Chia-Hung Lee

Surface charge-mediated rapid hepatobiliary excretion of mesoporous silica nanoparticles.

Nanoparticle-assisted drug delivery has been emerging as an active research area in recent years. The in vivo biodistribution of nanoparticle and its following mechanisms of biodegradation and/or excretion determine the feasibility and applicability of such a nano-delivery platform in the practical clinical translation. In this work we report the synthesis of the highly positive charge, near-infrared fluorescent mesoporous silica nanoparticles (MSNs) that demonstrate rapid hepatobiliary excretion, for use as traceable drug delivery platforms of high capacity. MSNs were incorporated with near-infrared fluorescent dye indocyanine green (ICG) via covalent or ionic bonding, to derive comparable constructs of significantly different net surface charge. In vivo fluorescence imaging and subsequent inductively coupled plasma-mass spectroscopy of harvested tissues, urine, and feces revealed markedly different uptake and elimination behaviors between the two conjugations; with more highly charged moieties (+34.4 mV at pH 7.4) being quickly excreted from the liver into the gastrointestinal tract, while less charged moieties (-17.6 mV at pH 7.4) remained sequestered within the liver. Taken together, these findings suggest that charge-dependent adsorption of serum proteins greatly facilitates the hepatobiliary excretion of silica nanoparticles, and that nanoparticle residence time in vivo can be regulated by manipulation of surface charge.

Tri-functionalization of mesoporous silica nanoparticles for comprehensive cancer theranostics—the trio of imaging, targeting and therapy

In this work we report the development of the tri-functionalized mesoporous silica nanoparticles (MSNs) for use as theranostic compounds that orchestrate the trio of imaging, target and therapy in a single particle. The MSNs are functionalized in sequence with (1) contrast agents that enable traceable imaging of particle targeting, (2) drug payloads for therapeutic intervention and, (3) biomolecular ligands for highly-targeted particle delivery. Traceable imaging of nanoparticles was accomplished by directly incorporating a near-infrared (NIR) fluorescent contrast agent, ATTO647N, into the silica framework of MSNs, to exploit the relative transparency of most tissues at NIR wavelengths and maximize MSN surface area available for the subsequent conjugating drugs and targeting ligands. An oxygen-sensing, palladium-porphyrin based photosensitizer (Pd-porphyrin; PdTPP) was incorporated into the MSNs nanochannels, to enable photodynamic therapy (PDT). cRGDyK peptides, tiling the outermost surfaces of MSNs, were used for targeting the overexpressed αvβ3 integrins of cancer cells, and to ensure the internalization of the photosensitizer PdTPP. In vitro cell evaluation of the theranostic platform demonstrated not only excellent targeting specificity and minimal collateral damage, but highly potent therapeutic effect as well.

Mesoporous silica nanoparticles functionalized with an oxygen-sensing probe for cell photodynamic therapy: potential cancer theranostics

Functionalization of mesoporous silica nanoparticles (MSNs) with Pd-porphyrins for cancer cell photodynamic therapy is reported. The composite platform, MSN–PdTPP, expands the role of Pd-porphyrins from their routine use as phosphorescence probes for oxygen sensing/imaging (diagnostics) to that of novel nano-photosensitizers for cancer cell phototherapy (therapeutics). The utility of MSN–PdTPP in the phototherapeutic treatment of MDA-MB-231 breast cancer cells is also evaluated, suggesting it is a promising cancer theranostic platform.

Enhanced Chemotherapy of Cancer Using pH-Sensitive Mesoporous Silica Nanoparticles to Antagonize P-Glycoprotein-Mediated Drug Resistance

Multidrug resistance (MDR) is the major clinical obstacle in the management of cancer by chemotherapy. Overexpression of ATP-dependent efflux transporter P-glycoprotein (PGP) is a key factor contributing to multidrug resistance of cancer cells. The purpose of the present study was to use the endosomal pH-sensitive MSN (mesoporous silica nanoparticles; MSN-Hydrazone-Dox) for controlled release of doxorubicin (Dox) in an attempt to overcome the PGP-mediated MDR. In vitro cell culture studies indicate that uptake of MSN-Hydrazone-Dox by the human uterine sarcoma MES-SA/Dox-resistant tumor (MES-SA/Dx-5) cell occurs through endocytosis, thus bypassing the efflux pump resistance. This improves the efficacy of the drug and leads to significant cytotoxicity and DNA fragmentation evidenced by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling and DNA laddering assays. In vivo studies show that the intratumor injection of MSN-Hydrazone-Dox induces significant apoptosis of MES-SA/Dox-resistant cancer cells. This is validated by active caspase-3 immunohistochemical analysis. However, MSN-Hydrazone, without doxorubicin conjugation, cannot induce apoptosis in vitro and in vivo. In conclusion, both in vitro and in vivo studies show that MSN could serve as an efficient nanocarrier entering cell avidly via endocytosis, thus bypassing the PGP efflux pump to compromise the PGP-mediated MDR. MSN-Hydrazone-Dox could further respond to endosomal acidic pH to release doxorubicin in a sustained manner. Besides the cell study, this is the first report that successfully shows the therapeutic efficacy of using MSN against MDR cancer in vivo. Mol Cancer Ther; 10(5); 761–9. ©2011 AACR.

