Branched α-helical peptides enhanced antitumor efficacy and selectivity.

Drug resistance to traditional chemotherapeutics is one of the main challenges in cancer treatment. Herein, cationic antimicrobial peptides (CAPs) were repurposed as anticancer agents to counter chemotherapy drug resistance. After a systematic study of de novo designed synthetic α-helical CAPs in various cell lines, the 4-arm branched peptide {[(LLKK)2]2κC}2 was found to exhibit better selectivity compared to its linear counterpart (LLKK)4, and was more effective than the 2-arm branched peptide [(LLKK)2]2κC. In particular, the 4-arm branched peptide could counter drug resistance and kill multiple drug resistant cells. Mechanism studies reveal that these α-helical peptides killed both the parent and resistant cancer cells based on the apoptotic pathway. The in vivo study in mice bearing breast tumors showed that branched peptides could be retained at the tumour sites after intratumoral injection and significantly reduced tumor growth while exhibiting minimal toxicity on main organs. These results indicate that the 4-arm branched peptide is a promising candidate for anticancer therapy.

Short Synthetic α-Helical-Forming Peptide Amphiphiles for Fungal Keratitis Treatment In Vivo.

The emergence of fungal keratitis is on the rise globally. However, current antifungal therapeutics are ineffective in severe keratomycosis. Previously reported α-helical peptides comprising 8-14 amino acids demonstrate broad-spectrum antimicrobial activity both in vitro and in vivo. Here, α-helical peptides of the optimized sequences are investigated for antifungal biofilm in vitro and in vivo using a fungal biofilm-caused mouse keratitis model. The peptides with the optimal composition demonstrate higher α-helical propensity and improve antifungal activity in dispersing Candida albicans biofilm in vitro. Moreover, the optimized α-helical peptides are not only effective in treating C. albicans biofilm-induced keratitis in mice, they are also nontoxic to the mice eyes. These peptides have the potential to be developed as antifungal agents for the treatment of C. albicans biofilm-caused keratitis.

Synthetic ß-sheet forming peptide amphiphiles for treatment of fungal keratitis.

Fungal keratitis is a leading cause of ocular morbidity. It is frequently misdiagnosed as bacterial keratitis, causing a delay in proper treatment. Furthermore, due to the lack of safe and effective anti-fungal agents for clinical use, treatment of fugal keratitis remains a challenge. In recent years, antimicrobial peptides (AMPs) have received considerable attention as potent and broad-spectrum antimicrobial agents with the potential to overcome antibiotics resistance. We previously reported the design of short synthetic ß-sheet forming peptides (IKIK)2-NH2 and (IRIK)2-NH2 with excellent antimicrobial activities and selectivities against various clinically relevant microorganisms, including Gram-positive Staphylococcus epidermidis and Staphylococcus aureus, Gram-negative Escherichia coli and Pseudomonas aeruginosa, and yeast Candida albicans (C. albicans). In this study, we evaluated the application of the two most promising synthetic ß-sheet forming peptide candidates for in vivo fungal keratitis treatment in comparison with the commercially available amphotericin B. It was found that topical solutions of the designed peptides are safe, and as effective as the clinically used amphotericin B. Compared to the costly and unstable amphotericin B, (IKIK)2-NH2 and (IRIK)2-NH2 are water-soluble, less expensive and stable. Thus, the synthetic ß-sheet forming peptides are presented as promising candidates for the treatment of fungal keratitis.

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.

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.

Prodrugs as self-assembled hydrogels: a new paradigm for biomaterials.

Prodrug-based self-assembled hydrogels represent a new class of active biomaterials that can be harnessed for medical applications, in particular the design of stimuli responsive drug delivery devices. In this approach, a promoiety is chemically conjugated to a known-drug to generate an amphiphilic prodrug that is capable of forming self-assembled hydrogels. Prodrug-based self-assembled hydrogels are advantageous as they alter the solubility of the drug, enhance drug loading, and eliminate the use of harmful excipients. In addition, self-assembled prodrug hydrogels can be designed to undergo controlled drug release or tailored degradation in response to biological cues. Herein we review the development of prodrug-based self-assembled hydrogels as an emerging class of biomaterials that overcome several common limitations encountered in conventional drug delivery.

