TMPyP-mediated photoinactivation of Pseudomonas aeruginosa improved in the presence of a cationic polymer.

Pseudomonas aeruginosa is one of the most refractory organisms to antibiotic treatment and appears to be one of the least susceptible to photodynamic treatment. TMPyP is effective in the photoinactivation of P. aeruginosa, and the co-administration with the cationic polymer Eudragit®-E100 (Eu) potentiates this effect against isolates both sensitive and resistant to antibiotics. The fluorescent population (>98%) observed by flow cytometry after exposure to Eu + TMPyP remained unchanged after successive washings, indicating a stronger interaction/internalization of TMPyP in the bacteria, which could be attributed to the rapid neutralization of surface charges. TMPyP and Eu produced depolarization of the cytoplasmic membrane, which increased when both cationic compounds were combined. Using confocal laser scanning microscopy, heterogeneously distributed fluorescent areas were observed after TMPyP exposure, while homogeneous fluorescence and enhanced intensity were observed with Eu + TMPyP. The polymer caused alterations in the bacterial envelopes that contributed to a deeper and more homogeneous interaction/internalization of TMPyP, leading to a higher probability of damage by cytotoxic ROS and explaining the enhanced result of photodynamic inactivation. Therefore, Eu acts as an adjuvant without being by itself capable of eradicating this pathogen. Moreover, compared with other therapies, this combinatorial strategy with a polymer approved for pharmaceutical applications presents advantages in terms of toxicity risks.

Characterization and Differential Cytotoxicity of Gramicidin Nanoparticles Combined with Cationic Polymer or Lipid Bilayer.

Gramicidin (Gr) nanoparticles (NPs) and poly (diallyl dimethyl ammonium) chloride (PDDA) water dispersions were characterized and evaluated against Gram-positive and Gram-negative bacteria and fungus. Dynamic light scattering for sizing, zeta potential analysis, polydispersity, and colloidal stability over time characterized Gr NPs/PDDA dispersions, and plating and colony-forming units counting determined their microbicidal activity. Cell viabilities of Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans in the presence of the combinations were reduced by 6, 7, and 7 logs, respectively, at 10 µM Gr/10 µg·mL-1 PDDA, 0.5 µM Gr/0. 5µg·mL-1 PDDA, and 0.5 µM Gr/0.5 µg·mL-1 PDDA, respectively. In comparison to individual Gr doses, the combinations reduced doses by half (S. aureus) and a quarter (C. albicans); in comparison to individual PDDA doses, the combinations reduced doses by 6 times (P. aeruginosa) and 10 times (C. albicans). Gr in supported or free cationic lipid bilayers reduced Gr activity against S. aureus due to reduced Gr access to the pathogen. Facile Gr NPs/PDDA disassembly favored access of each agent to the pathogen: PDDA suctioned the pathogen cell wall facilitating Gr insertion in the pathogen cell membrane. Gr NPs/PDDA differential cytotoxicity suggested the possibility of novel systemic uses for the combination.

Cationic polymer contributes to broaden the spectrum of vancomycin activity achieving eradication of Pseudomonas aeruginosa.

Vancomycin (VAN) is unable to penetrate the outer membrane of Gram-negative bacteria and reach the target site. One approach to overcome this limitation is to associate it with compounds with permeabilizing or antimicrobial properties. Eudragit E100® (Eu) is a cationic polymer insufficiently characterized for its potential antimicrobial action. Eu-VAN combinations were characterized, the antimicrobial efficacy against Pseudomonas aeruginosa was evaluated and previous studies on the effects of Eu on bacterial envelopes were extended. Time-kill assays showed eradication of P. aeruginosa within 3-6 h exposure to Eu-VAN, whilst VAN was ineffective. Eu showed regrowth in 24 h and delayed colony pigmentation. Although permeabilization of bacterial envelopes or morphological alterations observed by TEM and flow cytometry after exposure to Eu were insufficient to cause bacterial death, they allowed access of VAN to the target site, since Eu-VAN/Van-FL-treated cultures showed fluorescent staining in all bacterial cells, indicating Van-FL internalization. Consequently, Eu potentiated the activity of an otherwise inactive antibiotic against P. aeruginosa. Moreover, Eu-VAN combinations exhibited improved physicochemical properties and could be used in the development of therapeutic alternatives in the treatment of bacterial keratitis.

