Novel Core-Shell Polyamine Phosphate Nanoparticles Self-Assembled from PEGylated Poly(allylamine hydrochloride) with Low Toxicity and Increased In Vivo Circulation Time.

An approach for reducing toxicity and enhancing therapeutic potential of supramolecular polyamine phosphate nanoparticles (PANs) through PEGylation of polyamines before their assembly into nanoparticles is presented here. It is shown that the number of polyethylene glycol (PEG) chains for polyamine largely influence physico-chemical properties of PANs and their biological endpoints. Poly(allylamine hydrochloride) (PAH) are functionalized through carbodiimide chemistry with three ratios of PEG molecules per PAH chain: 0.1, 1, and 10. PEGylated PAH is then assembled into PANs by exposing the polymer to phosphate buffer solution. PANs decrease size and surface charge with increasing PEG ratios as evidenced by dynamic light scattering and zeta potential measurements, with the ten PEG/PAH ratio PANs having practically zero charge. Small angle X-ray scattering (SAXS) proves that PEG chains form a shell around a polyamine core, which is responsible for the screening of positive charges. MTT experiments show that the screening of amine groups decreases nanoparticle toxicity, with the lowest toxicity for the 10 PEG/PAH ratio. Fluorescence correlation spectroscopy (FCS) proves less interaction with proteins for PEGylated PANs. Positron emission tomography (PET) imaging of 18 F labelled PANs shows longer circulation time in healthy mice for PEGylated PANs than non-PEGylated ones.

Plasma Corona Protects Human Immune Cells from Structurally Nanoengineered Antimicrobial Peptide Polymers.

Safe and effective antimicrobials are needed to combat emerging antibiotic-resistant bacteria. Structurally nanoengineered antimicrobial peptide polymers (termed SNAPPs) interact with bacterial cell membranes to potently kill bacteria but may also interact at some level with human cell membranes. We studied the association of four different SNAPPs with six different white blood cells within fresh whole human blood by flow cytometry. In whole human blood, SNAPPs had detectable association with phagocytic cells and B cells, but not natural killer and T cells. However, without plasma proteins and therefore no protein corona on the SNAPPs, a greater marked association of SNAPPs with all white blood cell types was detected, resulting in cytotoxicity against most blood cell components. Thus, the formation of a protein corona around the SNAPPs reduced the association and prevented human blood cell cytotoxicity of the SNAPPs. Understanding the bio-nano interactions of these SNAPPs will be crucial to ensuring that the design of next-generation SNAPPs and other promising antimicrobial nanomaterials continues to display high efficacy toward antibiotic-resistant bacteria while maintaining a low toxicity to primary human cells.

Co-delivery of Sorafenib and CRISPR/Cas9 Based on Targeted Core-Shell Hollow Mesoporous Organosilica Nanoparticles for Synergistic HCC Therapy.

The rapid development of CRISPR/Cas9 systems has opened up tantalizing prospects to sensitize cancers to chemotherapy using efficient targeted genome editing, but safety concerns and possible off-target effects of viral vectors remain a major obstacle for clinical application. Thus, the construction of novel nonviral tumor-targeting nanodelivery systems has great potential for the safe application of CRISPR/Cas9 systems for gene-chemo-combination therapy. Here, we report a polyamidoamine-aptamer-coated hollow mesoporous silica nanoparticle for the co-delivery of sorafenib and CRISPR/Cas9. The core-shell nanoparticles had good stability, enabled ultrahigh drug loading, targeted delivery, and controlled-release of the gene-drug combination. The nanocomplex showed >60% EGFR-editing efficiency without off-target effects in all nine similar sites, regulating the EGFR-PI3K-Akt pathway to inhibit angiogenesis, and exhibited a synergistic effect on cell proliferation. Importantly, the co-delivery nanosystem achieved efficient EGFR gene therapy and caused 85% tumor inhibition in a mouse model. Furthermore, the nanocomplex showed high accumulation at the tumor site in vivo and exhibited good safety with no damage to major organs. Due to these properties, the nanocomplex provides a versatile delivery approach for efficient co-loading of gene-drug combinations, allowing for precise gene editing and synergistic inhibition of tumor growth without apparent side effects on normal tissues.

