Ching Li Tseng

Targeting efficiency and biodistribution of biotinylated-EGF-conjugated gelatin nanoparticles administered via aerosol delivery in nude mice with lung cancer

Lung cancer is the most malignant cancer today; in order to develop an effective drug delivery system for lung cancer therapy, gelatin nanoparticles (GPs) were modified with NeutrAvidin(FITC)-biotinylated epidermal growth factor (EGF) to form EGF receptor (EGFR)-seeking nanoparticles (GP-Av-bEGF). Aerosol droplets of the GP-Av-bEGF were generated by using a nebulizer and were delivered to mice model of lung cancer via aerosol delivery. Analysis of the aerosol size revealed that 99% of the nanoparticles after nebulization had a mass median aerodynamic diameter (MMAD) within the suitable range (0.5-5 microm) for lower airway deposition. The safety of inhaled nanoparticles was examined by lung edema and myeloperoxidase (MPO) activity assay. Theres no finding suggestive of acute lung inflammation following inhalation. The fluorescence images obtained from live mice showed that the GP-Av-bEGF could target the cancerous lungs in a more specific manner. Fluorescence analysis of the organs revealed that the GP-Av-bEGF was mainly distributed in cancerous lungs. In contrast, nanoparticle accumulation was lower in normal lungs. The histological results indicated that the fluorescent GP-Av-bEGF was colocalized with the anti-EGFR-immunostain due to EGFR binding. The results of this study revealed that GP-Av-bEGF could target to the EGFR-overexpression cancer cells in vivo and may prove to be beneficial drug carriers when administered by simple aerosol delivery for the treatment of lung cancer.

The use of biotinylated-EGF-modified gelatin nanoparticle carrier to enhance cisplatin accumulation in cancerous lungs via inhalation

To develop a polymer-anticancer drug conjugate, we employed gelatin nanoparticles (GPs) as carriers of cisplatin (CDDP) with anticipated improved therapeutic effect and reduced side effects. The anticancer activities of CDDP-incorporated in GPs (GP-Pt) with biotinylated-EGF (bEGF) modification (GP-Pt-bEGF) were studied. GP-Pt-bEGF with EGFR affinity produced much higher Pt concentrations in A549 cells (high EGFR expression) than in HFL1 cells (low EGFR expression). An in vitro anticancer study showed that GP-Pt-bEGF was more potent than free CDDP or GP-Pt because of its rapid effect on the cell cycle as well as a lower IC(50) (1.2microg/ml) that inhibits A549 cell growth. PI staining showed that cells treated with GP-Pt-bEGF for only 4h had the highest sub-G1 population. The CDDP formulations - free CDDP, GP-Pt, and GP-Pt-bEGF - were given by intratumorous injections to SCID mice in a subcutaneous model. This treatment showed that GP-Pt-bEGF had stronger anti-tumor activity and was less toxic than free CDDP in vivo. Mice treated with GP-Pt-bEGF showed slight body weight loss, whereas free CDDP treatment at the same dose caused a body weight loss of 20-30%. Furthermore, these formulations were given to mice with lung cancer via aerosol delivery. This treatment showed that inhaled GP-Pt-bEGF could target EGFR-overexpressing cells to achieve high cisplatin dosage in cancerous lungs. To summarize, gelatin nanoparticles loaded with CDDP and decorated with EGF tumor-specific ligand were successfully developed. Their in vitro and in vivo targeting ability and anticancer effect were confirmed. The aerosol delivery of the nanodrug carrier was demonstrated. Simple aerosol delivery of targeted drug carriers may prove useful for the clinical treatment of lung cancer patients.

An injectable oxidated hyaluronic acid/adipic acid dihydrazide hydrogel as a vitreous substitute

Vitrectomy is a common procedure for treating ocular-related diseases. The surgery involves removing the vitreous humor from the center of the eye, and vitreous substitutes are needed to replace the vitreous humor after vitrectomy. In the present study, we developed a colorless, transparent and injectable hydrogel with appropriate refractive index as a vitreous substitute. The hydrogel is formed by oxidated hyaluronic acid (oxi-HA) cross-linked with adipic acid dihydrazide (ADH). Hyaluronic acid (HA) was oxidized by sodium periodate to create aldehyde functional groups, which could be cross-linked by ADH. The refractive index of this hydrogel ranged between 1.3420 and 1.3442, which is quite similar to human vitreous humor (1.3345). The degradation tests demonstrated that the hydrogel could maintain the gel matrix over 35 days, depending on the ADH concentration. In addition, the cytotoxicity was evaluated on retina pigmented epithelium (RPE) cells cultivated following the ISO standard (tests for in vitro cytotoxicity), and the hydrogel was found to be non-toxic. In a preliminary animal study, the oxi-HA/ADH hydrogel was injected into the vitreous cavity of rabbit eyes. The evaluations of slit-lamp observation, intraocular pressure, cornea thickness and histological examination showed no significant abnormal biological reactions for 3 weeks. This study suggests that the injectable oxi-HA/ADH hydrogel should be a potential vitreous substitute.