Cationic Nanostructures as Adjuvants for Vaccines.

Spherical or discoidal lipid polymer nanostructures bearing cationic charges successfully adsorb a variety of oppositely charged antigens (Ag) such as proteins, peptides, nucleic acids, or oligonucleotides. This report provides instructions for the preparation and physical characterization of four different cationic nanostructures able to combine and deliver antigens to the immune system: (1) dioctadecyl dimethylammonium bromide (DODAB) bilayer fragments (DODAB BF); (2) polystyrene sulfate (PSS) nanoparticles (NPs) covered with one cationic dioctadecyl dimethylammonium bromide bilayer (DODAB) named (PSS/DODAB); (3) cationic NPs of biocompatible polymer poly(methyl methacrylate) (PMMA) prepared by emulsion polymerization of the methyl methacrylate (MMA) monomer in the presence of DODAB BF (PMMA/DODAB NPs); (4) antigen NPs (NPs) where the cationic polymer poly(diallyl dimethyl ammonium chloride) (PDDA) directly combined at nontoxic and low dose with the antigen (Ag); when the oppositely charged model antigen is ovalbumin (OVA), NPs are named PDDA/OVA. These nanostructures provide adequate microenvironments for carrying and delivering antigens to the antigen-presenting cells of the immune system.

Biocompatible Lipid Polymer Cationic Nanoparticles for Antigen Presentation.

Biocompatible lipid polymer nanoparticles (NPs) previously used as antimicrobial agents are explored here as immuno-adjuvants. Poly (methyl methacrylate) (PMMA)/dioctadecyldimethylammonium bromide (DODAB)/poly (diallyldimethylammonium chloride) (PDDA) nanoparticles (NPs) were prepared by emulsion polymerization of methyl methacrylate (MMA) in the presence of DODAB and PDDA, with azobisisobutyronitrile (AIBN) as the initiator. NPs characterization after dialysis by dynamic light-scattering yielded 225 ± 2 nm hydrodynamic diameter (Dz), 73 ± 1 mV zeta-potential (ζ), and 0.10 ± 0.01 polydispersity (P). Ovalbumin (OVA) adsorption reduced ζ to 45 ± 2 mV. Balb/c mice immunized with NPs/OVA produced enhanced OVA-specific IgG1 and IgG2a, exhibited moderate delayed type hypersensitivity reaction, and enhanced cytokines production (IL-4, IL-10, IL-2, IFN-γ) by cultured spleen cells. There was no cytotoxicity against cultured macrophages and fibroblasts. Advantages of the PMMA/DODAB/PDDA NPs were high biocompatibility, zeta-potential, colloidal stability, and antigen adsorption. Both humoral and cellular antigen-specific immune responses were obtained.

Biocompatible lipid polymer cationic nanoparticles for antigen presentation

Biocompatible lipid polymer nanoparticles (NPs) previously used as antimicrobial agents are explored here as immuno-adjuvants. Poly (methyl methacrylate) (PMMA)/dioctadecyldimethylammonium bromide (DODAB)/poly (diallyldimethylammonium chloride) (PDDA) nanoparticles (NPs) were prepared by emulsion polymerization of methyl methacrylate (MMA) in the presence of DODAB and PDDA, with azobisisobutyronitrile (AIBN) as the initiator. NPs characterization after dialysis by dynamic light-scattering yielded 225 ± 2 nm hydrodynamic diameter (Dz), 73 ± 1 mV zeta-potential (ζ), and 0.10 ± 0.01 polydispersity (P). Ovalbumin (OVA) adsorption reduced ζ to 45 ± 2 mV. Balb/c mice immunized with NPs/OVA produced enhanced OVA-specific IgG1 and IgG2a, exhibited moderate delayed type hypersensitivity reaction, and enhanced cytokines production (IL-4, IL-10, IL-2, IFN-γ) by cultured spleen cells. There was no cytotoxicity against cultured macrophages and fibroblasts. Advantages of the PMMA/DODAB/PDDA NPs were high biocompatibility, zeta-potential, colloidal stability, and antigen adsorption. Both humoral and cellular antigen-specific immune responses were obtained.