New Polyaminoisoprenyl Antibiotics Enhancers against Two Multidrug-Resistant Gram-Negative Bacteria from Enterobacter and Salmonella Species.

A series consisting of new polyaminoisoprenyl derivatives were prepared in moderate to good chemical yields varying from 32 to 64% according to two synthetic pathways: (1) using a titanium-reductive amination reaction affording a 50/50 mixture of cis and trans isomers and (2) a direct nucleophilic substitution leading to a stereoselective synthesis of the compounds of interest. These compounds were then successfully evaluated for their in vitro antibiotic enhancer properties against resistant Gram-negative bacteria of four antibiotics belonging to four different families. The mechanism of action against Enterobacter aerogenes of one of the most efficient of these chemosensitizing agents was precisely evaluated by using fluorescent dyes to measure outer-membrane permeability and to determine membrane depolarization. The weak cytotoxicity encountered led us to perform an in vivo experiment dealing with the treatment of mice infected with Salmonella typhimurium and affording preliminary promising results in terms of tolerance and efficiency of the polyaminoisoprenyl derivative 5r/doxycycline combination.

Autophagy induction by exogenous polyamines is an artifact of bovine serum amine oxidase activity in culture serum.

Polyamines are small polycationic alkylamines involved in many fundamental cellular processes, including proliferation, nucleic acid synthesis, apoptosis, and protection from oxidative damage. It has been proposed that in addition to these functions, elevated levels of polyamines promote longevity in various biological systems, including yeast, Drosophila, and murine models. A series of in vitro mechanistic studies by multiple investigators has led to the conclusion that addition of exogenous spermidine promotes longevity through autophagy induction; however, these experiments were confounded by the use of mammalian cell culture systems supplemented with fetal bovine serum. Using cell viability assays, LC3B immunoblots, and live-cell fluorescence microscopy, we report here that in the presence of ruminant serum, exogenously added polyamines are quickly oxidized by the copper-containing bovine serum amine oxidase. This polyamine oxidation resulted in the production of harmful byproducts including hydrogen peroxide, ammonia, and reactive aldehydes. Our data demonstrate that it is critically important to prevent confounding bovine serum amine oxidase-induced cytotoxicity in mechanistic studies of the roles of polyamines in autophagy.

ATP13A2 deficiency disrupts lysosomal polyamine export.

ATP13A2 (PARK9) is a late endolysosomal transporter that is genetically implicated in a spectrum of neurodegenerative disorders, including Kufor-Rakeb syndrome-a parkinsonism with dementia1-and early-onset Parkinson's disease2. ATP13A2 offers protection against genetic and environmental risk factors of Parkinson's disease, whereas loss of ATP13A2 compromises lysosomes3. However, the transport function of ATP13A2 in lysosomes remains unclear. Here we establish ATP13A2 as a lysosomal polyamine exporter that shows the highest affinity for spermine among the polyamines examined. Polyamines stimulate the activity of purified ATP13A2, whereas ATP13A2 mutants that are implicated in disease are functionally impaired to a degree that correlates with the disease phenotype. ATP13A2 promotes the cellular uptake of polyamines by endocytosis and transports them into the cytosol, highlighting a role for endolysosomes in the uptake of polyamines into cells. At high concentrations polyamines induce cell toxicity, which is exacerbated by ATP13A2 loss due to lysosomal dysfunction, lysosomal rupture and cathepsin B activation. This phenotype is recapitulated in neurons and nematodes with impaired expression of ATP13A2 or its orthologues. We present defective lysosomal polyamine export as a mechanism for lysosome-dependent cell death that may be implicated in neurodegeneration, and shed light on the molecular identity of the mammalian polyamine transport system.

