configuration for generating a high shear stress was used to prepare VST SD formulations. The thermodynamic state of the drug and its dispersion in the polymers were evaluated by solid-state studies, including Fourier-transform infrared, X-ray diffraction, and differential scanning calorimetry. Drug release from the SD formulations was assessed at pH values of 1.2, 4.0, and 6.8. Pharmacokinetic study was performed in rats to estimate the oral absorption of VST. RESULTS: HME with a high shear rate produced by the twin-screw system was successfully applied to prepare VST-loaded SD formulations. Drug amorphization and its molecular dispersion in the polymer matrix were verified by several solid-state studies. Drug release from SD formulations was improved, compared to the pure drug, particularly at pH 6.8. Oral absorption of drug in rats was also enhanced in SP and TPGS-based SD groups compared to that in the pure drug group. CONCLUSION: SP and TPGS-based SDs, prepared by the HME process, could be used to improve aqueous solubility, dissolution, and oral absorption of poorly water-soluble drugs.

Angelica gigas Nakai and Soluplus-Based Solid Formulations Prepared by Hot-Melting Extrusion: Oral Absorption Enhancing and Memory Ameliorating Effects.

Oral solid formulations based on Angelica gigas Nakai (AGN) and Soluplus were prepared by the hot-melting extrusion (HME) method. AGN was pulverized into coarse and ultrafine particles, and their particle size and morphology were investigated. Ultrafine AGN particles were used in the HME process with high shear to produce AGN-based formulations. In simulated gastrointestinal fluids (pH 1.2 and pH 6.8) and water, significantly higher amounts of the major active components of AGN, decursin (D) and decursinol angelate (DA), were extracted from the HME-processed AGN/Soluplus (F8) group than the AGN EtOH extract (ext) group (p < 0.05). Based on an in vivo pharmacokinetic study in rats, the relative oral bioavailability of decursinol (DOH), a hepatic metabolite of D and DA, in F8-administered mice was 8.75-fold higher than in AGN EtOH ext-treated group. In scopolamine-induced memory-impaired mice, F8 exhibited a more potent cognitive enhancing effect than AGN EtOH ext in both a Morris water maze test and a passive avoidance test. These findings suggest that HME-processed AGN/Soluplus formulation (F8) could be a promising therapeutic candidate for memory impairment.

New investigation of distribution imaging and content uniformity of very low dose drugs using hot-melt extrusion method.

The content uniformity of low dose drugs in dosage forms is very important for quality assurance. The aim of this study was to prepare uniformly and homogeneously distributed dosage forms of very low-dose drugs using twin screw hot-melt extrusion (HME) and to investigate the distribution of drugs using instrumental analyses. For the feasibility of HME method, a very low amount of coumarin-6, a fluorescent dye, was used to visualize distribution images using confocal laser scanning microscope (CLSM). Limaprost, tamsulosin and glimepiride were then used as low-dose model drugs to study the applicability of HME for content uniformity and distribution behaviors. Hydrophilic thermosensitive polymers with low melting point, such as Poloxamer188 and polyethylene glycol (PEG) 6000, were chosen as carriers. The melt extrusion was carried out around 50°C, at which both carriers were easily dissolved but model drugs remained in solid form. The physicochemical properties of the hot-melt extrudates, including differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD) and Fourier transform infrared spectroscopy (FT-IR), were measured. Content uniformity of the drugs was also checked by HPLC. CLSM imaging showed that model drugs were well distributed throughout the hot-melt extrudate, giving better content uniformity with low batch-to-batch variations compared with simple physical mixtures. DSC, PXRD and FT-IR data showed that there was no interaction or interference between model drugs and thermosensitive polymers. The current HME methods could be used to prepare uniformly distributed and reproducible solid dosage forms containing very low dose drugs for further pharmaceutical applications.

Ultrafine Angelica gigas powder normalizes ovarian hormone levels and has antiosteoporosis properties in ovariectomized rats: particle size effect.

The root of Angelica gigas (Korean angelica) is traditionally used to treat women's ailments that are caused by an impairment of menstrual blood flow and cycle irregularities. This study evaluated the effect particle size of Korean angelica powder on its efficacy for treating estrogen-related symptoms of menopause. Initially, Korean angelica roots were pulverized into ultrafine powder, and orally administered to the rats at a concentration of 500 mg/kg body weight for 8 weeks. The effects of Korean angelica powder particle size on extraction yield, contents of bioactive compounds (decursin and decursinol angelate), levels of serum ovarian hormones (estradiol and progesterone), reproductive hormones (luteinizing hormone and follicle-stimulating hormone), and experimental osteoporosis parameters (mineral density, strength, and histological features) were determined. A significant increase (fivefold) in the contents of decursin and decursinol angelate in the extract of the ultrafine Korean angelica powder was observed compared to coarse Korean angelica powder. Rats were divided into sham-operated or ovariectomized (OVX) groups that were fed coarse (CRS) or ultrafine (UF) ground Korean angelica root. The serum levels of estradiol in the OVX_UF group were 19.2% and 54.1% higher than that of OVX_CRS group. Serum bone-alkaline phosphatase/total-alkaline phosphatase index in the OVX_UF group was half that of the OVX_CRS group. In addition, less trabecular bone loss and thick cortical areas were observed in rats administered ultrafine powder. Therefore, ultrafine grinding may enhance the bioactivity of herbal medicines and be especially useful when their extracted forms lose bioactivity during processing, storage, and oral intake.