Cytochrome c covalently immobilized on mesoporous silicas as a peroxidase: Orientation effect

Cytochrome c (cyt c), a heme protein with positive electric charge and global dimensions of 2.5 × 2.5 × 3.7 nm, is immobilized by covalent bonding in the nanochannels and on the surface of IBN4 (pore size = 5.3–7.1 nm) mesoporous silicas. The composite material behaves as a peroxidase in the oxidation of organic molecules in the presence of hydrogen peroxide. The surface of IBN4 was first modified with three different linkers, glutaric anhydride (GAC), glutaraldehyde (GAH) and succinimido-3-maleimidopropanoate (SMP), to facilitate the binding (bioconjugation) with cyt c. Different linkers expose the catalytic active site (Fe-heme) to different environments, which allows us to examine the orientation effect imposed by the binding linker. Molecular modeling further allows us to assess the orientation effect on the catalytic activity arising from the distribution of electric charges of cyt c immobilized in different surface-modified nanochannels. The accessibility of the Fe active center in immobilized cyt c is found to be in the following order: IBN4-N-SMP-cyt c > IBN4-N-GAH-cyt c > IBN4-N-GAC-cyt c, which is correlated to the measured trend of the initial specific peroxidase-like activities of immobilized cyt c in three different modified surfaces towards the oxidation of 4-aminoantipyrine. The surface-modified nanochannels of mesoporous silica provide the confining spaces that could prevent cyt c from protein unfolding and orient the active site in a favorable location in the pores to facilitate its activity. However, there is much more structural decay after hydrothermal treatment and the activities diminish accordingly: the IBN4-N-GAH-cyt c sample lost most of its activity, the IBN4-N-SMP-cyt c lost its activity due to less protection of the active center of Fe-heme, and the IBN4-N-GAC-cyt c retained good activity. These temperature effects are further confirmed in the UV-Vis spectra and EPR studies. Cyt c immobilized on functionalized IBN4 surfaces exists in high spin state, as inferred from EPR and UV-Vis studies, which differs from the primarily low spin state of native cyt c. The high spin state arises from the replacement of Met-80 ligands of heme Fe(III) by water or silanol groups on the silica surface, which could open up the heme groove for easy access of oxidants to the iron center and facilitate the catalytic activity. Finally, we apply the covalently immobilized cyt c in the oxidation of a representative polycyclic aromatic hydrocarbon (PAH)—pyrene. The trend in activity can be understood from the design principle we learned in this work.

pH-Triggered Controllable Release of Silver–Indole-3 Acetic Acid Complexes from Mesoporous Silica Nanoparticles (IBN-4) for Effectively Killing Malignant Bacteria

An efficient approach for the antimicrobial agent delivery specifically at acidic pH has been proposed. At the outset, functionalized mesoporous nanoparticles (NPs) were examined to verify the success of synthesis while considering the structural properties by various characterizations. The NPs were immobilized with silver-indole-3 acetic acid hydrazide (IAAH-Ag) complexes via a pH-sensitive hydrazone bond, which functioned as a model drug. When the transitional metal complexes with IBN-4-IAAH-Ag were exposed to acidic pH (near pH 5.0), the silver ions were preferentially released (70%) in a controlled manner up to 12 h by pH-sensitive denial of hydrazone bonds. In contrary, a low drug release (about 25%) was seen in physiological buffer (pH 7.4) demonstrating the pH sensitive release of this drug. Furthermore, the antibacterial efficacy of this unique structured sample was tested against the planktonic cells and biofilms of Gram-positive and Gram-negative bacteria with field emission scanning electron microscope in turn measuring the growth curves, formation of lethal reactive oxygen species, protein leakage, and DNA damage. The synthesized pH-sensitive IAAH-Ag complex was found to have high antimicrobial efficacy against multidrug resistant clinical isolates both in planktonic and biofilm states. Going forward, the synthesized nanoconjugates proved a good in vivo efficacy in treating the bacterial infection of mice. These new metal complex-conjugated NPs through a pH-sensitive hydrazone bond opened up a new avenue for the design and synthesis of the next generation antibacterial agents, which would act as an alternative to antibiotics.