Naked Poly(amidoamine) Dendrimer Nanoparticles Exhibit Intrinsic Embryotoxicity During the Early Stages of Normal Development.

To investigate the impact of poly(amidoamine) dendrimers (PAMAMs) in the embryo, we explored the outcome of different generations (G4 and G6) on the early stages of embryogenesis using the chicken embryo as a model. We also monitored their effect on angiogenesis in the chorioallantoic membrane (CAM). Our data revealed that cationic PAMAMs provoke substantial embryotoxicity, as they significantly induce death (up to 50%, p < 0 05) and inhibit angiogenesis of the CAM (up to 30%, p < 0 05) in a generation-dependent manner in comparison to controls and other types of PAMAMs (anionic and neutral). Moreover, cationic PAMAMs alter the expression of genes related to cell survival, cell cycle, proliferation, transcription factor, apoptosis, and angiogenesis, as shown by RT-PCR analysis. Our data suggest that PAMAM dendrimers exhibit intrinsic toxicity in embryos at the early stages and inhibits angiogenesis of the CAM. Thus, future studies are necessary to illustrate the exact mechanism of PAMAM dendrimers in embryotoxicity.

A Simple LC-MS Method for the Quantitation of Alkaloids in Endophyte-Infected Perennial Ryegrass.

The rapid identification and quantitation of alkaloids produced by Epichloë endophyte-infected pasture grass is important for the agricultural industry. Beneficial alkaloids, such as peramine, provide the grass with enhanced insect protection. Conversely, ergovaline and lolitrem B can negatively impact livestock. Currently, a single validated method to measure these combined alkaloids in planta does not exist. Here, a simple two-step extraction method was developed for Epichloë-infected perennial ryegrass (Lolium perenne L.). Peramine, ergovaline and lolitrem B were quantified using liquid chromatography-mass spectrometry (LC-MS). Alkaloid linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, precision, selectivity, recovery, matrix effect and robustness were all established. The validated method was applied to eight different ryegrass-endophyte symbiota. Robustness was established by comparing quantitation results across two additional instruments; a triple quadruple mass spectrometer (QQQ MS) and by fluorescence detection (FLD). Quantitation results were similar across all three instruments, indicating good reproducibility. LOQ values ranged from 0.8 ng/mL to 6 ng/mL, approximately one hundred times lower than those established by previous work using FLD (for ergovaline and lolitrem B), and LC-MS (for peramine). This work provides the first highly sensitive quantitative LC-MS method for the accurate and reproducible quantitation of important endophyte-derived alkaloids.

Microbial Metabolites Determine Host Health and the Status of Some Diseases.

The gastrointestinal (GI) tract is a highly complex organ composed of the intestinal epithelium layer, intestinal microbiota, and local immune system. Intestinal microbiota residing in the GI tract engages in a mutualistic relationship with the host. Different sections of the GI tract contain distinct proportions of the intestinal microbiota, resulting in the presence of unique bacterial products in each GI section. The intestinal microbiota converts ingested nutrients into metabolites that target either the intestinal microbiota population or host cells. Metabolites act as messengers of information between the intestinal microbiota and host cells. The intestinal microbiota composition and resulting metabolites thus impact host development, health, and pathogenesis. Many recent studies have focused on modulation of the gut microbiota and their metabolites to improve host health and prevent or treat diseases. In this review, we focus on the production of microbial metabolites, their biological impact on the intestinal microbiota composition and host cells, and the effect of microbial metabolites that contribute to improvements in inflammatory bowel diseases and metabolic diseases. Understanding the role of microbial metabolites in protection against disease might offer an intriguing approach to regulate disease.

Nuclear localization signal peptide enhances transfection efficiency and decreases cytotoxicity of poly(agmatine/N,N'-cystamine-bis-acrylamide)/pDNA complexes.

