Dramatic reduction of toxicity of Poly(hexamethylene guanidine) disinfectant by charge neutralization.

The current study aimed to investigate the toxicity of positively charged polyhexamethylene guanidine (PHMG) polymer and its complexation with different anionic natural polymers such as k-carrageenan (kCG), chondroitin sulfate (CS), sodium alginate (Alg.Na), polystyrene sulfonate sodium (PSS.Na) and hydrolyzed pectin (HP). The physicochemical properties of the synthesized PHMG and its combination with anionic polyelectrolyte complexes (PECs) namely PHMG:PECs were characterized using zeta potential, XPS, FTIR, and TG analysis. Furthermore, cytotoxic behavior of the PHMG and PHMG:PECs, respectively, were evaluated using human liver cancer cell line (HepG2). The study results revealed that the PHMG alone had slightly higher cytotoxicity to the HepG2 cells than the prepared polyelectrolyte complexes such as PHMG:PECs. The PHMG:PECs showed a significant reduction of cytotoxicity to the HepG2 cells than the pristine PHMG alone. A reduction of PHMG toxicity was observed may be due to the facile formation of complexation between the positively charged PHMG and negatively charged anionic natural polymers such as kCG, CS, Alg. Na, PSS.Na and HP, respectively, via charge balance or neutralization. The experimental results indicate that the suggested method might significantly lower PHMG toxicity while improving biocompatibility.

Antibacterial and clusteroluminogenic hypromellose-graft-chitosan-based polyelectrolyte complex films with high functional flexibility for food packaging.

Food packaging can extend the shelf life of food products and enhance the safety and quality of the food. This study reports food-grade polyelectrolyte complex films generated via electrostatic interactions between two cellulose-based agents [viz., hypromellose-graft-chitosan, and carmellose sodium]. At optimal conditions, our films show good barrier properties, high transparency, and high efficiency in post-production agent loading. They also demonstrate intrinsic antibacterial effects against both Gram-negative and Gram-positive bacteria. By using frozen chicken breasts as a model, the films enable real-time monitoring of the status of the frozen food due to the property of clusterisation-triggered emission. Along with their negligible toxicity, our films warrant further development as multi-functional films for effective and self-indicating food packaging.

Efficient near-infrared anionic conjugated polyelectrolyte for photothermal therapy.

In this work, an anionic conjugated polyelectrolyte (PCP-SO3K), in which the backbone contains alternating 4,4-bis-alkyl-4H-cyclopenta-[2,1-b;3,4-b']-dithiophene and benzene structural units and the charges are provided by pendant sulfonate groups, was synthesized. The ionic nature of PCP-SO3K renders it soluble in water, and PCP-SO3K aqueous solution exhibits good photostability, with two main absorbance bands centered at 490 nm and 837 nm before and after laser irradiation. Its NIR absorption in water, negligible photoluminescence and insignificant intersystem crossing endow PCP-SO3K with efficient photothermal therapy performance, and an effective photothermal conversion efficiency of 56.7% was realized. Thus, PCP-SO3K aqueous solution can be used as an effective photothermal agent for in vivo applications as its photoactivity can be triggered by NIR light and can convert laser energy into thermal energy in a water environment. Of particular importance is the fact that complete tumor remission without recurrence in 4T1 tumor-bearing mice was realized after intravenous injection of PCP-SO3K aqueous solution and laser irradiation (2.0 W cm-2, 808 nm). The results indicate that the application of anionic conjugated polyelectrolytes as photothermal agents in photothermal therapy provides a new platform for the design of photothermal agents for clinical cancer treatment.

Stimulus-Responsive Polyzwitterionic Surfaces Made from Itaconic Acid: Self-Triggered Antimicrobial Activity, Protein Repellency, and Cell Compatibility.

A functional monomer carrying a carboxylate and a protected primary ammonium group is synthesized from itaconic acid. When copolymerized with dimethyl acrylamide and 4-methacryloyloxybenzophenone, cross-linkable polyzwitterions are obtained. These are converted to surface-attached polyzwitterion networks by simultaneous UV-triggered C,H insertion reactions. The resulting polyzwitterion-coated substrates were studied by surface plasmon resonance spectroscopy measurements, ζ potential and various biological assays. They were (expectedly) protein repellent, yet at the same time (and unexpectedly) cell-adhesive and antimicrobially active. This was attributed to stimulus-responsiveness of the polyzwitterion (confirmed by the ζ potential measurements), which enables charge adjustment at different pH values. When protonated, the polyzwitterions become amphiphilic polycations and, in this state, kill bacteria upon contact like their parent structures (polymer-based synthetic mimics of antimicrobial peptides, SMAMPs).

