ABSTRACT
Mycobacterial infectious diseases, including tuberculosis (TB), severely threaten global public health. Nonreplicating Mycobacterium tuberculosis (Mtb) is extremely difficult to eradicate using current TB drugs that primarily act on replicating cells. Novel TB drugs acting on unconventional targets are needed to combat TB efficiently. Although membrane-disrupting antimicrobial peptides and their synthetic mimics exhibit the potential to kill persisters, the lack of microbe selectivity, especially toward mycobacteria, has been a concern. Here, we report that the recently developed poly(guanylurea)-piperazine (PGU-P) shows fast and selective mycobactericidal effects. Using a nonpathogenic model organism, Mycobacterium smegmatis (Msm), we have found that the mycobactericidal effects of PGU-P are correlated to the disruption of the mycobacterial membrane potential and bioenergetics. Accordingly, PGU-P also potentiates bedaquiline, an oxidative phosphorylation-targeting TB drug disturbing mycobacterial bioenergetics. Importantly, PGU-P also exhibits a promising activity against pathogenic Mtb with a minimum inhibitory concentration of 37 µg/mL. Our results support that PGU-P is a novel class of antimycobacterial biomaterial, and the unique structural feature can contribute to developing novel antimycobacterial drugs.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis , Humans , Antitubercular Agents/pharmacology , Proton-Motive Force , Polymers/pharmacology , Tuberculosis/drug therapy , Microbial Sensitivity TestsABSTRACT
We present the importance of functional group isomerism on intracellular protein delivery using polymers containing different isomeric side chains. While the physical properties of polymer/protein complexes are relatively similar, different planarity of the isomers greatly influences the cellular entry efficiency.
Subject(s)
Polymers , Isomerism , Polymers/chemistryABSTRACT
Despite the high potential of controlling cellular processes and treating various diseases by intracellularly delivered proteins, current delivery systems exhibit poor efficiency due to poor serum stability, cellular entry, and cytosolic availability of proteins. Here, we report a novel functional group, phenyl carbamoylated guanidine (Ph-CG), that greatly enhances the delivery efficiency to various types of cells. Owing to the substantially lowered pKa , the hydrophobic Ph-CG offers optimized inter-macromolecular interactions via enhanced hydrogen-bonding and hydrophobic interactions. The coplanarity of Ph-CG also leads to the better intracellular entry of protein complexes. Intracellularly delivered apoptosis-inducing enzymes and antibodies significantly induce cell viability inhibitions in a serum-containing medium. The newly developed Ph-CG can be introduced to various existing carriers, leading to the realization of future therapeutic protein delivery.
Subject(s)
Polymers , Proteins , Cytosol/metabolism , Drug Delivery Systems , Guanidine/chemistry , Hydrophobic and Hydrophilic Interactions , Polymers/chemistry , Proteins/chemistryABSTRACT
We demonstrate a successful target gene knockdown in ex vivo normal human bronchial epithelium (NHBE) cells covered with mucus layers using the guanylurea functionalization technique modulating the chemical environment at the positive charge of a gene carrier.
Subject(s)
Drug Carriers/chemistry , Guanidines/chemistry , Polymers/chemistry , RNA, Small Interfering/administration & dosage , Respiratory Mucosa/metabolism , Urea/chemistry , Cells, Cultured , Drug Carriers/chemical synthesis , Epithelial Cells/metabolism , Gene Knockdown Techniques , Guanidines/chemical synthesis , Histone Deacetylases/genetics , Humans , Molecular Structure , Polymers/chemical synthesis , RNA Interference , RNA, Small Interfering/genetics , Urea/chemical synthesisABSTRACT
Fluorescent macromolecules were developed for intracellular labeling in live cells. Coupling rigid rod phenyleneethynylene trimers with flexible amphiphilic diamines via the imine-bond formation chemistry yielded rigid-flexible [2+2] macromolecules showing nucleic acid selectivity and nontoxicity in live cells.
