Patent Grant
10278395
This invention relates generally to the preparation and applications of internally and/or externally functionalized environmentally benign nanoparticles (EbNPs), which are produced by a three-step procedure: (1) synthesis of native EbNPs, (2) functionalization with active agents, and (3) additional surface property customization via one or more modifier(s).
Engineered nanoparticles exhibit unique and useful physical, chemical, and biological particle-specific attributes that may help to solve pressing challenges of mankind in industries including life sciences, energy, and health care. However, nanoparticle waste has been recognized as a potential health hazard,1 as the post-utilization activity of engineered nanoparticles combined with their persistence may result in short and long-term toxicity in humans and the environment.2 For example, it has been found that the physical and chemical characteristics of persistent nanoparticles (PNPs), and therefore, their activity, may not change even after high-temperature treatments in solid-waste incineration plants.3 One way to minimize the post-utilization hazard of nanoparticles is to minimize their residence time and presence in the environment. Employing degradable nanoparticles with matching functionality to PNPs may serve as a suitable solution.
Lignin, the most abundant aromatic polymer in nature,4 has an amorphous structure and is biodegradable. Lignin covalently crosslinks the cell walls of plants, and plays a vital role in plant health, growth, and development.5 When extracted from biomass, the structure of modified lignin varies depending on the initial plant source and the method of isolation. Lignin obtained via the organosolv process, such as High Purity Lignin (HPL), is strongly hydrophobic, does not incorporate any sulfur containing groups, and therefore, preserves best the structure of native lignin of all processed lignins.6 However, the most common extraction method is the Kraft pulping processes.7 INDULIN AT lignin (IAT), a modified lignin that contains a small number of hydrophilic thiol groups, is recovered by this process. In aqueous systems, matrixes of IAT have shown high absorbance capabilities of heavy metal ions for environmental remediation purposes.8, 9 Cationic metal ions are electrostatically attracted to IAT, which is negatively charged in aqueous solution due to deprotonation of its main functional groups (Table 6). Recently, the synthesis of pH-stable IAT-based environmentally benign nanoparticles (EbNPs) in ethylene glycol was reported.10 Hence, we propose that by infusing IAT EbNPs with functional metal ions, it will be possible to synthesize degradable nanoparticles that match the nanoparticle functionality of the respective metal PNPs, while increasing post-utilization safety.
Silver nanoparticles (AgNPs) are among the most widely employed PNPs, as their broad-spectrum antimicrobial properties allow them to combat bacteria strains exhibiting antibiotic resistance,11 which are reported in human pathogens including Escherichia coli (E. coli)12 and Pseudomonas aeruginosa (P. aeruginosa).13 As infection control measures can minimize the spread of drug-resistant bacteria,14 and therefore the potential for nosocomial infections,15 silver-containing products may find increasing utilization in the medical sector to prevent bacteria growth on catheters,16 prostheses,17 and dental materials,18 and to reduce the infection potential of burn wounds.19 In addition, with the emergence of antimicrobial PNPs in textiles,20 water filters,21 and other consumer products, the human exposure potential to PNPs with their associated risks increases. Human skin exposure studies indicate that AgNPs can be released from antibacterial fabrics into liquids resembling “sweat”.22 Studies on commercially available wound dressings proved that dressings containing AgNPs exhibit stronger cytotoxic effects toward keratinocytes than do PNP-free counterparts.23 In vitro studies on mammalian fibroblasts have revealed that AgNP can induce apoptosis. In this context, AgNP may potentially affect human health.24, 25, 26
Several methods for the preparation of antimicrobial silver-based nanoparticle systems have been reported. Most procedures employ highly reactive reducing agents such as sodium borohydride (NaBH4) or hydrazine (N2H4) to reduce silver ions to metallic silver. Green synthesis methods of producing AgNP can reduce the environmental impact during fabrication given that no harsh solvents or reducing agents are employed.27, 28 However, due to the persistent nature of AgNPs, the problem of post-utilization toxicity associated with non-degradable nanoparticles remains unaddressed.
In particular non-limiting embodiments, the present invention provides:
The schematic in
Here, we report non-limiting, exemplary data on the synthesis of native HPL and IAT EbNPs, the infusion of native IAT EbNPs with silver ions, the surface charge modification of the system with PDADMAC, and the quantification of antimicrobial efficiencies for opportunistic human pathogens E. coli and P. aeruginosa. In addition, we provide a hypothesis to explain the antimicrobial mechanism associated with Ag-EbNPs-PDADMAC.