Phosphonium Polyelectrolyte Complexes for the Encapsulation and Slow Release of Ionic Cargo.

Polyelectrolyte complexation, the combination of anionically and cationically charged polymers through ionic interactions, can be used to form hydrogel networks. These networks can be used to encapsulate and release cargo, but the release of cargo is typically rapid, occurring over a period of hours to a few days and they often exhibit weak, fluid-like mechanical properties. Here we report the preparation and study of polyelectrolyte complexes (PECs) from sodium hyaluronate (HA) and poly[tris(hydroxypropyl)(4-vinylbenzyl)phosphonium chloride], poly[triphenyl(4-vinylbenzyl)phosphonium chloride], poly[tri(n-butyl)(4-vinylbenzyl)phosphonium chloride], or poly[triethyl(4-vinylbenzyl)phosphonium chloride]. The networks were compacted by ultracentrifugation, then their composition, swelling, rheological, and self-healing properties were studied. Their properties depended on the structure of the phosphonium polymer and the salt concentration, but in general, they exhibited predominantly gel-like behavior with relaxation times greater than 40 s and self-healing over 2-18 h. Anionic molecules, including fluorescein, diclofenac, and adenosine-5'-triphosphate, were encapsulated into the PECs with high loading capacities of up to 16 wt %. Fluorescein and diclofenac were slowly released over 60 days, which was attributed to a combination of hydrophobic and ionic interactions with the dense PEC network. The cytotoxicities of the polymers and their corresponding networks with HA to C2C12 mouse myoblast cells was investigated and found to depend on the structure of the polymer and the properties of the network. Overall, this work demonstrates the utility of polyphosphonium-HA networks for the loading and slow release of ionic drugs and that their physical and biological properties can be readily tuned according to the structure of the phosphonium polymer.

In vivo Studies on Pharmacokinetics, Toxicity and Immunogenicity of Polyelectrolyte Nanocapsules Functionalized with Two Different Polymers: Poly-L-Glutamic Acid or PEG.

BACKGROUND: The functionalization of a nanoparticle surface with PEG (polyethylene glycol) is an approach most often used for extending nanomaterial circulation time, enhancing its delivery and retention in the target tissues, and decreasing systemic toxicity of nanocarriers and their cargos. However, because PEGylated nanomedicines were reported to induce immune response including production of anti-PEG antibodies, activation of the complement system as well as hypersensitivity reactions, hydrophilic polymers other than PEG are gaining interest as its replacement in nanomaterial functionalization. Here, we present the results of in vivo evaluation of polyelectrolyte nanocapsules with biodegradable, polyelectrolyte multilayer shells consisting of poly-l-lysine (PLL) and poly-l-glutamic (PGA) acid as a potential drug delivery system. We compared the effects of nanocapsules functionalized with two different "stealth" polymers as the external layer of tested nanocapsules was composed of PGA (PGA-terminated nanocapsules, NC-PGA) or the copolymer of poly-l-lysine and polyethylene glycol (PEG-terminated nanocapsules, NC-PEG). METHODS: Nanocapsules pharmacokinetics, biodistribution and routes of eliminations were analysed postmortem by fluorescence intensity measurement. Toxicity of intravenously injected nanocapsules was evaluated with analyses of blood morphology and biochemistry and by histological tissue analysis. DNA integrity was determined by comet assay, cytokine profiling was performed using flow cytometer and detection of antibodies specific to PEG was performed by ELISA assay. RESULTS: We found that NC-PGA and NC-PEG had similar pharmacokinetic and biodistribution profiles and both were eliminated by hepatobiliary and renal clearance. Biochemical and histopathological evaluation of long-term toxicity performed after a single as well as repeated intravenous injections of nanomaterials demonstrated that neither NC-PGA nor NC-PEG had any acute or chronic hemato-, hepato- or nephrotoxic effects. In contrast to NC-PGA, repeated administration of NC-PEG resulted in prolonged increased serum levels of a number of cytokines. CONCLUSION: Our results indicate that NC-PEG may cause undesirable activation of the immune system. Therefore, PGA compares favorably with PEG in equipping nanomaterials with stealth properties. Our research points to the importance of a thorough assessment of the potential influence of nanomaterials on the immune system.