Subject(s)
Alkynes/chemistry , Ethers/chemistry , Macrocyclic Compounds/chemistry , Nucleic Acids/chemistry , Polymers/chemistry , HeLa Cells , Humans , Proton Magnetic Resonance SpectroscopyABSTRACT
In-depth understanding of the biophysicochemical interactions at the nano-bio interface is important for basic cell biology and applications in nanomedicine and nanobiosensors. Here, the extracellular surface potential and topography changes of live cell membranes interacting with polymeric nanomaterials using a scanning ion conductance microscopy-based potential imaging technique are investigated. Two structurally similar amphiphilic conjugated polymer nanoparticles (CPNs) containing different functional groups (i.e., primary amine versus guanidine) are used to study incubation time and functional group-dependent extracellular surface potential and topographic changes. Transmembrane pores, which induce significant changes in potential, only appear transiently in the live cell membranes during the initial interactions. The cells are able to self-repair the damaged membrane and become resilient to prolonged CPN exposure. This study provides an important observation on how the cells interact with and respond to extracellular polymeric nanomaterials at the early stage. This study also demonstrates that extracellular surface potential imaging can provide a new insight to help understand the complicated interactions at the nano-bio interface and the following cellular responses.
Subject(s)
Cell Membrane/physiology , Ion Transport/physiology , Membrane Potentials/physiology , Microscopy/methods , Nanoparticles/metabolism , Cell Line, Tumor , HeLa Cells , Humans , Polymers/chemistryABSTRACT
Bacterial infections are serious health threats. Emerging drug resistance in bacteria further poses serious challenges to the treatment options involving traditional antibiotics. Antimicrobial polymers disrupt the physical cell membrane integrity of bacteria to address the drug resistance problems. Here, we introduce a conceptually new class of antimicrobial polymers containing positively charged guanylurea backbones for enhanced antimicrobial effects. The initial structure-activity relationship studies demonstrate that poly(guanylurea piperazine)s (PGU-Ps) exhibit excellent antimicrobial activity against different types of bacteria with high selectivity. The new design concept of using a positively charged guanylurea backbone will contribute to the development of future biocompatible, specific, and selective antimicrobial polymers.
Subject(s)
Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Guanidines/chemical synthesis , Guanidines/pharmacology , Polymers/chemical synthesis , Polymers/pharmacology , Urea/analogs & derivatives , Anti-Bacterial Agents/chemistry , Bacterial Infections/drug therapy , Guanidines/chemistry , Humans , Microbial Sensitivity Tests , Polymers/chemistry , Structure-Activity Relationship , Urea/chemical synthesis , Urea/chemistry , Urea/pharmacologyABSTRACT
Biodegradable conjugated polymer nanoparticles (CPNs) were prepared for high mitochondrial targeting in live cancer cells. The degradable CPNs are nontoxic and specifically localized to the mitochondria of live tumor cells through macropinocytosis followed by intracellular degradation and trafficking.
Subject(s)
Mitochondria/metabolism , Nanoparticles/chemistry , Polymers/chemistry , Microscopy, Confocal , PinocytosisABSTRACT
Positively charged conjugated polymer nanoparticles (CPNs) are emerging biomaterials exhibiting high levels of cellular entry. High rate of cellular entry efficiency is believed that the amphiphilic CPNs interact efficiently with the negatively charged hydrophobic cellular membranes. For the first time, the cell surface morphological changes of human cervical cancer cells treated with CPNs using a scanning probe microscopy technique, scanning ion conductance microscopy (SICM) are imaged. After 1 h of CPN incubation, distinct changes are observed in cell surface morphology such as interconnected protrusions and pits with sub-micrometer sizes, which are not observed from cells treated with positively charged polyethyleneimine (PEI) under the same treatment conditions. The change on cell surface morphology is quantified by surface roughness ratio, which is increased as CPN concentration increases, while the ratio first increases and then decreases as the incubation time increases. These results suggest that cells respond actively toward CPN with both positive charges on the side chain and the hydrophobicity from rigid aromatic backbone, which leads to subsequent endocytosis. In conclusion, it is demonstrated that SICM is a suitable imaging technique to reveal the dynamic alternations on the cell surface morphology at the early stage of nanoparticles endocytosis with high resolution.
Subject(s)
Endocytosis/physiology , Nanoparticles/metabolism , HeLa Cells , Humans , Hydrophobic and Hydrophilic Interactions , Microscopy/methods , Nanoparticles/chemistry , Nanoparticles/ultrastructure , Polyethyleneimine/chemistry , Static Electricity , Surface PropertiesABSTRACT
Four different conjugated polymer nanoparticles (CPNs) were used to differentiate structurally similar glycosaminoglycans (GAGs) in a urine simulant. Unique emission response patterns of CPNs were analyzed by linear discriminant analysis (LDA), confirming that structurally diverse CPNs are sensitive and effective at differentiating GAGs in a complex biological medium.