Efficient Non-Viral Gene Modification of Mesenchymal Stromal Cells from Umbilical Cord Wharton's Jelly with Polyethylenimine.

Mesenchymal stromal cells (MSC) derived from human umbilical cord Wharton's jelly (WJ) have a wide therapeutic potential in cell therapy and tissue engineering because of their multipotential capacity, which can be reinforced through gene therapy in order to modulate specific responses. However, reported methodologies to transfect WJ-MSC using cationic polymers are scarce. Here, WJ-MSC were transfected using 25 kDa branched- polyethylenimine (PEI) and a DNA plasmid encoding GFP. PEI/plasmid complexes were characterized to establish the best transfection efficiencies with lowest toxicity. Expression of MSC-related cell surface markers was evaluated. Likewise, immunomodulatory activity and multipotential capacity of transfected WJ-MSC were assessed by CD2/CD3/CD28-activated peripheral blood mononuclear cells (PBMC) cocultures and osteogenic and adipogenic differentiation assays, respectively. An association between cell number, PEI and DNA content, and transfection efficiency was observed. The highest transfection efficiency (15.3 ± 8.6%) at the lowest toxicity was achieved using 2 ng/µL DNA and 3.6 ng/µL PEI with 45,000 WJ-MSC in a 24-well plate format (200 µL). Under these conditions, there was no significant difference between the expression of MSC-identity markers, inhibitory effect on CD3+ T lymphocytes proliferation and osteogenic/adipogenic differentiation ability of transfected WJ-MSC, as compared with non-transfected cells. These results suggest that the functional properties of WJ-MSC were not altered after optimized transfection with PEI.

Cationic Nanostructures for Vaccines Design.

Subunit vaccines rely on adjuvants carrying one or a few molecular antigens from the pathogen in order to guarantee an improved immune response. However, to be effective, the vaccine formulation usually consists of several components: an antigen carrier, the antigen, a stimulator of cellular immunity such as a Toll-like Receptors (TLRs) ligand, and a stimulator of humoral response such as an inflammasome activator. Most antigens are negatively charged and combine well with oppositely charged adjuvants. This explains the paramount importance of studying a variety of cationic supramolecular assemblies aiming at the optimal activity in vivo associated with adjuvant simplicity, positive charge, nanometric size, and colloidal stability. In this review, we discuss the use of several antigen/adjuvant cationic combinations. The discussion involves antigen assembled to 1) cationic lipids, 2) cationic polymers, 3) cationic lipid/polymer nanostructures, and 4) cationic polymer/biocompatible polymer nanostructures. Some of these cationic assemblies revealed good yet poorly explored perspectives as general adjuvants for vaccine design.

Simple Nanoparticles from the Assembly of Cationic Polymer and Antigen as Immunoadjuvants.