Polycation Architecture and Assembly Direct Successful Gene Delivery: Micelleplexes Outperform Polyplexes via Optimal DNA Packaging.

Cellular delivery of biomacromolecules is vital to medical research and therapeutic development. Cationic polymers are promising and affordable candidate vehicles for these precious payloads. However, the impact of polycation architecture and solution assembly on the biological mechanisms and efficacy of these vehicles has not been clearly defined. In this study, four polymers containing the same cationic poly(2-(dimethylamino)ethyl methacrylate) (D) block but placed in different architectures have been synthesized, characterized, and compared for cargo binding and biological performance. The D homopolymer and its diblock copolymer poly(ethylene glycol)-block-poly(2-(dimethylamino) ethyl methacrylate) (OD) readily encapsulate pDNA to form polyplexes. Two amphiphilic block polymer variants, poly(2-(dimethylamino)ethyl methacrylate)-block-poly(n-butyl methacrylate) (DB) and poly(ethylene glycol)-block-poly(2-(dimethylamino)ethyl methacrylate)-block-poly(n-butyl methacrylate) (ODB), self-assemble into micelles, which template pDNA winding around the cationic corona to form micelleplexes. Micelleplexes were found to have superior delivery efficiency compared to polyplexes and detailed physicochemical and biological characterizations were performed to pinpoint the mechanisms by testing hypotheses related to cellular internalization, intracellular trafficking, and pDNA unpackaging. For the first time, we find that the higher concentration of amines housed in micelleplexes stimulates both cellular internalization and potential endosomal escape, and the physical motif of pDNA winding into micelleplexes, reminiscent of DNA compaction by histones in chromatin, preserves the pDNA secondary structure in its native B form. This likely allows greater payload accessibility for protein expression with micelleplexes compared to polyplexes, which tightly condense pDNA and significantly distort its helicity. This work provides important guidance for the design of successful biomolecular delivery systems via optimizing the physicochemical properties.

Polyelectrolyte Carboxymethyl Cellulose for Enhanced Delivery of Doxorubicin in MCF7 Breast Cancer Cells: Toxicological Evaluations in Mice Model.

PURPOSE: Chemotherapy as an important tool for cancer treatment faces many obstacles such as multidrug resistance and adverse toxic effects on healthy tissues. Drug delivery systems have opened a new window to overcome these problems. METHODS: A polyelectrolyte carboxymethyl cellulose polymer as a magnetic nanocarrier was synthesized for enhancing delivery and uptake of doxorubicin in MCF7 breast cancer cells and decreasing the adverse toxic effects to healthy tissues. RESULTS: The physicochemical properties of developed nanocarrier showed that it can be used in drug delivery purposes. The efficiency of the delivery system was assessed by loading and release studies. Besides, biological assays including protein-particle interaction, hemolysis assay, cytotoxicity study, cellular uptake, and apoptosis analysis were performed. All results persuaded us to investigate the cytotoxic effects of nanocarrier in an animal model by determining the biochemical parameters attributed to organ injuries, and hematoxylin and eosin (H&E) staining for histopathological manifestations. We observed that the nanocarrier has no toxic effect on healthy tissues, while, it is capable of reducing the toxic side effects of doxorubicin by more cellular internalization. CONCLUSION: Chemical characterizations and biological studies confirmed that developed nanocarrier with permanent cationic groups of imidazolium and anionic carboxylic acid groups is an effective candidate for anticancer drug delivery.

Chitosan-Chondroitin sulfate based polyelectrolyte complex for effective management of chronic wounds.

Acute and chronic wound remain an unresolved clinical problem among various demographic groups. Traditional marketed products focus mainly on inhibition of bacterial growth at the wound site neglecting the tissue repair, which significantly affect the healing rate. It would be highly beneficial if a wound healing material can be developed which has both antibacterial as well as tissue regenerating potential. We have prepared a polyelectrolyte complex (PEC) using chitosan (CH) and chondroitin sulfate (CS) which can form an in-situ scaffold by spontaneous mixing. The fabrication of CH-CS PEC was optimized using Quality-By-Design (QbD) approach. The prepared PEC showed very high swelling and porosity property. It was found to be non-hemolytic with good blood compatibility and low blood clotting index. It also exhibited good antibacterial activity against both gram-positive and gram-negative bacteria. The cell proliferation study exhibited good cytocompatibility and almost four-fold increase in cell density when treated with CH-CS PEC compared to control. In summary, we demonstrated that the prepared CH-CS PEC showed good blood compatibility, high antibacterial effect, and promoted wound healing potentially by stimulating fibroblast growth, making it an ideal wound dressing material.