We synthesized native HPL EbNPs via the solvent-antisolvent precipitation method. As illustrated in
The data on the effect of the initial HPL loading in the solvent on the final EbNP size are shown in
We investigated the effect of the stock solution dilution rate on the HPL EbNP size. As reported in
The stability of HPL EbNPs against pH change was tested in dialyzed 0.10 wt % HPL EbNP samples, which were diluted down to 0.05 wt %. To adjust the pH of the EbNP suspensions, callibrated amounts of HNO3 or NaOH solution were added. As indicated in
Native IAT EbNPs were synthesized via the organic solvent water-based pH-drop method in ethylene glycol and the data are reported in
As reported in
We performed an ionic strength study to investigate the effect of increasing ionic strength on the stability of IAT EbNP suspensions, and to determine if the IAT EbNP suspensions may exhibit colloidal stability at ionic strength levels equivalent to the ones found in physiological testing media used in biocidal testing.
We modeled the interaction energy W(D) according to DLVO theory in selected IAT EbNPs at three chosen ionic strengths. At an ionic strength of 0.25 mol/L, we determined a Debye length k−1/=0.61 nm. With ξ-potential of −15.0 mV at that ionic strength, we calculated a surface potential Ψ0=−40.8 mV. We determined the electrostatic repulsion energy W(Delec) and the van der Walls attraction energy W(DVDW) as a function of the separation distance D to evaluate the total interaction energy W(D). As shown in
We obtained IAT EbNPs with a hydrodynamic diameter of 72 nm with a polydispersity index of 0.230 and ξ-potential of −23.5 mV. The DLS equipment measured a conductivity of 0.139 mS/cm, and an electrophoretic mobility of −1.403 μm cm/V s. As depicted in
We functionalized negatively charged IAT EbNPs with Ag+ ions in aqueous solution. We chose a common soluble salt, AgNO3, as an Ag+ ion source.
Ag+ reference solutions in the range of 0.25 ppm to 500 ppm were prepared from 1000 ppm Ag+ standard, and the corresponding potential in mV was recorded using an Ag+ ion selective electrode in conjunction with a multimeter. Each reading was obtained after 2.5 minutes of equilibrium time.
We prepared additional AgNO3 standards with 40 ppm Ag+, 100 ppm Ag+, 200 ppm Ag+, and 800 ppm Ag+, and added 0.5 ml of each of these standards to 9.5 ml of previously prepared 0.0526 wt % IAT EbNP suspensions to infuse the particles with Ag+ ions. To determine the Ag+ ion content adsorbed on the EbNPs, we first determined the residual Ag+ ions in the supernatant in each sample, and then closed the Ag+ ion balance to estimate the amount of Ag+ ions adsorbed on the particles. The ion content in the supernatant was determined with an Ag+ ion selective electrode (ISE).
Table 1 summarizes the Ag+ ion infused samples with various amounts of Ag+ ion loadings. The initial loading corresponds to the overall Ag+ ion content in the 10 ml sample at the time of infusion. The Ag+ ion content in the supernatant was calculated from the mV reading at the ISE with the following equation.
We measured a negative-potential for the Ag+ infused IAT EbNPs.
We modeled the Ag+ ion adsorption equilibria with a Langmuir adsorption isotherm and normalized the Ag+ uptake capabilities per surface area. The Langmuir adsorption isotherm is described by the following equation:
We determined a maximal adsorption Γmax=8688 ppm Ag+/m2 particle surface area, and a K value of 0.000115 in
To allow electrostatic attraction between negatively charged bacteria in aqueous solution and the Ag+ ion functionalized EbNPs, we reversed the surface charge of Ag+ infused EbNPs from negative to positive. We modified the surface properties of the EbNP system through adsorption of PDADMAC, a positively charged polyelectrolyte. To find a suitable PDADMAC concentration for the EbNP surface modification, we prepared samples with the initial PDADMAC concentrations reported in Table 2. The particles were coated in 5 ml batches of IAT EbNP 0.05 wt % suspension. For the surface modification step, 5 ml of polyelectrolyte solution with the previously reported wt % was added rapidly to the IAT EbNP suspension. The final concentration of IAT in the sample was 0.025 wt %. To investigate the stability and the change of properties of the coated samples, we measured the z-averages and the ξ-potential with DLS.
The magnitude of the positive surface potential, obtained after coating the IAT EbNPs, is dependent on the polyelectrolyte used. Similarly to the samples with PDADMAC coating reported previously, polyallylamine hydrochloride (PAH) coated IAT EbNPs were synthesized to prove the possibility to customize the surface charge magnitude by choice of suitable polyelectrolytes. The z-averages and ξ-potentials were measured and are reported in Table 3. The corresponding trends are illustrated in
A suitable sample obtained was Ag-EbNPs-PDADMAC (d=72 nm) with a final IAT EbNP concentration of 0.025 wt %, an Ag+ ion content on the particles of 0.71 ppm, an Ag+ ion amount in the supernatant of 1.79 ppm, and a PDADMAC concentration of 0.01 wt % in the colloidal suspension. The surface potential was reversed from −25.0 mV to +32.4 mV with the addition of PDADMAC −0.01 wt % in the final sample. The final sample pH was 5.5. Other samples were prepared accordingly.