Utilization of Enzyme-Immobilized Mesoporous Silica Nanocontainers (IBN-4) in Prodrug-Activated Cancer Theranostics

To develop a carrier for use in enzyme prodrug therapy, Horseradish peroxidase (HRP) was immobilized onto mesoporous silica nanoparticles (IBN-4: Institute of Bioengineering and Nanotechnology), where the nanoparticle surfaces were functionalized with 3-aminopropyltrimethoxysilane and further conjugated with glutaraldehyde. Consequently, the enzymes could be stabilized in nanochannels through the formation of covalent imine bonds. This strategy was used to protect HRP from immune exclusion, degradation and denaturation under biological conditions. Furthermore, immobilization of HRP in the nanochannels of IBN-4 nanomaterials exhibited good functional stability upon repetitive use and long-term storage (60 days) at 4 °C. The generation of functionalized and HRP-immobilized nanomaterials was further verified using various characterization techniques. The possibility of using HRP-encapsulated IBN-4 materials in prodrug cancer therapy was also demonstrated by evaluating their ability to convert a prodrug (indole-3-acetic acid (IAA)) into cytotoxic radicals, which triggered tumor cell apoptosis in human colon carcinoma (HT-29 cell line) cells. A lactate dehydrogenase (LDH) assay revealed that cells could be exposed to the IBN-4 nanocomposites without damaging their membranes, confirming apoptotic cell death. In summary, we demonstrated the potential of utilizing large porous mesoporous silica nanomaterials (IBN-4) as enzyme carriers for prodrug therapy.

Effect of spin configuration on the reactivity of cytochrome c immobilized in mesoporous silica

Cytochrome c (cytc), a heme protein with a positive electric charge, is immobilized in the nanochannels of mesoporous silica (MPS) by either electrostatic forces or covalent bonding. The electrostatic interaction between cytc and MPS arises from the introduction of aluminum into the framework of MPS to produce a negative charge on the porous surface (Al-MPS). The covalent bonding arises from the binding between the heme iron center and the –SH group of mercaptotriethoxysilane in thiol-functionalized MPS (MPS-SH). The nanochannels of MPS provide a confining space that can prevent cytc from protein unfolding and preserve its activity. Cytc immobilized in Al-MPS exists in a high-spin state, as inferred from ESR and UV–Vis studies. This is different from native cytc, which shows primarily the low-spin state. The high-spin state arises from the replacement of the Met-80 ligands of heme Fe(III) by water or silanol groups on the silica surface, which can open up the heme groove for ready access of oxidants to the iron center and facilitate catalytic activity. MPS-SH-supported cytc can exist in both high- and low-spin states. The low-spin state arises from the replacement of the axial ligands of heme Fe(III) by the –SH group, which can result in poisoning of the active site and reduce the catalytic activity with respect to the decomposition of hydroperoxides. In ESR spin trapping experiments, we show that cytc catalyzes homolytic cleavage of the O–O bond of hydroperoxide and generates a protein cation radical (g = 2.00). A possible mechanism for the MPS-cytc catalytic oxidation of hydroperoxide is proposed based on a spectroscopic characterization of the system.

Synthesis and Characterization of Chitosan-Coated Near-Infrared (NIR) Layered Double Hydroxide-Indocyanine Green Nanocomposites for Potential Applications in Photodynamic Therapy.

We designed a study for photodynamic therapy (PDT) using chitosan coated Mg–Al layered double hydroxide (LDH) nanoparticles as the delivery system. A Food and Drug Administration (FDA) approved near-infrared (NIR) fluorescent dye, indocyanine green (ICG) with photoactive properties was intercalated into amine modified LDH interlayers by ion-exchange. The efficient positively charged polymer (chitosan (CS)) coating was achieved by the cross linkage using surface amine groups modified on the LDH nanoparticle surface with glutaraldehyde as a spacer. The unique hybridization of organic-inorganic nanocomposites rendered more effective and successful photodynamic therapy due to the photosensitizer stabilization in the interlayer of LDH, which prevents the leaching and metabolization of the photosensitizer in the physiological conditions. The results indicated that the polymer coating and the number of polymer coats have a significant impact on the photo-toxicity of the nano-composites. The double layer chitosan coated LDH–NH2–ICG nanoparticles exhibited enhanced photo therapeutic effect compared with uncoated LDH–NH2–ICG and single layer chitosan-coated LDH–NH2–ICG due to the enhanced protection to photosensitizers against photo and thermal degradations. This new class of organic-inorganic hybrid nanocomposites can potentially serve as a platform for future non-invasive cancer diagnosis and therapy.

High loading of C60 in nanochannels of mesoporous MCM-41 materials

Very high loadings of C60 and Canions in MCM-41 mesoporous materials have been achieved by modifying the channel surface with amino-functional group. Detailed physical properties of these mesoporous materials containing C60 were characterized by EPR, NMR, XRD, FTIR and UV-vis spectroscopic techniques. The surface area and pore size of mesoporous materials were further determined by N2 adsorption-desorption isotherms. The mesopores of MCM-41 show sieving behavior in excluding the larger sized C120O while absorbing the smaller C60 as indicated in the EPR studies. 2002 Elsevier Science Inc. All rights reserved.