Rationally designed α-helical broad-spectrum antimicrobial peptides with idealized facial amphiphilicity.

A series of 12-amino acid peptide analogs is designed using point mutation strategy based on an α-helical peptide template. The first mutation in the series, KL12, has an idealized facial amphiphilicity. Subsequent mutations are performed to increase hydrophobic or cationic contents. Idealized facial amphiphilicity show enhanced antimicrobial activity and selectivity against most of the tested microbes. Increasing hydrophobic contents further enhance antimicrobial potency; however, selectivity of the most hydrophobic analog is impaired due to non-specific interactions with mammalian cell membrane. This study demonstrates that facial amphiphilicity and hydrophobic content are strongly correlated with antimicrobial activity and selectivity of antimicrobial peptides.

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.

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.

Cationic amphiphilic alpha-helical peptides for the treatment of carbapenem-resistant Acinetobacter baumannii infection.

The emergence of multidrug-resistant Gram-negative bacteria, in particular Acinetobacter baumannii and Pseudomonas aeruginosa, is a critical clinical problem worldwide. Antimicrobial peptides (AMPs) have received increasing attention due to their ability to overcome multidrug-resistant microbes. We recently reported that cysteine-functionalized alpha-helical peptides LLKKLLKKC and CLLKKLLKKC effectively eradicated Gram-negative bacteria in vitro. In this study, the antibacterial properties of these peptides against carbapenem-resistant clinical isolates of A. baumannii were studied both in vitro and in vivo. The minimum inhibitory concentrations (MICs) of the peptides against 20 clinical isolates of carbapenem-resistant A. baumannii were determined in comparison with imipenem. The results showed that the A. baumannii isolates were more susceptible to (LLKK)(2)C than to C(LLKK)(2)C in vitro, and 90% of the 20 tested strains had an MIC of lower than or equal to 36.8 and 63.1 µmol/L, respectively. However, the bactericidal effect of C(LLKK)(2)C was much faster than that of (LLKK)(2)C. Furthermore, these peptides also showed excellent potency in mouse models of peritonitis and pneumonia infections caused by carbapenem-resistant A. baumannii. Importantly, both peptides had a high therapeutic index (>25), but caused no significant adverse effects on the liver and kidney functions and the balance of electrolytes in the blood. These peptides can be a promising alternative treatment modality to traditional antibiotics for nosocomial bacterial infections caused by multidrug-resistant Gram-negative bacteria, especially carbapenem-resistant A. baumannii.

Oligomerized alpha-helical KALA peptides with pendant arms bearing cell-adhesion, DNA-binding and endosome-buffering domains as efficient gene transfection vectors.

In this study, a number of KALA-based α-helical peptides were designed and synthesized as non-viral gene carriers. The effects of lysine and histidine residues in the pendant arms and cell-binding RGD motif on DNA binding, particle size, zeta potential, cytotoxicity and gene expression efficiency were first explored. Increasing the lysine and histidine residues reduced particle size and increased zeta potential of DNA complexes, leading to greater gene expression efficiency. In addition, the introduction of RGD group further improved gene expression level. The peptide with optimal compositions, RGDN(3)K(6)H(3)CKHLAKALAKALAC (RC29), was then oligomerized to form di-, tri- and tetra-RC29 via disulfide linkage. Upon oligomerization, RC29 attained a 3-dimensional long α-helical structure with pendant arm(s) extending transversally outwards. Each arm contains a cell-adhesion motif (RGD), DNA-binding and endosome-buffering domains(.) The α-helicity of the oligomerized peptides was evaluated by circular dichroism (CD) spectroscopy, which showed that an increased oligomerization degree led to a stronger α-helical structure. These peptides form complexes with DNA efficiently. The minimum size and maximum zeta potential of tri-RC29/DNA complexes was about 200 nm and 32.5 mV, respectively. In comparison, RC29 formed DNA complexes with a similar zeta potential, but particle size was significantly larger (355 nm). DNA complexes formed at pH 7.0 yielded higher gene expressions than those formed at pH 5.5 and 6.5. Among all the oligomerized peptides, tri-RC29 provided the highest gene expression efficiency, and its peak luciferase level was 1.5 times higher than that yielded by PEI at its optimal N/P ratio (i.e. 10). Moreover, oligomerized RC29/DNA complexes were less cytotoxic than PEI/DNA complexes. These α-helical peptides can be promising carrier for delivery of therapeutic genes in the treatment of genetic disorders.