Since antigens are negatively charged, they combine well with positively charged adjuvants. Here, ovalbumin (OVA) (0.1 mg·mL-1) and poly (diallyldimethylammonium chloride) (PDDA) (0.01 mg·mL-1) yielded PDDA/OVA assemblies characterized by dynamic light scattering (DLS) and scanning electron microscopy (SEM) as spherical nanoparticles (NPs) of 170 ± 4 nm hydrodynamic diameter, 30 ± 2 mV of zeta-potential and 0.11 ± 0.01 of polydispersity. Mice immunization with the NPs elicited high OVA-specific IgG1 and low OVA-specific IgG2a production, indicating a Th-2 response. Delayed-type hypersensitivity reaction (DTH) was low and comparable to the one elicited by Al(OH)3/OVA, suggesting again a Th-2 response. PDDA advantages as an adjuvant were simplicity (a single-component adjuvant), low concentration needed (0.01 mg·mL-1 PDDA) combined with antigen yielding neglectable cytotoxicity, and high stability of PDDA/OVA dispersions. The NPs elicited much higher OVA-specific antibodies production than Al(OH)3/OVA. In vivo, the nano-metric size possibly assured antigen presentation by antigen-presenting cells (APC) at the lymph nodes, in contrast to the location of Al(OH)3/OVA microparticles at the site of injection for longer periods with stimulation of local dendritic cells. In the future, it will be interesting to evaluate combinations of the antigen with NPs carrying both PDDA and elicitors of the Th-1 response.

Simple Nanoparticles from the Assembly of Cationic Polymer and Antigen as Immunoadjuvants

Since antigens are negatively charged, they combine well with positively charged adjuvants. Here, ovalbumin (OVA) (0.1 mg·mL-1) and poly (diallyldimethylammonium chloride) (PDDA) (0.01 mg·mL-1) yielded PDDA/OVA assemblies characterized by dynamic light scattering (DLS) and scanning electron microscopy (SEM) as spherical nanoparticles (NPs) of 170 ± 4 nm hydrodynamic diameter, 30 ± 2 mV of zeta-potential and 0.11 ± 0.01 of polydispersity. Mice immunization with the NPs elicited high OVA-specific IgG1 and low OVA-specific IgG2a production, indicating a Th-2 response. Delayed-type hypersensitivity reaction (DTH) was low and comparable to the one elicited by Al(OH)3/OVA, suggesting again a Th-2 response. PDDA advantages as an adjuvant were simplicity (a single-component adjuvant), low concentration needed (0.01 mg·mL-1 PDDA) combined with antigen yielding neglectable cytotoxicity, and high stability of PDDA/OVA dispersions. The NPs elicited much higher OVA-specific antibodies production than Al(OH)3/OVA. In vivo, the nano-metric size possibly assured antigen presentation by antigen-presenting cells (APC) at the lymph nodes, in contrast to the location of Al(OH)3/OVA microparticles at the site of injection for longer periods with stimulation of local dendritic cells. In the future, it will be interesting to evaluate combinations of the antigen with NPs carrying both PDDA and elicitors of the Th-1 response

Microbicidal Dispersions and Coatings from Hybrid Nanoparticles of Poly (Methyl Methacrylate), Poly (Diallyl Dimethyl Ammonium) Chloride, Lipids, and Surfactants.

Hybrid and antimicrobial nanoparticles (NPs) of poly (methyl methacrylate) (PMMA) in the presence of poly (diallyl dimethyl ammonium) chloride (PDDA) were previously obtained by emulsion polymerization in absence of surfactant with low conversion. In the presence of amphiphiles such as cetyl trimethyl ammonium bromide (CTAB), dioctadecyl dimethyl ammonium bromide (DODAB) or soybean lecithin, we found that conversion increased substantially. In this work, the effect of the amphiphiles on the NPs core-shell structure and on the antimicrobial activity of the NPs was evaluated. NPs dispersions casted on silicon wafers, glass coverslips or polystyrene substrates were also used to obtain antimicrobial coatings. Methods for characterizing the dispersions and coatings were based on scanning electron microscopy, dynamic light scattering, determination of thickness, rugosity, and wettability for the coatings and determination of colony-forming unities (log CFU/mL) of microbia after 1 h interaction with the coatings or dispersions. The amphiphiles used during PMMA/PDDA/amphiphile NPs synthesis reduced the thickness of the NPs PDDA shell surrounding each particle. The antimicrobial activity of the dispersions and coatings were due to PDDA-the amphiphiles were either washed out by dialysis or remained in the PMMA polymeric core of the NPs. The most active NPs and coatings were those of PMMA/PDDA/CTAB-the corresponding coatings showed the highest rugosity and total surface area to interact with the microbes. The dispersions and coatings obtained by casting of the NPs dispersions onto silicon wafers were hydrophilic and exhibited microbicidal activity against Escherichia coli, Staphylococcus aureus, and Candida albicans. In addition, a major effect of reduction in particle size revealed the suitability of nanometric and cationic NPs (sizes below 100 nm) represented by PMMA/PDDA/CTAB NPs to yield maximal microbicidal activity from films and dispersions against all microbia tested. The reduction of cell viability by coatings and dispersions amounted to 6-8 logs from [PDDA] ≥ minimal microbicidal concentration.