At present, nonviral gene vectors develop rapidly, especially cationic polymers. A series of bioreducible poly(amide amine) (PAA) polymers containing guanidino groups have been synthesized by our research team. These novel polymer vectors demonstrated significantly higher transfection efficiency and lower cytotoxicity than polyethylenimine (PEI)-25kDa. However, compared with viral gene vectors, relatively low transfection efficiency, and high cytotoxicity are still critical problems confronting these polymers. In this study, poly(agmatine/N,N'-cystamine-bis-acrylamide) p(AGM-CBA) was selected as a model polymer, nuclear localization signal (NLS) peptide PV7 (PKKKRKV) with good biocompatibility and nuclear localization effect was introduced to investigate its impact on transfection efficiency and cytotoxicity. NLS peptide-mediated in vitro transfection was performed in NIH 3T3 cells by directly incorporating NLS peptide with the complexes of p(AGM-CBA)/pDNA. Meanwhile, the transfection efficiency and cytotoxicity of these complexes were evaluated. The results showed that the transfection efficiency could be increased by 5.7 times under the appropriate proportion, and the cytotoxicity brought by the polymer vector could be significantly reduced.

Hyperbranched polymeric nanomaterials impair the freshwater crustacean Daphnia magna.

Hyperbranched polymers are nanomaterials belonging to the class of dendritic architectures with increasing applications in many diverse fields. We studied the toxicity of two hyperbranched polymers to the freshwater crustacean Daphnia magna. A hyperbranched hydroxyl-terminated polyester and a commercial hyperbranched polyamidoamine, Helux-3316 were tested for the acute immobilization of daphnids, the overproduction of reactive oxygen species and the activity of the antioxidant enzymes catalase and glutathione S-transferase. The effect for D. magna immobilization was higher for the hyperbranched polyamidoamine Helux-3316, which was attributed to the presence of primary amino groups on its surface. Following exposure to both hyperbranched polymers, a clear overproduction of reactive oxygen species took place accompanied by concentration-dependent enzymatic antioxidant response. Our results showed that the overproduction of reactive oxygen species activated antioxidant defence mechanisms and was responsible for the immobilization of daphnids exposed to both hyperbranched polymers. We showed evidence of the uptake of fluorescently labelled Helux-3316 that accumulated into the gastrointestinal tract of D. magna, and its removal via excretion within fecal pellets. This is the first work reporting the internalization of hyperbranched polymers in aquatic organisms.

Lactate Dehydrogenase Assay for Assessment of Polycation Cytotoxicity.

Cellular toxicity and/or cell death entail complex mechanisms that require multifaceted characterization. A detailed mechanistic assessment of cytotoxicity is essential for design and construction of more effective polycations for nucleic acid delivery. A single toxicity assay cannot stand alone in determining the type and extent of damage or cell death mechanism. In this chapter we describe a lactate dehydrogenase (LDH) assay for high-throughput screening that can be used as a starting point for further detailed cytotoxicity determination. LDH release is considered an early event in necrosis but a late event in apoptosis. An accurate temporal assessment of the toxic responses is crucial as late apoptosis may convert into necrosis as well as in situations where cell death is initiated without any visible cell morphological changes or responses in assays measuring late events, resulting in early ongoing toxicity being overlooked.

Determination of Polycation-Mediated Perturbation of Mitochondrial Respiration in Intact Cells by High-Resolution Respirometry (Oxygraph-2k, OROBOROS).

A better understanding of the molecular basis of polycation-mediated impairment of mitochondrial bioenergetics might improve the design and synthesis of more efficient and safer polymeric transfectants. Here we utilize the phosphorylation control protocol for studying the effect of polycations on mitochondrial respiration in intact mammalian cells using Oxygraph-2k (OROBOROS). The protocol offers an opportunity to comprehensively monitor mitochondrial respiration through consecutive additions of various cell membrane permeable compounds that alter mitochondrial respiration, thus providing useful information on different states of mitochondrial respiration. Furthermore, we demonstrate how to analyze the data obtained with the phosphorylation control protocol and how to calculate the respiratory flux ratios, which can be used as indicators of respiratory functionality and mitochondrial health.

