Shih-Jer Huang

Folic acid–Pluronic F127 magnetic nanoparticle clusters for combined targeting, diagnosis, and therapy applications

Superparamagnetic iron oxides possess specific magnetic properties in the presence of an external magnetic field, which make them an attractive platform as contrast agents for magnetic resonance imaging (MRI) and as carriers for drug delivery. In this study, we investigate the drug delivery and the MRI properties of folate-mediated water-soluble iron oxide incorporated into micelles. Pluronic F127 (PF127), which can be self-assembled into micelles upon increasing concentration or raising temperatures, is used to decorate water-soluble polyacrylic acid-bound iron oxides (PAAIO) via a chemical reaction. Next, the hydrophobic dye Nile red is encapsulated into the hydrophobic poly(propylene oxide) compartment of PF127 as a model drug and as a fluorescent agent. Upon encapsulation, PAAIO retains its superparamagnetic characteristics, and thus can be used for MR imaging. A tumor-specific targeting ligand, folic acid (FA), is conjugated onto PF127-PAAIO to produce a multifunctional superparamagnetic iron oxide, FA-PF127-PAAIO. FA-PF127-PAAIO can be simultaneously applied as a diagnostic and therapeutic agent that specifically targets cancer cells that overexpress folate receptors in their cell membranes. PF127-PAAIO is used as a reference group. Based on FTIR and UV-vis absorbance spectra, the successful synthesis of PF127-PAAIO and FA-PF127-PAAIO is realized. The magnetic nanoparticle clusters of PF127-PAAIO and FA-PF127-PAAIO are visualized by transmission electron microscope (TEM). FA-PF127-PAAIO, together with a targeting ligand, displays a higher intracellular uptake into KB cells. This result is confirmed by laser confocal scanning microscopy (CLSM), flow cytometry, and atomic absorption spectroscopy (AAS) studies. The hysteresis curves, generated by using a superconducting quantum interference device (SQUID) magnetometer analysis, demonstrate that the magnetic nanoparticles are superparamagnetic with insignificant hysteresis. The MTT assay explains the negligible cell cytotoxicity of PF127-PAAIO and FA-PF127-PAAIO. In KB cells, the in vitro MRI study indicates the better T(2)-weighted images in FA-PF127-PAAIO than in PF127-PAAIO.

Folate-mediated chondroitin sulfate-Pluronic® 127 nanogels as a drug carrier

Pluronic F127 (PF127), one of the polymers which can inhibit drug efflux transporters in cancer therapy, was used to produce amphiphilic nanocarriers for doxorubicin (DOX). In order to stabilize the nanocarriers, the hydroxyl groups on both termini of PF127 were acrylated and reacted with methacrylated chondroitin sulfate (CSMA) to form CS-PF127 nanogel. The introducing CSMA has carboxylic acid groups which can be used to react with a folic acid-polyethylene glycol (FA-PEG). Folic acid, having high binding affinity to tumor-associated folate receptors (FR), provides a selective delivery of doxorubicin (DOX) to FR-positive tumor cells. DOX was loaded either in a cationic DOX.HCl form through the electrostatic interactions with the negative charges of chondroitin sulfate, or in a free DOX form by solubilization into the PPO core compartment of PF127. The loading efficiency and release behavior of DOX prepared from two different formulations are compared. The synthesis of CS-PF127 and FA-PEG grafted CS-PF127 (FA-CS-PF127) was characterized by nuclear magnetic resonance spectrometry (NMR), ultraviolet/visible spectroscopy (UV), and X-ray photoelectron spectroscopy (XPS). With a fluorescent probe technique, the critical aggregation concentrations (CAC) are 7.5 x 10(-2)mg/mL for CS-PF127 and 7.9 x 10(-2)mg/mL for FA-CS-PF127, respectively. The spherical images of nanogels were visualized with the use of the transmission electron microscope (TEM). The particle diameters measured by dynamic light scattering (DLS) are 299.6+/-8.2nm for CS-PF127 and 138.3+/-12.3 for FA-CS-PF127, neither aggregation nor change in sizes in double deionized (DD) water after 20 days. The better cellular uptake of FA-CS-PF127 in KB cells was evidenced by confocal laser scanning microscopy (CLSM) and flow cytometry upon loading Rhodamine123 as a probe.

Succinated chitosan as a gene carrier for improved chitosan solubility and gene transfection.

UNLABELLED Chitosan (CHI), a linear polysaccharide, has been intensively studied as a nonviral gene delivery vector. The low physiological solubility of CHI has limited its gene transfection efficiency. Here we report the synthesis of different substitution degrees of succinated chitosans (CHI-succ) to increase water solubility. According to the proton nuclear magnetic resonance spectra, the degree of deacetylation of hydrolyzed CHI was roughly 88% and the degrees of succinylation in three CHI-succ polymers were approximately 5, 10, and 20%. Various weight ratios of CHI/DNA and CHI-succ/DNA polyplexes were prepared for gel electrophoresis retardation, particle size, zeta potential, and morphology studies. The results suggest that the plasmid DNA is readily entrapped at a CHI-succ/DNA weight ratio of 20; the sizes and zeta potentials were between 110 and 140 nm and ±1-5 mV, and the polyplexes exhibited low cytotoxicity against HEK 293T cells. CHI-succ with 5 and 10% degrees of substitution showed improved transfection efficiency as compared with nascent CHI. FROM THE CLINICAL EDITOR Chitosan, a cationic polysacchride with gene therapy potential, has inherently poor water solubility, which is improved by partial succinylation according to this report. The new DNA/Chitosan polyplexes exhibit improved safety against HEK 293T cells.