Structural exploration of hydrophobic core in polycationic micelles for improving siRNA delivery efficiency and cell viability.

Improving siRNA delivery efficiency often encounters a dilemma with poor or decreased biocompatibility for polycationic micelles. To address this dilemma, this work focused on a structural exploration of the hydrophobic core in amphiphilic polycationic micelles by preparing two amphiphilic polycations with block or random hydrophobic segments, poly(ethylene glycol)-block-poly(aminoethyl methacrylate)-block-poly(2-diamylamine ethyl methacrylate)-block-poly(2-diethylamine ethyl methacrylate) (mPEG-PAMA-PD5A-PDEA, PADE) and poly(ethylene glycol)-block-poly(aminoethyl methacrylate)-block-poly(2-diamylamine ethyl methacrylate-co-2-diethylamine ethyl methacrylate) (mPEG-PAMA-P(D5A/DEA), PA(D/E)). The properties of the two copolymers and their self-assembly micelles were characterized, including structure, morphology, size and zeta potential. Cytotoxicity, siRNA silencing efficiency and cellular uptake of PADE/siRNA and PA(D/E)/siRNA complexes were evaluated in HepG2 and MDA-MB-231 cells in vitro. The endosome escape and intracellular distribution of PADE/siRNA and PA(D/E)/siRNA in HepG2 cells were also observed by CLSM. Significantly, the results indicated that PA(D/E)/siRNA showed not only better gene silencing efficiency but also lower cytotoxicity, which may be attributed to the homogeneous morphology of the hydrophobic core of PA(D/E) micelles. Therefore, this work provides a new pathway to overcome the dilemma between siRNA delivery efficiency and biocompatibility for the development of efficient polycation carriers.

In vitro toxicity studies of biodegradable, polyelectrolyte nanocapsules.

BACKGROUND: Toxicity of nanomaterials is one of the most important factors limiting their medical application. Evaluation of in vitro nanotoxicity allows for the identification and elimination of most of the toxic materials prior to animal testing. The current knowledge of the possible side effects of biodegradable nanomaterials, such as liposomes and polymeric organic nanoparticles, is limited. Previously, we developed a potential drug delivery system in the form of nanocapsules with polyelectrolyte, biodegradable shells consisting of poly-l-lysine and poly-l-glutamic acid (PGA), formed by the layer-by-layer adsorption technique. METHODS: Hemolysis assay, viability tests, flow cytometry analysis of vascular cell adhesion molecule-1 expression on endothelium, analysis of nitric oxide production, measurement of intracellular reactive oxygen species levels, detection of antioxidant enzyme activity, and analysis of DNA damage with comet assay were performed to study the in vitro toxicity of nanocapsules. RESULTS: In this work, we present the results of an in vitro analysis of toxicity of five-layer positively charged poly-l-lysine-terminated nanocapsules (NC5), six-layer negatively charged PGA-terminated nanocapsules (NC6) and five-layer PEGylated nanocapsules (NC5-PEG). PGA and polyethylene glycol (PEG) were used as two different "stealth" polymers. Of all the polyelectrolyte nanocapsules tested for blood compatibility, only cationic NC5 showed acute toxicity toward blood cells, expressed as hemolysis and aggregation. Neither NC6 nor NC5-PEG had proinflammatory activity evaluated through changes in the expression of NF-κB-dependent genes, iNOS and vascular cell adhesion molecule-1, induced oxidative stress, or promoted DNA damage in various cells. CONCLUSION: Our studies clearly indicate that PGA-coated (negatively charged) and PEGylated polyelectrolyte nanocapsules do not show in vitro toxicity, and their potential as a drug delivery system may be safely studied in vivo.

Electrostatic self-assembled nanoparticles based on spherical polyelectrolyte brushes for magnetic resonance imaging.

Magnetic resonance imaging (MRI) is one of the most important medical imaging techniques for clinical diagnosis. Contrast agents (CAs) are commonly necessarily used to enhance the imaging quality of MRI and differentiate diseased tissues from normal ones. Herein, we introduced a macromolecular carrier, spherical polyelectrolyte brushes (SPBs), which consists of a solid polystyrene (PS) core and polyacrylic acid (PAA) chains as a brush layer to host Gd(iii) complexes. The cationic Gd(iii) complex Gd-DTPA-NO-C4 was synthesized through a 6-step approach and then formed electrostatic self-assemblies with SPBs to afford magnetic assemblies. The regular appearance of the core-shell type structure of the assemblies was confirmed by TEM and SEM. Besides, a remarkable enhancement in relaxivity up to 62 mM-1 s-1 of these assemblies was determined, much higher than that of clinically used small molecule CAs (4-5 mM-1 s-1). It is noteworthy that the assemblies exhibit non-cytotoxicity even at the concentration of Gd(iii) up to 150 µM, showing great potential for clinical MRI applications.