We compared the antimicrobial activity of Ag-EbNPs-PDADMAC with that of positively charged branched polyethylene imine AgNPs (BPEI AgNPs) and AgNO3 solutions (see supplemental information for BPEI AgNP and AgNO3 sample preparations). We performed quantitative antimicrobial tests on Gram-negative E. coli BL21 (DE3), a common human pathogen, and qualitative tests on Gram-negative P. aeruginosa, a human pathogen not susceptible to antimicrobial amines such as BPEI and PDADMAC. Therefore, any antimicrobial activity in the P. aeruginosa tests will predominantly stem from silver.
The activity of each active agent was determined by comparing the number of colony forming units (CFU) of a reference plate with the CFU of a test plate as depicted in
The maximum antimicrobial reduction efficiency of 100% was reached when no CFU could be determined on the test plate. We quantified by the antimicrobial reduction efficiency “E” with the following equation
The schematic in
Table 4 compare the quantitative antimicrobial efficiency of each active agent in the E. coli tests. The reduction efficiency of six different samples with increasing Ag ppm equivalent ranging from 0 ppm to 54 ppm was investigated. The graphs show the reduction efficiency at two time points, 1 minute and 30 minutes. The weight percentages of the control samples and the silver contents in the active agents were chosen to show antimicrobial thresholds and to facilitate comparisons between the samples. Native IAT EbNPs without Ag+ functionalization and surface modification did not result in any observable reduction in CFU (not reported), which suggests that the native IAT EbNPs are benign. Also, IAT EbNPs with Ag+ functionalization but without PDADMAC coating did not result in significant reduction of CFU after 1 minute (0%) and 30 minutes (5%). We suggest that the low antimicrobial efficiency may be attributed to the negative surface charge of these EbNPs, which may hinder them from overcoming the electrostatic barrier between the particles and the bacteria. IAT EbNPs coated with PDADMAC resulted in strong reduction of CFU after an exposure time of 30 minutes, which may be attributed to the antimicrobial effect of the quarterly amine PDADMAC. PDADMAC solution alone (not reported) exhibited strong bactericidal effects towards E. coli as well, comparable to the efficiency of Ag-EbNPs-PDADMAC or 100% after 30 minutes exposure time. Ag-EbNPs-PDADMAC exhibited strong reduction in CFU, prevalent after 1 minute exposure time. The corresponding supernatant of Ag-EbNPs-PDADMAC exhibited no observable effect after 1 minute. The reduction of CFU in the supernatant after 30 minutes exposure time may be explained by residue active agent in the solution. BPEI AgNPs and AgNO3 solutions exhibited antimicrobial effects at 20 ppm Ag and 40 ppm Ag respectively. Overall, the Ag-EbNPs-PDADMAC sample outperformed the BPEI AgNPs and AgNO3 samples in terms of antimicrobial efficiency normalized on Ag ppm equivalent.
As mentioned previously, the qualitative antimicrobial test on P. aeruginosa can distinguish the antimicrobial effect of PDADMAC from the effect of silver.
Pseudomonas
aeruginosa
Pseudomonas
aeruginosa
We developed a new class of nanomaterials with increased efficiency and potentially improved nanoparticle post-utilization safety. Functionalized environmentally benign nanoparticles (EbNPs) exhibit locally confined and temporarily limited bioactivity. Other than their persistent counterparts, they are predominately made from biodegradable and sustainable materials, and are synthesized via green chemistry. As these EbNPs may lose their activity due to depletion of agent, dissolution of the EbNP system, or degradation of the lignin-based matrix by the environment, they can minimize any potential nanomaterial waste hazards. In addition to the beneficial post-utilization performance, EbNPs may deliver higher efficiency in terms of active agent employed in comparison to persistent nanoparticle system. In biocidal tests on the human pathogens E. coli and P. aeruginosa, we proved that silver ion infused EbNPs with positive surface charge (Ag-EbNP-PDADMAC) exhibit significantly higher antimicrobial activities in terms of Ag equivalent than silver nanoparticles. The increased efficiency of EbNPs with functional equivalent to their persistent counterparts, may favor substitution of a wide range of applied metal nanoparticles. Moreover, the benign nature of f-EbNPs opens opportunities for new applications of nanoparticles in the agriculture, home and personal care, and pharmaceutical industry.
DLS (Malvern Instruments Ltd., Nano ZS, λ=633 nm, max. 5 mW)
Syringe pump (New Area Pump Systems, NE-4000)
UV-Vis spectrometer (Jasco UV/Vis V-550 spectrophotometer)
UV lamp (Uvitron, Sunray 400SM)
Multimeter (Mettler Toledo, S80)
Materials and Chemicals Used in EbNP Synthesis.
Lignin.
We obtained INDULIN AT lignin (IAT) powder (lot MB05) and supporting documentation from MeadWestVaco (MWV) Charleston, S.C. We estimated the distribution of the main functional groups per 100 aromatic units according to the literature provided by MWV, and assigned pKa values from tables. We obtained High Purity Lignin (HPL) powder and supporting documentation from Lignol Burnaby, BC, