Nikken Wiradharma

Self-assembled oligopeptide nanostructures for co-delivery of drug and gene with synergistic therapeutic effect.

In this study, oligopeptide amphiphile containing three blocks of amino acids, Ac-(AF)(6)-H(5)-K(15)-NH(2) (FA32), were synthesized and evaluated as carriers for co-delivery of drug and gene. Doxorubicin (DOX), luciferase reporter gene, and p53 gene were used as a model drug and genes. The peptide amphiphile self-assembled into cationic core-shell nanostructures (i.e. micelles), with a CMC value of around 0.042 mg/mL, estimated by fluorescent spectroscopy technique. FA32 nanostructures had an average size of 102+/-19 nm, and a zeta potential of 22.8+/-0.2 mV. These nanostructures had a high capacity for DOX encapsulation, with a DOX loading level of up to 22%. In addition, DOX release from the micelles was sustained without obvious initial burst. DOX-loaded micelles were effectively taken up by HepG2 cells, with an IC(50) of 1.8 mg/L for DOX-loaded FA32, which was higher than that of free DOX (0.25 mg/L). In addition, FA32 micelles condensed DNA efficiently to form small complexes with net positive charge on the surface. In vitro gene transfection studies showed that FA32 induced comparable gene expression level to polyethylenimine. Co-delivery of drug and gene using FA32 micelles was demonstrated via confocal imaging, luciferase expression in the presence of DOX, and synergy in cytotoxic effect between p53 gene and DOX. It was shown that through simultaneous delivery of both p53 gene and DOX using FA32 micelles, an increase in p53 mRNA expression level as well as end point cytotoxicity towards HepG2 cells was achieved. FA32 micelles, therefore, have a great potential in delivering hydrophobic anticancer drug and gene simultaneously for improved cancer therapy.

Synthesis of a family of amphiphilic glycopolymers via controlled ring-opening polymerization of functionalized cyclic carbonates and their application in drug delivery

Polymers bearing pendant carbohydrates have a variety of biomedical applications especially in the area of targeted drug delivery. Here we report the synthesis of a family of amphiphilic block glycopolymers containing d glucose, d galactose and d mannose via metal-free organocatalyzed ring-opening polymerization of functional cyclic carbonates generating narrowly dispersed products of controlled molecular weight and end-group fidelity, and their application in drug delivery. These glycopolymers self-assemble into micelles having a high density of sugar molecules in the shell, a size less than 100 nm with narrow size distribution even after drug loading, and little cytotoxicity, which are important for drug delivery. Using galactose-containing micelles as an example, we demonstrate their strong targeting ability towards ASGP-R positive HepG2 liver cancer cells in comparison with ASGP-R negative HEK293 cells although the galactose is attached to the carbonate monomer at 6-position. The enhanced uptake of DOX-loaded galactose-containing micelles by HepG2 cells significantly increases cytotoxicity of DOX as compared to HEK293. This new family of amphiphilic block glycopolymers has great potential as carriers for targeted drug delivery.

Synthetic cationic amphiphilic α-helical peptides as antimicrobial agents

Antimicrobial peptides (AMPs) secreted by the innate immune system are prevalent as the effective first-line of defense to overcome recurring microbial invasions. They have been widely accepted as the blueprints for the development of new antimicrobial agents for the treatment of drug resistant infections. However, there is also a growing concern that AMPs with a sequence that is too close to the host organisms AMP may inevitably compromise its own natural defense. In this study, we design a series of synthetic (non-natural) short α-helical AMPs to expand the arsenal of the AMP families and to gain further insights on their antimicrobial activities. These cationic and amphiphilic peptides have a general sequence of (XXYY)(n) (X: hydrophobic residue, Y: cationic residue, and n: the number of repeat units), and are designed to mimic the folding behavior of the naturally-occurring α-helical AMPs. The synthetic α-helical AMPs with 3 repeat units, (FFRR)(3), (LLRR)(3), and (LLKK)(3), are found to be more selective towards microbial cells than rat red blood cells, with minimum inhibitory concentration (MIC) values that are more than 10 times lower than their 50% hemolytic concentrations (HC(50)). They are effective against Gram-positive B. subtilis and yeast C. albicans; and the studies using scanning electron microscopy (SEM) have elucidated that these peptides possess membrane-lytic activities against microbial cells. Furthermore, non-specific immune stimulation assays of a typical peptide shows negligible IFN-α, IFN-γ, and TNF-α inductions in human peripheral blood mononuclear cells, which implies additional safety aspects of the peptide for both systemic and topical use. Therefore, the peptides designed in this study can be promising antimicrobial agents against the frequently-encountered Gram-positive bacteria- or yeast-induced infections.