Gelatin nanoparticles as gene carriers for transgenic chicken applications

To develop a safe and effective nonviral gene delivery system for transgenic chicken manipulation, we developed gelatin nanocarriers using a reporter plasmid (pEGFP-C1; enhanced green fluorescence protein, EGFP) that expressed EGFP. pEGFP-C1-containing gelatin nanoparticles (GP/pEGFP) were prepared using a water–ethanol solvent displacement method and characterized by size, surface charge, DNA loading, and DNA protection ability. For gene delivery, pEGFP-C1 was stably and efficiently encapsulated in GPs that were approximately 300 nm in diameter with a slight negative surface charge, which was prepared from gelatin solution at pH 8.0. Approximately, 85% of the plasmid DNA was encapsulated in the GPs. Electrophoresis results showed that the GPs provided protection against DNase I digestion. We used the GP/pEGFP as a vector to transfect cells and chicken embryos. The vector was nontoxic to cells, and GFP expression was effectively expressed 24 h after HeLa cell transfection. Direct injection was adapted for vector transport to the chicken embryo; injection in the area opaca (Ao) of the egg resulted in the highest hatching rate without affecting embryo development. GFP gene expression in embryo sections was observed 4 days after injection. The results of this study demonstrate that GPs are a suitable nonviral vector for delivering exogenous genes for transgenic chicken manipulation.

Development of lattice-inserted 5-Fluorouracil-hydroxyapatite nanoparticles as a chemotherapeutic delivery system.

Developing an effective vehicle for cancer treatment, hydroxyapatite nanoparticles were fabricated for drug delivery. When 5-Fluorouracil, a major chemoagent, is combined with hydroxyapatite nanocarriers by interclay insertion, the modified hydroxyapatite nanoparticles have superior lysosomal degradation profiles, which could be leveraged as controlled drug release. The decomposition of the hydroxyapatite nanocarriers facilitates the release of 5-Fluorouracil into the cytoplasm causing cell death. Hydroxyapatite nanoparticles with/without 5-Fluorouracil were synthesized and analyzed in this study. Their crystallization properties and chemical composition were examined by X-ray diffraction and Fourier transforms infrared spectroscopy. The 5-Fluorouracil release rate was determined by UV spectroscopy. The biocompatibility of hydroxyapatite-5-Fluorouracil extraction solution was assessed using 3T3 cells via a WST-8 assay. The effect of hydroxyapatite-5-Fluorouracil particles which directly work on the human lung adenocarcinoma (A549) cells was evaluated by a lactate dehydrogenase assay via contact cultivation. A 5-Fluorouracil-absorbed hydroxyapatite particles were also tested. Overall, hydroxyapatite-5-Fluorouracils were prepared using a co-precipitation method wherein 5-Fluorouracil was intercalated in the hydroxyapatite lattice as determined by X-ray diffraction. Energy dispersive scanning examination showed the 5-Fluorouracil content was higher in hydroxyapatite-5-Fluorouracil than in a prepared absorption formulation. With 5-Fluorouracil insertion in the lattice, the widths of the a and c axial constants of the hydroxyapatite crystal increased. The extraction solution of hydroxyapatite-5-Fluorouracil was nontoxic to 3T3 cells, in which 5-Fluorouracil was not released in a neutral phosphate buffer solution. In contrast, at a lower pH value (2.5), 5-Fluorouracil was released by the acidic decomposition of hydroxyapatite. Finally, the results of the lactate dehydrogenase assay revealed that 5-Fluorouracil-hydroxyapatite was highly toxic to A549 cells through direct culture, this phenomenon may result from lysosomal decomposition of particles causing 5-Fluorouracil releasing. The pH-responsive hydroxyapatite-5-Fluorouracil nanoparticles have the potential to be part of a selective drug-delivery system in chemotherapy for cancer treatment.