Subject(s)
Biomimetic Materials/chemistry , Glycosaminoglycans/chemistry , Nanoparticles/chemistry , Polymers/chemistry , Urine , Absorption, PhysicochemicalABSTRACT
Core-shell conjugated polymer nanoparticles (CPNs) were fabricated by complexing a semi-flexible, primary amine-containing conjugated polymer (CP) with hyaluronic acid (HA). Flexibility introduced in the rigid rod conjugated backbone allows backbone reorganization to increase π-π interaction under ionic complexation, resulting in core-shell nanoparticles with a hydrophobic CP core wrapped with a HA shell. The core-shell nanoparticles exhibited no cellular toxicity and high cancer cell specificity with minimal binding to normal cells.
ABSTRACT
The subcellular localization and toxicity of conjugated polymer nanoparticles (CPNs) are dependent on the chemical structure of the side chains and backbone. Primary amine-containing CPNs exhibit high Golgi localization with no toxicity. Incorporation of short ethylene oxide and tertiary amine side chains contributes to decreased Golgi localization and increased toxicity, respectively.
Subject(s)
Nanoparticles/chemistry , Polymers/chemistry , Amines/chemistry , Cell Survival/drug effects , Ethylene Oxide/chemistry , Golgi Apparatus/metabolism , HeLa Cells , Humans , Nanoparticles/toxicityABSTRACT
Understanding the cellular entry pathways of synthetic biomaterials is highly important to improve overall labeling and delivery efficiency. Herein, cellular entry mechanisms of conjugated polymer nanoparticles (CPNs) are presented. CPNs are intrinsic fluorescent materials used for various biological applications. While CPNs cause no toxicity, decreased CPN uptake is observed from cancer cells pretreated with genistein, which is an inhibitor of caveolae-mediated endocytosis (CvME). CvME is further confirmed by high co-localization with caveolin-1 proteins found in the caveolae and caveosomes. Excellent photophysical properties, non-toxicity, and non-destructive delivery pathways support that CPNs are promising multifunctional carriers minimizing degradation of contents during delivery.
Subject(s)
Drug Delivery Systems , Endocytosis , Nanoparticles/chemistry , Polymers/chemistry , Biocompatible Materials/chemistry , Caveolae/chemistry , Caveolin 1/chemistry , Caveolin 1/metabolism , Cell Membrane Permeability/drug effects , Cells, Cultured , Fluorescent Dyes/chemistry , Humans , Nanoparticles/administration & dosage , Polymers/administration & dosageABSTRACT
We developed a new synthetic approach to high molecular weight poly(p-phenylenebutadiynylene) s (PPBs) by increasing backbone flexibility. The introduction of a small amount of flexible units along the backbone improved both the physical and photophysical properties of the polymers. These materials were successfully fabricated into conjugated polymer nanoparticles (CPNs) and used for fluorescent live cell imaging for the first time.
ABSTRACT
Fluorescence and phosphorescence lifetime imaging are powerful techniques for studying intracellular protein interactions and for diagnosing tissue pathophysiology. While lifetime-resolved microscopy has long been in the repertoire of the biophotonics community, current implementations fall short in terms of simultaneously providing 3D resolution, high throughput, and good tissue penetration. This report describes a new highly efficient lifetime-resolved imaging method that combines temporal focusing wide-field multiphoton excitation and simultaneous acquisition of lifetime information in frequency domain using a nanosecond gated imager from a 3D-resolved plane. This approach is scalable allowing fast volumetric imaging limited only by the available laser peak power. The accuracy and performance of the proposed method is demonstrated in several imaging studies important for understanding peripheral nerve regeneration processes. Most importantly, the parallelism of this approach may enhance the imaging speed of long lifetime processes such as phosphorescence by several orders of magnitude.