Advanced Materials for Co-Delivery of Drugs and Genes in Cancer Therapy

With cancer being the major cause of mortality worldwide, the continued development of safe and efficacious treatments is warranted. A better understanding of the molecular mechanism and genetic basis of tumor initiation and progression, coupled with advances in chemistry, molecular biology and engineering have led to discovery of a wide range of therapeutic agents for cancer therapy. However, multidrug-resistance, which is mainly caused by malfunction of genes, has become a major problem in chemotherapy. To overcome this problem, the simultaneous delivery of genes to cancer cells has been proposed to correct the malfunctioned genes to sensitize the cells to chemotherapeutics. This progress report summarizes key advances in drug and gene delivery with focus on the development of polymers, peptides, liposomes and inorganic materials as nanocarriers for co-delivery of small molecular drugs and macromolecular genes or proteins. In addition, challenges and future perspectives in the design of nanocarriers for the co-delivery of therapeutic drugs and genes are discussed.

Cationic micelles self-assembled from cholesterol-conjugated oligopeptides as an efficient gene delivery vector.

Cholesterol-conjugated H(5)R(10) and H(10)R(10) oligopeptides (HR15-Chol and HR20-Chol) were designed and synthesized. These amphiphilic oligopeptides were able to self-assemble into cationic micelles in aqueous solution at low concentrations, and their critical micelle concentrations in sodium acetate buffer (20mM, pH 5.0) were 17.8 and 28.2mg/L respectively. The micelle formation was further evidenced via SEM and dynamic light scattering analyses. The average hydrodynamic size of HR15-Chol and HR20-Chol micelles was about 425 and 435 nM in diameter with zeta potential of 64 and 66 mV respectively. The formation of micelles increased local concentration of cationic charge, leading to higher DNA binding efficiency as compared to the control peptides HR15 and HR20. The minimum size observed for HR15-Chol/DNA and HR20-Chol/DNA complexes was about 175-176 nM, and the maximum zeta potential was around 61-62 mV. In comparison, HR15 and HR20 formed DNA complexes with a similar size but significantly lower zeta potential (i.e. about 31-40 mV). In particular, after being challenged by DMEM medium, the size of peptide/DNA complexes was increased significantly and their surface charge was neutralized. Nevertheless, the size of the micelle/DNA complexes formed from HR15-Chol and HR20-Chol was still about 200 nM with positive charge of around 20 mV at high N/P ratios. The micelles induced much higher overall gene expression (i.e. luciferase expression) levels than the peptides in both HepG2 and HEK293 cell lines. Increasing the histidine residue from 0 to 5 to 10 further increased gene expression efficiency. In particular, HR20-Chol micelles yielded 95% GFP-positive HepG2 cells at N/P 50, much higher than that induced by PEI at its optimal N/P ratio (i.e. 10), which was 6.8%. In 4T1 cells, HR20-Chol induced 2 times higher luciferase expression level than PEI at their optimal N/P ratios. Moreover, HR20-Chol micelle/DNA complexes were less cytotoxic than PEI/DNA complexes. These micelles may be a promising carrier for delivery of therapeutic genes.

Strategies employed in the design and optimization of synthetic antimicrobial peptide amphiphiles with enhanced therapeutic potentials.

Antimicrobial peptides (AMPs) which predominantly act via membrane active mechanisms have emerged as an exciting class of antimicrobial agents with tremendous potential to overcome the global epidemic of antibiotics-resistant infections. The first generation of AMPs derived from natural sources as diverse as plants, insects and humans has provided a wealth of compositional and structural information to design novel synthetic AMPs with enhanced antimicrobial potencies and selectivities, reduced cost of production due to shorter sequences and improved stabilities under physiological conditions. In this review, we will first discuss the common strategies employed in the design and optimization of synthetic AMPs, followed by highlighting the various approaches utilized to enhance the therapeutic potentials of designed AMPs under physiological conditions. Lastly, future perspectives on the development of improved AMPs for therapeutic applications will be presented.