Diaminododecane-based cationic bolaamphiphile as a non-viral gene delivery carrier.

The advancement in gene therapy relies upon the discovery of safe and efficient delivery agents and methods. In this study, we report the design and synthesis of a cationic bolaamphiphile as a non-viral gene delivery agent. The bolaamphiphile is composed of 1,12-diaminododecane as the central hydrophobic unit linked to the hydrophilic pentaethylenehexamine via thioether-based glycidyl units. This bolaamphiphile condensed DNA efficiently into nanoparticles of sizes around 150-200 nm with positive zeta potential of 30-35 mV. In vitro luciferase expression levels and percentage of GFP expressing cells induced by the bolaamphiphile/DNA complexes were higher than those mediated by the often used "golden" standard of non-viral systems, polyethyleneimine (PEI, branched, 25 kDa) at its optimal N/P ratio in HEK293, HepG2, NIH3T3, HeLa and 4T1 cells. In vitro cytotoxicity testing revealed that the DNA complexes fabricated from this cationic bolaamphiphile displayed marginal toxicity towards all the cell lines tested. In addition, in vivo transfection studies carried out in a 4T1 mouse breast cancer model showed that the cationic bolaamphiphile delivered DNA more efficiently than PEI. This cationic bolaamphiphile may make a promising gene delivery vector for future gene therapy.

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.

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 organism's 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.

Branched disulfide-based polyamidoamines capable of mediating high gene transfection.

DNA condensation, endosomal escape of DNA/polymeric complexes, and unpacking of DNA are the key steps in the process of non-viral gene delivery. Amongst these steps, currently the unpacking of the DNA cargo from the DNA/polymeric nanocomplexes is the most challenging and arguably the most crucial if one wants to achieve high gene transfection with minimum cytotoxicity in the target cell. In this report we review current and past examples in the literature that demonstrate concerted efforts in designing and synthesizing various forms of cationic polymeric vectors having "built in" features. Such features can be certain types of chemical functional groups, such as amines and acids or other degradable bonds like esters, carbonates and disulfides, which allow for breakdown of polymeric vectors in certain cellular compartments. This may lead the DNA cargo to dissociate from the DNA/polymer complexes so as to maximize intracellular gene delivery. Furthermore, we provide further evidence that it is possible to achieve the goal of high gene transfection coupled with low cytotoxicity via rational design and formulation of branched polyamidoamines containing disulfide bonds. The DNA binding ability of these polymers and particle size as well as zeta potential of their DNA complexes were investigated. The cytotoxicity of pure polymer and polymer/DNA complexes at various polymer concentrations was studied in HEK293 human embryonic kidney, HepG2 human liver carcinoma, 4T1 mouse breast cancer and HeLa human cervical cancer cell lines. In vitro gene transfection efficiency induced by polymer/DNA complexes was explored in these cell lines by using luciferase and GFP reporter genes in comparison with PEI.

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.

Brush-Like Amphoteric Poly[isobutylene-alt-(maleic acid)-graft-oligoethyleneamine)]/DNA Complexes for Efficient Gene Transfection.