Antitumor Efficacy of Doxorubicin Released from Crosslinked Nanoparticulate Chondroitin Sulfate/Chitosan Polyelectrolyte Complexes

It is demonstrated that nanoparticulate PEC with a crosslinked shell sustains DOX release and increases DOX activity against cancer cells. CSMA was synthesized to prepare PEC with chitosan. The double bonds among CSMA were used to form a shell crosslink. The released DOX from DOX-loaded PECs against human cancer KB cells and A549 cells were qualitatively traced by confocal laser scanning microscopy and flow cytometry, and quantitatively measured by capillary electrophoresis. All the results implied the DOX-loaded PEC with a crosslinked shell had the best anti-cancer potency of free DOX and the DOX-loaded PEC prepared from pure chondroitin sulfate and chitosan in both the cell lines.

One-pot synthesis of PDMAEMA-bound iron oxide nanoparticles for magnetofection

Superparamagnetic iron oxide nanoparticles (SPIONs) have been widely used for multiple biomedical applications. Magnetic-assisted transfection (magnetofection) using SPION is an attractive gene vector candidate. In this work, poly(2-dimethylamino)ethyl methacrylate-bound iron oxide nanoparticles (IO-PDMAEMA) were generated using a grafting-from approach via atom transfer radical polymerization (ATRP) for use as a gene vector. Preparing an iron oxide-initiator (IO-initiator) is a typical and important step. We designed a simple method to produce an IO-initiator containing bromide groups (IO-Br). The IO-Br was synthesized by reacting iron oxide nanoparticles with 2-bromoisobutyric acid using a one-pot solvothermal method at a high temperature. We optimized IO-PDMAEMA by controlling the PDMAEMA molecular weight, allowing higher gene expression with lower cytotoxicity. The hydrodynamic diameter of IO-Br was 76.7 nm, which increased to 361.7 nm after polymerization. Transversal relaxivity studies suggested that IO-PDMAEMA can be a contrast agent for magnetic resonance imaging. The magnetofection efficacy of IO-PDMAEMA/pDNA was measured in HEK 293T cells with or without fetal bovine serum (FBS). The IO-PDMAEMA/pDNA magnetoplexes exhibited remarkably improved gene expression in the presence of a magnetic field and 10% FBS compared with a commercial product, PolyMag/pDNA. No significant cytotoxicity of IO-PDMAEMA/pDNA was observed with different incubation time periods with or without the magnetic field. Confocal laser scanning microscopic images showed that the amount of internalized plasmid DNA increased in the assisted magnetic field.

Pentablock copolymers of pluronic F127 and modified poly(2-dimethyl amino)ethyl methacrylate for internalization mechanism and gene transfection studies

Cationic polymers are one of the major nonviral gene delivery vectors investigated in the past decade. In this study, we synthesized several cationic copolymers using atom transfer radical polymerization (ATRP) for gene delivery vectors: pluronic F127-poly(dimethylaminoethyl methacrylate) (PF127-pDMAEMA), pluronic F127-poly (dimethylaminoethyl methacrylate-tert-butyl acrylate) (PF127-p(DMAEMA-tBA)), and pluronic F127-poly(dimethylaminoethyl methacrylate-acrylic acid) (PF127-p(DMAEMA-AA)). The copolymers showed high buffering capacity and efficiently complexed with plasmid deoxyribonucleic acid (pDNA) to form nanoparticles 80–180 nm in diameter and with positive zeta potentials. In the absence of 10% fetal bovine serum, PF127-p(DMAEMA-AA) showed the highest gene expression and the lowest cytotoxicity in 293T cells. After acrylic acid groups had been linked with a fluorescent dye, the confocal laser scanning microscopic image showed that PF127-p(DMAEMA-AA)/pDNA could efficiently enter the cells. Both clathrin-mediated and caveolae-mediated endocytosis mechanisms were involved. Our results showed that PF127-p(DMAEMA-AA) has great potential to be a gene delivery vector.