Combined Fluorimetric Caspase-3/7 Assay and Bradford Protein Determination for Assessment of Polycation-Mediated Cytotoxicity.

Cationic polyplexes and lipoplexes are widely used as artificial systems for nucleic acid delivery into the cells, but they can also induce cell death. Mechanistic understanding of cell toxicity and biological side effects of these cationic entities is essential for optimization strategies and design of safe and efficient nucleic acid delivery systems. Numerous methods are presently available to detect and delineate cytotoxicity and cell death-mediated signals in cell cultures. Activation of caspases is part of the classical apoptosis program and increased caspase activity is therefore a well-established hallmark of programmed cell death. Additional methods to monitor cell-death related signals must, however, also be carried out to fully define the type of cell toxicity in play. These may include methods that detect plasma membrane damage, loss of mitochondrial membrane potential, phosphatidylserine exposure, and cell morphological changes (e.g., membrane blebbing, nuclear changes, cytoplasmic swelling, cell rounding). Here we describe a 96-well format protocol for detection of caspase-3/7 activity in cell lysates, based on a fluorescent caspase-3 assay, combined with a method to simultaneously determine relative protein contents in the individual wells.

New polyamine drugs as more effective antichagas agents than benznidazole in both the acute and chronic phases.

Despite the continuous research effort that has been made in recent years to find ways to treat the potentially life threatening Chagas disease (CD), this remains the third most important infectious disease in Latin America. CD is an important public health problem affecting 6-7 million people. Since the need to search for new drugs for the treatment of DC persists, in this article we present a panel of new polyamines based on the tripodal structure of tris(2-aminomethyl)amine (tren) that can be prepared at low cost with high yields. Moreover, these polyamines present the characteristic of being water-soluble and resistant to the acidic pH values of stomach, which would allow their potential oral administration. In vitro and in vivo assays permitted to identify the compound with the tren moiety functionalized with one fluorene unit (7) as a potential antichagas agent. Compound 7 has broader spectrum of action, improved efficacy in acute and chronic phases of the disease and lower toxicity than the reference drug benznidazole. Finally, the action mechanisms studied at metabolic and mitochondrial levels shows that the trypanocidal activity of compound 7 could be related to its effect at the glycosomal level. Therefore, this work allowed us to select compound 7 as a promising candidate to perform preclinical evaluation studies.

A Study of the Cytotoxic Effect of Microcapsules and Their Constituent Polymers on Macrophages and Tumor Cells.

We studied the effect of different concentrations of polyelectrolytes poly(allylamine hydrochloride) (PAH) and polystyrene sulfonate (PSS) as well as the effects of microcapsules coated with these polymers on survival of Ehrlich ascites carcinoma cells and mouse peritoneal macrophages and on ROS production by phagocytes. PAH reduced viability of Ehrlich ascites carcinoma in a concentration-dependent manner (LD50=12-15 µg/ml). This effect was presumably determined by its ability to bind phosphates, thereby depleting the culture medium. At the same time, PAH did not affect the viability of macrophages. PSS produced no cytotoxic effect on the examined cells. Polyelectrolyte capsules with the shell architectonics (PAH/PSS)3 and (PAH/PSS)3PAH in the examined concentration range had no effect on the viability of macrophages and tumor cells. PAH microcapsules with positively charged surface much more rapidly and more intensively activated macrophages. The chemiluminescence response directly depended on the amount of capsules in the solution.

Robust and Biocompatible Functionalization of ZnS Nanoparticles by Catechol-Bearing Poly(2-methyl-2-oxazoline)s.

Zinc sulfide (ZnS) nanoparticles (NPs) are particularly interesting materials for their electronic and luminescent properties. Unfortunately, their robust and stable functionalization and stabilization, especially in aqueous media, has represented a challenging and not yet completely accomplished task. In this work, we report the synthesis of colloidally stable, photoluminescent and biocompatible core-polymer shell ZnS and ZnS:Tb NPs by employing a water-in-oil miniemulsion (ME) process combined with surface functionalization via catechol-bearing poly-2-methyl-2-oxazoline (PMOXA) of various molar masses. The strong binding of catechol anchors to the metal cations of the ZnS surface, coupled with the high stability of PMOXA against chemical degradation, enable the formation of suspensions presenting excellent colloidal stability. This feature, combined with the assessed photoluminescence and biocompatibility, make these hybrid NPs suitable for optical bioimaging.