The effect of thiol functional group incorporation into cationic helical peptides on antimicrobial activities and spectra

Antimicrobial peptides (AMP) have been proposed as blueprints for the development of new antimicrobial agents for the treatment of drug resistant infections. A series of synthetic AMPs capable of forming α-helical structures and containing free-sulfhydryl groups are designed in this study ((LLKK)(2)C, C(LLKK)(2)C, (LLKK)(3)C, C(LLKK)(3)C). In particular, the AMP with 2 cysteine residues at the terminal ends of the peptide and 2 repeat units of LLKK, i.e., C(LLKK)(2)C, has been demonstrated to have high selectivity towards a wide range of microbes from Gram-positive Bacillus subtilis, Gram-negative Escherichia coli, Pseudomonas aerogenosa, and yeast Candida albicans over red blood cells. At the MIC levels, this peptide does not induce significant hemolysis, and its MIC values occur at the concentration of more than 10 times of their corresponding 50% hemolysis concentrations (HC(50)). Microscopy studies suggest that this peptide kills microbial cells by inducing pores of ∼20-30 nm in size in microbial membrane on a short time scale, which further develops to grossly damaged membrane envelope on a longer time scale. Multiple treatments of microbes with this peptide at sub MIC concentration do not induce resistance, even up to passage 10. However, the same treatment with conventional antibiotics penicillin G or ciprofloxacin easily develop resistance in the treated microbes. In addition, the peptides are shown not to induce secretion of IFN-γ and TNF-α in human monocytes as compared to lipopolysaccharide, which implies additional safety aspects of the peptides to be used as both systemic and topical antimicrobial agents. Therefore, this study provides an excellent basis to develop promising antimicrobial agents that possess a broad range of antimicrobial activities with less susceptibility for development of drug resistance.

Broad-spectrum antimicrobial supramolecular assemblies with distinctive size and shape

With the increased prevalence of antibiotic-resistant infections, there is an urgent need for innovative antimicrobial treatments. One such area being actively explored is the use of self-assembling cationic polymers. This relatively new class of materials was inspired by biologically pervasive cationic host defense peptides. The antimicrobial action of both the synthetic polymers and naturally occurring peptides is believed to be complemented by their three-dimensional structure. In an effort to evaluate shape effects on antimicrobial materials, triblock polymers were polymerized from an assembly directing terephthalamide-bisurea core. Simple changes to this core, such as the addition of a methylene spacer, served to direct self-assembly into distinct morphologies-spheres and rods. Computational modeling also demonstrated how subtle core changes could directly alter urea stacking motifs manifesting in unique multidirectional hydrogen-bond networks despite the vast majority of material consisting of poly(lactide) (interior block) and cationic polycarbonates (exterior block). Upon testing the spherical and rod-like morphologies for antimicrobial properties, it was found that both possessed broad-spectrum activity (Gram-negative and Gram-positive bacteria as well as fungi) with minimal hemolysis, although only the rod-like assemblies were effective against Candida albicans.

Supramolecular high-aspect ratio assemblies with strong antifungal activity

Efficient and pathogen-specific antifungal agents are required to mitigate drug resistance problems. Here we present cationic small molecules that exhibit excellent microbial selectivity with minimal host toxicity. Unlike typical cationic polymers possessing molecular weight distributions, these compounds have an absolute molecular weight aiding in isolation and characterization. However, their specific molecular recognition motif (terephthalamide-bisurea) facilitates spontaneous supramolecular self-assembly manifesting in several polymer-like properties. Computational modelling of the terephthalamide-bisurea structures predicts zig-zag or bent arrangements where distal benzyl urea groups stabilize the high-aspect ratio aqueous supramolecular assemblies. These nanostructures are confirmed by transmission electron microscopy and atomic force microscopy. Antifungal activity against drug-sensitive and drug-resistant strains with in vitro and in vivo biocompatibility is observed. Additionally, despite repeated sub-lethal exposures, drug resistance is not induced. Comparison with clinically used amphotericin B shows similar antifungal behaviour without any significant toxicity in a C. albicans biofilm-induced mouse keratitis model.

Delivery of reprogramming factors into fibroblasts for generation of non-genetic induced pluripotent stem cells using a cationic bolaamphiphile as a non-viral vector

Protein delivery allows a clinical effect to be directly realized without genetic modification of the host cells. We have developed a cationic bolaamphiphile as a non-viral vector for protein delivery application. The relatively low toxicity and efficient protein delivery by the cationic bolaamphiphile prompted us to test the system for the generation of induced pluripotent stem cells (iPSCs) as an alternative to the conventional vector-based genetic approach. Studies on the kinetics and cytotoxicity of the protein delivery system led us to use an optimized cationic bolaamphiphile-protein complex ratio of 7:1 (wt/wt) and a 3 h period of incubation with human fibroblasts, to ensure complete and non-toxic protein delivery of the reprogramming proteins. The reprogrammed cells were shown to exhibit the characteristics of embryonic stem cells, including expression of pluripotent markers, teratoma formation in SCID mice, and ability to be differentiated into a specific lineage, as exemplified by neuronal differentiation.