Controlled immobilization of chondroitin sulfate in polyacrylic acid networks

Novel semi-interpenetrating polymer networks (semi-IPNs) of chondroitin sulfate (ChS) and acrylic acid (AA) were prepared with the aim of obtaining a hydrogel for use as a colon-specific drug carrier. By controlling the concentrations of cross-linking agent, diethylene glycol dimethacrylate (DEGDA), as well as the reaction solvent, high swelling percentages were obtained (approx. 1600%). However, the highest sol percent obtained for these hydrogels was approx. 70%, and most of the chondroitin sulfate remained soluble and could be extracted. Therefore, an alternative approach was adopted: methacrylate-grafted ChS (ChSMA) was synthesized and then co-polymerized with acrylic acid (AA) at a molar ratio of 1:5 with various concentrations of AA. The sol content of these ChSMA-AA hydrogels was reduced to approx. 20%, and the cross-linking densities were almost 100-fold higher than those of the semi-IPNs. FT-IR spectra showed that the H-bonding interactions between ChS and PAA and the spectra of the semi-IPNs were similar to that of PAA itself after sol extraction. In contrast, the FT-IR spectra of ChSMA-AA remained intact after sol extraction. Ketoprofen was used as a model drug to test the sustained release behavior of these hydrogels.

Angiopep-pluronic F127-conjugated superparamagnetic iron oxide nanoparticles as nanotheranostic agents for BBB targeting

Pluronic® F127-modified water-dispersible poly(acrylic acid)-bound iron oxide (PF127-PAAIO) nanoparticles have been prepared as diagnostic agents. A blood-brain-barrier penetrating peptide, angiopep-2 (ANG), was further conjugated onto the surface of the PF127-PAAIO (ANG-PF127-PAAIO) for brain targeting. The ANG-PF127-PAAIO shows negligible cell cytotoxicity, better cellular uptake, and higher T2-weighted image enhancement than the PF127-PAAIO in U87 cells. Using an ex vivo blood-brain barrier (BBB) model, we showed that the ANG-PF127-PAAIO shows better permeability to bypass the BBB. This is because the ANG-PF127-PAAIO has a dual-targeting ability, recognition of the low-density lipoprotein receptor-related protein and clathrin-mediated receptor on the U87 surface. Thus, the ANG-PF127-PAAIO is a potential nanotheranostic agent for brain dysfunction.

Retinol-encapsulated water-soluble succinated chitosan nanoparticles for antioxidant applications

The aim of this study was to stabilize all-trans-retinol (RE) by complexification with chitosan derivatives through H-bonding. Succinated chitosan (CHI-succ) with three different degrees (5, 10, 20 mol%) of succinylation were synthesized to form complexes with RE. Various weight ratios (w/w) of CHI-succ/RE complexes were prepared and characterized to produce stable complexes in nanometer size. The CHI-succ(0.20)/RE complex with approximate 250 nm in diameter was obtained using a CHI-succ(0.20) concentration of 0.005% (w/v) in double deionized water with various contents of RE. From fine-tuning the degree of succinylation and the weight ratio of the CHI-succ and RE, the formation of supramolecular complexes simultaneously improved water solubility and stability of RE. The cell viability of CHI-succ polymers and their RE complexes in 3T3 cells were all > 85% relative to the control. The antioxidant ability of the CHI-succ(0.20)/RE complexes was significantly greater than that of pure RE using a 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay (p < 0.01).

The synthesis and comparison of poly(methacrylic acid)–poly(ε-caprolactone) block copolymers with and without symmetrical disulfide linkages in the center for enhanced cellular uptake

A self-assembled poly(methacrylic acid)–poly(e-caprolactone) block copolymer with a disulfide linkage, PMAA-b-PCL-SS-PCL-b-PMAA (S-PCL-PMAA)2, was synthesized for enhanced cellular uptake due to a reduction response to glutathione (GSH) and pH-sensitive characteristics. For comparison, a reduction-insensitive PMAA-b-PCL-CC-PCL-b-PMAA (C-PCL-PMAA)2, using a carbon–carbon linkage as a symmetrical center was also synthesized. These block copolymers were synthesized via the combination of ring opening polymerization (ROP) and atom transfer radical polymerization (ATRP) followed by hydrolysis. The similar number of MAA repeating units was controlled in the copolymers containing either disulfide or carbon–carbon linkages. Copolymers could self-assemble into core–shell micelles in an aqueous solution owing to amphiphilicity. The molecular weight of (S-PCL-PMAA)2 increases linearly with reaction time at both reaction temperatures of 40 and 80 °C. Critical micelle concentrations range within 3.09 × 10−3 to 6.31 × 10−3 mg mL−1 at pH 5 and 3.16 × 10−2 to 3.98 × 10−2 mg mL−1 at pH 8. The average hydrodynamic diameters of micelles are ∼200 nm. The cellular uptake of (S-PCL-PMAA)2 increases with incubation time and is higher in the medium with GSH than without in CRL-5802 cells. In contrast, the cellular uptake of (C-PCL-PMAA)2 is insensitive to the presence of GSH. The higher internalization of the micelle containing disulfides in the presence of GSH is attributable to all three pinocytosis pathways involved, including macropinocytosis-, caveolae-, clathrin-mediated endocytosis, but in the absence of GSH clathrin-mediated endocytosis is only involved. The nascent (S-PCL-PMAA)2 is non-cytotoxic to four cell lines. However, the paclitaxel-encapsulated (S-PCL-PMAA)2 shows slightly higher cell-killing ability than free paclitaxel against CRL-5802 cells. Thus, the GSH-responsive (S-PCL-PMAA)2 is a potential drug delivery system.