Relationship between Degree of Polymeric Ionisation and Hydrolytic Degradation of Eudragit® E Polymers under Extreme Acid Conditions.

The commercial copolymers Eudragit® E 100 and Eudragit® PO are widely used materials in the pharmaceutical field as coating systems. Such materials derived from amino-methacrylate groups under acidulated conditions may acquire an ionisable fraction or undergo hydrolytic degradation of the polymeric structure. This work focused on establishing the chemical, physical, and surface changes of two reprocessed polymeric materials, here named as EuCl-E-100 and EuCl-E-PO, which were obtained from the commercial Eudragit® E 100 and Eudragit® E PO, respectively. The commercial materials were exposed to extreme acid conditions, where the polymers were solubilised and subsequently dried by the refractance window method. The materials obtained were chemically characterised by potentiometric titration, nuclear magnetic resonance spectroscopy (1H NMR and 13C NMR) in one and two dimensions (COSY, HSQC, and HMBC), infrared spectroscopy, X-ray diffraction, and differential scanning calorimetry. Changes in the physical properties of the materials were evaluated through studies of flowability, compactability, and their ability to gain and lose humidity. Surface thermodynamic studies were carried out through contact angle measurements using the sessile drop method. The results showed that the processed polymeric materials acquired a substantial degree of ionisation without undergoing hydrolysis of the esterified groups. Furthermore, such changes improved the flow characteristics of the material and the solubility in aqueous media at pH > 5, while also maintaining the hydrophobicity degree of the polymeric surface.

Synthesis of zeolite membranes on calcium silicate support and their bioactive response.

The synthesis of calcium silicate supported zeolite membrane was carried out by second growth method. The chemical nature of the functionalizing agent on the formation of homogenous zeolite membrane was evaluated. One monomer and two cationic polymers were used: 3-aminopropyltriethoxysilane (APS), polyethylenimine (PEI) and polydiallyldimethylammonium chloride (PDDA). The support was subjected to chemical functionalization and then it was rubbed with zeolite crystals. The W zeolite was used as zeolite seed in two different Si/Al ratios. The functionalized and rubbed supports were submitted to hydrothermal treatment at 150 °C for 48 h. The bioactivity of the homogeneous zeolite membranes was evaluated by the biomimetic method through the membranes soaking in a simulated body fluid (SBF) at 37 °C for 21 days. Two immersion methods were evaluated. The products were characterized by XRD and SEM techniques. The results indicated that the supported functionalization with PDDA and the Si/Al ratio (higher than 1.8) of zeolite enhanced the interaction between the support and the zeolite precursor enhancing the formation of homogeneous zeolite membrane on the surface. The presence of the functional groups of PDDA on the membrane was detected by FTIR. After immersion in SBF, the zeolite membrane was stable and led to the formation of Ca-P layer on its surface. The re-immersion method led to the formation of richer Ca/P layer (1.36). These findings allowed generating a zeolite membrane with combined properties of calcium silicate and the controllable porosity of zeolitic material making it potentially useful for bone regeneration and drug releasing.