PREPARATION OF GELATIN NANOPARTICLES WITH EGFR SELECTION ABILITY VIA BIOTINYLATED-EGF CONJUGATION FOR LUNG CANCER TARGETING

Lung cancer is the most malignant cancer today, and specific drug delivery has been developed for superior outcome. In this study, gelatin nanoparticles (GPs) were firstly employed as native carriers. Second, NeutrAvidinFITC was then grafted on the particle surface (GP-Av); finally much more amount of biotinylated EGF were able to be conjugated with NeutrAvidinFITC forming ligand- binding nanoparticles (GP-Av-bEGF) to enhance the targeting efficiency. These nanoparticles were applied as EGFR-seeking agents to detect lung cancer cells. Results of particle characterization show that the modification process had no influence on size (230 nm). Round and smooth nanoparticles were observed by AFM. The surface property of nanoparticles was characterized by surface plasmon resonance (SPR) and flowcytometry analysis as well as by examining the interaction of the modified EGF on particle surface with the ability to recognize EGFR. The binding ability of GPs with or without EGF modification is different. SPR assay showed that EGF-conjugated particles (GP-Av-bEGF) have stronger and faster bonding signal than the unmodified one (GP-Av). Free EGF competition results from SPR and A549 cell (lung adenocarcinoma cells) culture also confirmed the EGF receptormediated endocytosis mechanism for nanoparticles with EGF-modified binding. The in vitro targeting ability was confirmed by the uptake rate of different cells via flow cytometry assay. GP-Av-bEGF resulted in higher entrance efficiency on A549 than on normal lung cells (HFL1) and U2-OS (osteosarcoma cells) due to A549 possessing more amounts of EGFR. The targeting ability of GP-Av-bEGF nanoparticles with specific EGFR tracing ability was proved, which holds promise for further anticancer drug applications.

Preparation of arginine–glycine–aspartic acid-modified biopolymeric nanoparticles containing epigalloccatechin-3-gallate for targeting vascular endothelial cells to inhibit corneal neovascularization

Neovascularization (NV) of the cornea can disrupt visual function, causing ocular diseases, including blindness. Therefore, treatment of corneal NV has a high public health impact. Epigalloccatechin-3-gallate (EGCG), presenting antiangiogenesis effects, was chosen as an inhibitor to treat human vascular endothelial cells for corneal NV treatment. An arginine–glycine–aspartic acid (RGD) peptide–hyaluronic acid (HA)-conjugated complex coating on the gelatin/EGCG self-assembly nanoparticles (GEH-RGD NPs) was synthesized for targeting the αvβ3 integrin on human umbilical vein endothelial cells (HUVECs) in this study, and a corneal NV mouse model was used to evaluate the therapeutic effect of this nanomedicine used as eyedrops. HA-RGD conjugation via COOH and amine groups was confirmed by 1H-nuclear magnetic resonance and Fourier-transform infrared spectroscopy. The average diameter of GEH-RGD NPs was 168.87±22.5 nm with positive charge (19.7±2 mV), with an EGCG-loading efficiency up to 95%. Images of GEH-RGD NPs acquired from transmission electron microscopy showed a spherical shape and shell structure of about 200 nm. A slow-release pattern was observed in the nanoformulation at about 30% after 30 hours. Surface plasmon resonance confirmed that GEH-RGD NPs specifically bound to the integrin αvβ3. In vitro cell-viability assay showed that GEH-RGD efficiently inhibited HUVEC proliferation at low EGCG concentrations (20 μg/mL) when compared with EGCG or non-RGD-modified NPs. Furthermore, GEH-RGD NPs significantly inhibited HUVEC migration down to 58%, lasting for 24 hours. In the corneal NV mouse model, fewer and thinner vessels were observed in the alkali-burned cornea after treatment with GEH-RGD NP eyedrops. Overall, this study indicates that GEH-RGD NPs were successfully developed and synthesized as an inhibitor of vascular endothelial cells with specific targeting capacity. Moreover, they can be used in eyedrops to inhibit angiogenesis in corneal NV mice.