Oxidative degradation of polyamines by serum supplement causes cytotoxicity on cultured cells.

Serum is a common supplement for cell culture due to it containing the essential active components for the growth and maintenance of cells. However, the knowledges of the active components in serum are incomplete. Apart from the direct influence of serum components on cultured cells, the reaction of serum components with tested drugs cannot be ignored, which usually results in the false conclusion on the activity of the tested drugs. Here we report the toxicity effect of polyamines (spermidine and spermine) on cultured cells, especially on drug-resistant cancer cell lines, which resulted from the oxidative degradation of polyamines by amine oxidases in serum supplement. Upon adding spermidine or spermine, high concentration of H2O2, an enzyme oxidation product of polyamines, was generated in culture media containing ruminant serum, such as fetal bovine serum (FBS), calf serum, bovine serum, goat serum or horse serum, but not in the media containing human serum. Drug-resistant cancer cell lines showed much higher sensitivity to the oxidation products of polyamines (H2O2 and acrolein) than their wild cell lines, which was due to their low antioxidative capacity.

2-Aminoimidazole facilitates efficient gene delivery in a low molecular weight poly(amidoamine) dendrimer.

Functional groups have shown great potential in gene delivery. However, a number of the reported functional groups can only overcome one certain physiological barrier, resulting in limited transfection efficiencies. Based on the structure-activity relationships of both imidazolyl and guanidyl, we designed a novel multifunctional group, 2-aminoimidazole (AM), for gene delivery. On modifying with the AM group, the transfection efficiency of low molecular weight poly(amidoamine) (G2) was 200 times greater than the parent dendrimer in vitro. In contrast, the transfection efficiency of G2 showed a decreasing trend when it was grafted with imidazole. Assays revealed that the AM group played multiple roles in gene delivery, including condensing DNA into monodisperse nanoparticles of 80-90 nm in diameter, achieving nearly ten times higher cellular-uptake efficacy, and enhancing the abilities of endosome/lysosome escape and nuclear localization. What's more, AM showed low toxicity. These results demonstrate that the AM group could be a promising tool in non-viral gene delivery.

Comparison of Gene Transfection and Cytotoxicity Mechanisms of Linear Poly(amidoamine) and Branched Poly(ethyleneimine) Polyplexes.

PURPOSE: This study aimed to further explore the mechanisms behind the ability of certain linear polyamidoamines (PAAs) to transfect cells with minimal cytotoxicity. METHODS: The transfection efficiency of DNA complexed with a PAA of a molecular weight over 10 kDa or 25 kDa branched polyethyleneimine (BPEI) was compared in A549 cells using a luciferase reporter gene assay. The impact of endo/lysosomal escape on transgene expression was investigated by transfecting cells in presence of bafilomycin A1 or chloroquine. Cytotoxicity caused by the vectors was evaluated by measuring cell metabolic activity, lactate dehydrogenase release, formation of reactive oxygen species and changes in mitochondrial membrane potential. RESULTS: The luciferase activity was ~3-fold lower after transfection with PAA polyplexes than with BPEI complexes at the optimal polymer to nucleotide ratio (RU:Nt). However, in contrast to BPEI vectors, PAA polyplexes caused negligible cytotoxic effects. The transfection efficiency of PAA polyplexes was significantly reduced in presence of bafilomycin A1 while chloroquine enhanced or decreased transgene expression depending on the RU:Nt. CONCLUSIONS: PAA polyplexes displayed a pH-dependent endo/lysosomal escape which was not associated with cytotoxic events, unlike observed with BPEI polyplexes. This is likely due to their greater interactions with biological membranes at acidic than neutral pH.