Preparation, characterization and in vitro release of ß-galactosidase loaded polyelectrolyte nanoparticles.

Improving encapsulation efficacy (EE) and bioavailability of ß-galactosidase (ß-gal) is always a challenge due to its fragility. In this work, ß-gal loaded ß-chitosan (CS) nanoparticles (NPs) were successfully prepared based on ionic gelation technique and electrostatic attraction for improving its EE and in vitro releasing capacity. The particle size of ß-gal loaded low and high molecular weight (LMW and HMW) ß-CS NPs reached 584.37 and 652.46nm, with Zeta-potential (ZP) of 26.37 and 16.46mV under the optimal conditions, respectively. In vitro release study conducted at pH4.5 and 7.4 showed that ß-gal loaded LMW and HMW ß-CS NPs with EE of 68.32 and 58.64% sustained the release of the ß-gal over 12h. The ß-gal incorporated into ß-CS NPs was confirmed with the results of physicochemical and structural properties of ß-gal loaded ß-CS NPs, and prepared NPs had hardly any cytotoxicity in the range of 0.1-1.0mg/mL. The results indicated that ß-gal loaded ß-CS NPs could serve as non-toxic delivery carriers for the treatment of lactose intolerance.

Is the aquatic toxicity of cationic polyelectrolytes predictable from selected physical properties?

Cationic acrylamide-based polyelectrolytes (cPAM) are widely used in industry. They can be designed for optimal performance in a specific application, but this opportunity means the environmental safety of all different alternatives needs to be addressed. Both the inclusion of environmental toxicity as a design variable and the establishment of relationships between structure and ecotoxicity are thus current challenges. The aim of this study was to assess whether structural variables such as molecular weight, charge density and the integrative intrinsic viscosity parameter can be used to predict the environmental safety of cPAMs, as well as if these relationships are stable when the biological models change. Five cPAMs comprising molecular weight and charge density gradients were tested against bacteria, microalgae, macrophytes and daphnids. While correlations were found between physical properties of cPAMs as expected, no clear ecotoxicity patterns could be identified. All cPAMs can be classified as harmful to aquatic life on the basis of the responses elicited in the most sensitive organisms, microalgae and daphnids. Unicellular bacteria were the least sensitive eco-receptors possibly due to cell wall structure or the protective effect of the ionic strength of the test medium. The macrophytes were also tolerant to cPAMs exposure, which may be related to exposure avoidance mechanisms. The order of toxicity of cPAMs depended on the test organism, preventing the establishment of stable structure-ecotoxicity relationships. Therefore, the study leads to the overall generalist recommendation of relying on the most sensitively responding test organisms when developing new (eco)safe-by-design cPAMs.

Mucoadhesive nanostructured polyelectrolytes complexes modulate the intestinal permeability of methotrexate.

Nanostructured polyelectrolytes complexes (nano PECs) loaded with methotrexate (MTX) were obtained by the polyelectrolyte complexation of chitosan (CS) and hyaluronic acid (HA), further incorporating hypromellose phthalate (HP). The mean diameter of nano PECs ranged from 325 to 458nm, with a narrow size distribution. Zeta potential was close to +30mV, decreasing to +21mV after the incorporation of HP, a range of values that favour the physical stability of system as the interaction with cationic biological membranes. The electrostatic interactions between the different components were indicated by the FTIR data. The mucoadhesiveness of nano PECs was demonstrated and MTX and HP influenced this property. The cell viability assays showed the biosafety of the isolated polymers and nano PECs in intestinal HT29-MTX and Caco-2 cell lines at 4h of test. The permeability values of MTX loaded in CS/HA nano PECs were 7.6 and 4-fold higher than those of CS/HA/HP nano PECS and free drug, respectively, in the Caco-2 monoculture. In mucus secreting co-culture cell model these values were 3 and 6.5 fold, respectively. Such features indicate that nano PECs developed in this work can be promising carriers for MTX in the treatment of local or systemic diseases.