Calcium phosphate cement chamber as an immunoisolative device for bioartificial pancreas: In vitro and preliminary in vivo study

Objectives: This study examined a calcium phosphate cement (CPC) chamber as an immunoisolative device to facilitate the use of xenogeneic cell sources without immunosuppression for the bioartificial pancreas (BAP). Methods: Mouse insulinoma cells were encapsulated in agarose gel and then enclosed in a CPC chamber to create a BAP. Bioartificial pancreas were evaluated by cell viability, live-dead cell ratio, and cytokine-mediated cytotoxicity assay and implanted into the peritoneal cavity of diabetic rats. Nonfasting blood glucose and serum insulin levels were analyzed perioperatively; BAPs were also retrieved for histological examination. Results: Insulinoma cells enclosed in the CPC chamber had normal viability, cell survival, and insulin secretion that was even cultured in media with cytokines. The nonfasting blood glucose level of rats was decreased from 460 ± 50 to 132 ± 43 mg/dL and maintained euglycemia for 22 days; serum insulin level was increased from 0.34 ± 0.11 to 1.43 ± 0.30 &mgr;g/dL after operation. Histological examination revealed the fibrous tissue envelopment, and immune-related cells that competed for oxygen resulting in hypoxia could be attributed to the dysfunction of BAPs. Conclusions: This study proved the feasibility for using a CPC chamber as an immunoisolative device for the BAP. An alternative implanted site should be considered to extend the functional longevity of BAPs in further study.

In vitro study of root fracture treated by CO2 laser and DP-bioactive glass paste

BACKGROUND/PURPOSE An ideal material has yet to be discovered that can successfully treat vertical root fracture. Therefore, the purpose of this study was to use a continuous-wave CO2 laser of medium-energy density to fuse DP-bioactive glass paste (DPGP) to vertical root fracture. METHODS The DP-bioglass powder was based on a Na2O-CaO-SiO2-P2O5 system and it was mixed with phosphoric acid (65% concentration) with a powder/liquid ratio of 2 g/4 mL to form DPGP. The interaction of DPGP and dentin was analyzed by means of X-ray diffractometer (XRD) and differential thermal analysis/thermogravimetric analysis (DTA/TGA). Root fracture line was filled with DPGP followed by CO2 laser irradiation and the result was examined by scanning electron microscopy (SEM). RESULTS The main crystal phase of DPGP was monocalcium phosphate monohydrate (Ca(H2PO4)2.H2O) and the phase transformed to dicalcium phosphate dihydrate (CaHPO4.2H2O) after mixing DPGP with dentin powder (DPG-D). Additionally, gamma-Ca2P2O7 and beta-Ca2P2O7 were identified when DPG-D was lased by CO2 laser. The reaction temperature was between 500 degrees C and 1100 degrees C. SEM results demonstrated that the fracture line was effectively sealed by DPGP. CONCLUSION The chemical reaction of DPGP and dentin indicated that DPGP combined with CO2 laser is a potential regimen for the treatment of vertical root fracture.

Herbal supplement in a buffer for dry eye syndrome treatment

Dry eye syndrome (DES) is one of the most common types of ocular diseases. There is a major need to treat DES in a simple yet efficient way. Artificial tears (AT) are the most commonly used agents for treating DES, but are not very effective. Herbal extractions of ferulic acid (FA), an anti-oxidant agent, and kaempferol (KM), an anti-inflammatory reagent, were added to buffer solution (BS) to replace ATs for DES treatment. The cytotoxicity and anti-inflammatory effects were examined in vitro by co-culture with human corneal epithelial cells (HCECs) to obtain the optimal concentration of KM and FA for treating HCECs. Physical properties of BS, such as pH value, osmolality, and refractive index were also examined. Then, rabbits with DES were used for therapeutic evaluation. Tear production, corneal damage, and ocular irritation in rabbits’ eyes were examined. The non-toxic concentrations of KM and FA for HCEC cultivation over 3 days were 1 µM and 100 µM, respectively. Live/dead stain results also show non-toxicity of KM and FA for treating HCECs. Lipopolysaccharide-stimulated HCECs in inflammatory conditions treated with 100 µM FA and 1 µM KM (FA100/KM1) showed lower IL-1B, IL-6, IL-8, and TNFα expression when examined by real-time PCR. The BS with FA100/KM1 had neutral pH, and a similar osmolality and refractive index to human tears. Topical delivery of BS + FA100/KM1 showed no irritation to rabbit eyes. The corneal thickness in the BS + FA100/KM1 treated group was comparable to normal eyes. Results of DES rabbits treated with BS + FA100/KM1 showed less corneal epithelial damage and higher tear volume than the normal group. In conclusion, we showed that the combination of FA (100 µM) and KM (1 µM) towards treating inflamed HCECs had an anti-inflammatory effect, and it is effective in treating DES rabbits when BS is added in combination with these two herbal supplements and used as a topical eye drop.