Subject(s)
Cytoplasm/ultrastructure , Fluorescence , Imaging, Three-Dimensional , Microscopy, Fluorescence/methods , Photons , HumansABSTRACT
Understanding and controlling aggregation structures of conjugated polymers (CPs) in aqueous solutions is critical to improving the physical and photophysical properties of CPs for biological applications. Here, we present spectroscopic evidence, including nuclear magnetic resonance (NMR) spectroscopic results, that different organic acid treatment induces different aggregation structures and photophysical properties of CPs in water. Conjugated polymer nanoparticles (CPNs) were fabricated by treating a non-aqueous soluble, primary amine-containing poly(phenylene ethynylene) (PPE-NH(2)) with organic acids followed by dialysis. CPNs formed by acetic acid (AA) treatment (CPN-AAs) exhibit characteristics of loose aggregation with minimal π-π stacking, while CPNs formed by tartaric acid (TA) treatment (CPN-TAs) exhibit a high degree of π-π stacking among PPE-NH(2) chains. The controlled aggregation for a specific application was demonstrated by comparing the fluorescence quenching abilities of the CPN-AAs and the CPN-TAs. A doubled Stern-Volmer constant was obtained from the densely packed CPN-TAs compared to that of the loosely aggregated CPN-AAs.
ABSTRACT
Loosely aggregated conjugated polymer nanoparticles (CPNs) were used as nontoxic and efficient small interfering RNA (siRNA) delivery vehicles with delivery visualization. A significant down regulation (94%) of a target gene was achieved by transfection of HeLa cells with the CPNs/siRNA complexes, supporting CPN as a promising siRNA delivery carrier.
Subject(s)
Nanoparticles/chemistry , Polymers/chemistry , RNA, Small Interfering/metabolism , Actins/antagonists & inhibitors , Actins/genetics , Actins/metabolism , HeLa Cells , Humans , RNA InterferenceABSTRACT
BACKGROUND: Post transcriptional gene silencing (PTGS) is a mechanism harnessed by plant biologists to knock down gene expression. siRNAs contribute to PTGS that are synthesized from mRNAs or viral RNAs and function to guide cellular endoribonucleases to target mRNAs for degradation. Plant biologists have employed electroporation to deliver artificial siRNAs to plant protoplasts to study gene expression mechanisms at the single cell level. One drawback of electroporation is the extensive loss of viable protoplasts that occurs as a result of the transfection technology. RESULTS: We employed fluorescent conjugated polymer nanoparticles (CPNs) to deliver siRNAs and knockdown a target gene in plant protoplasts. CPNs are non toxic to protoplasts, having little impact on viability over a 72 h period. Microscopy and flow cytometry reveal that CPNs can penetrate protoplasts within 2 h of delivery. Cellular uptake of CPNs/siRNA complexes were easily monitored using epifluorescence microscopy. We also demonstrate that CPNs can deliver siRNAs targeting specific genes in the cellulose biosynthesis pathway (NtCesA-1a and NtCesA-1b). CONCLUSIONS: While prior work showed that NtCesA-1 is a factor involved in cell wall synthesis in whole plants, we demonstrate that the same gene plays an essential role in cell wall regeneration in isolated protoplasts. Cell wall biosynthesis is central to cell elongation, plant growth and development. The experiments presented here shows that NtCesA is also a factor in cell viability. We show that CPNs are valuable vehicles for delivering siRNAs to plant protoplasts to study vital cellular pathways at the single cell level.
Subject(s)
Nanoparticles/chemistry , Polymers/chemistry , Protoplasts/metabolism , RNA, Small Interfering/genetics , Cell Wall/metabolism , Cells, Cultured , Flow Cytometry , Fluorescent Dyes/chemistry , Gene Expression Regulation, Plant , Microscopy, Confocal , Microscopy, Fluorescence , Plant Proteins/genetics , Plant Proteins/metabolism , Protoplasts/cytology , Pyridinium Compounds/chemistry , Quaternary Ammonium Compounds/chemistry , RNA Interference , RNA, Small Interfering/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Nicotiana/cytology , Nicotiana/genetics , Nicotiana/metabolism , Transfection/methodsABSTRACT
Decarboxylative coupling of sp-sp2 carbons is possible by palladium catalyst. Employing propiolic acid (1) as a difunctional alkyne, and using the consecutive reactions of the Sonogashira reaction and the decarboxylative coupling, unsymmetrically substituted diaryl alkynes were obtained in moderate to good yield.