Synthetic gene delivery vectors, especially cationic polymers have attracted enormous attention in recent decades because of their ease of manufacture, targettability, and scaling up. However, certain issues such as high cytotoxicity and low transfection efficiency problems have hampered the advance of nonviral gene delivery. In this study, we designed and synthesized brush-like amphoteric poly[isobutylene-alt-(maleic acid)-graft-oligoethyleneamine] capable of mediating highly efficient gene transfection. The polymers are composed of multiple pendant oligoethyleneimine molecules with alternating carboxylic acid moiety grafted onto poly[isobutylene-alt-(maleic anhydride)]. The polymer formed from pentaethylenehexamine {i.e., poly[isobutylene-alt-(maleic acid)-graft-pentaethylenehexamine)]} was able to condense DNA efficiently into nanoparticles of size around 200 nm with positive zeta potential of about 28-30 mV despite its amphoteric nature. Luciferase expression level and percentage of GFP expressing cells induced by this polymer was higher than those mediated with polyethyleneimine (branched, $\overline M _{\rm w} $ 25 kDa) by at least one order of magnitude at their optimal N/P ratios on HEK293, HepG2, and 4T1 cells. In vitro cytotoxicity testing revealed that the polymer/DNA complexes were less cytotoxic than those of PEI, and the viability of the cells after being incubated with the polymer/DNA complexes at the optimal N/P ratios was higher than 85%. This polymer can be a promising gene delivery carrier for gene therapy.

Design and evaluation of Peptide amphiphiles with different hydrophobic blocks for simultaneous delivery of drugs and genes.

In this study, peptides with six different compositions were designed and synthesized to study the effect of the structure of the hydrophobic block in triblock oligopeptide amphiphiles ((A(m) X)(n) H(5) K(15) ) on self-assembly properties, drug loading capacity and gene expression efficiency. The peptides were synthesized using a standard Fmoc-solid phase peptide synthesis protocol, which are A(12) H(5) K(15) , A(11) FH(5) K(15) , (A(5) F)(2) H(5) K(15) , (A(3) F)(3) H(5) K(15) , (AF)(6) H(5) K(15) and (AL)(6) H(5) K(15) . A(12) H(5) K(15) , A(11) FH(5) K(15) , (A(5) F)(2) H(5) K(15) and (A(3) F)(3) H(5) K(15) , formed micelles at concentrations above 200 mg · L(-1) with large size or aggregation. However, (AF)(6) H(5) K(15) and (AL)(6) H(5) K(15) had CMC values of 42 and 49 mg · L(-1) respectively, and the resulting micelles were much smaller in size as compared to the other peptide designs (108 and 233 nm for (AF)(6) H(5) K(15) and (AL)(6) H(5) K(15) , respectively). In addition, both peptides were able to load paclitaxel (PTX) into nanoparticles although PTX-loaded (AF)(6) H(5) K(15) nanoparticles had a smaller size (278 versus 295 nm, respectively). Encapsulation efficiency of PTX with (AF)(6) H(5) K(15) nanoparticles was 74%. Gene transfection studies showed that luciferase expression level induced by (AF)(6) H(5) K(15) micelles in HepG2 cell line was much higher than that mediated by (AL)(6) H(5) K(15) micelles at their respective optimal N/P ratios (3.2 × 10(8) versus 3.9 × 10(7) RLU · mg proteins(-1) ). Therefore, simultaneous delivery of PTX and luciferase-encoding plasmid was conducted using (AF)(6) H(5) K(15) micelles against HepG2 cells, and the results demonstrated that the co-delivery of PTX at 0.01 mg · L(-1) further increased luciferase expression level from 3.2 × 10(8) to 8.0 × 10(8) RLU · mg proteins(-1) at the optimal N/P ratio (i.e., 18). In summary, it is important to optimize the hydrophobic block of the oligopeptide amphiphiles in order to achieve desired properties for co-delivery of drugs and genes.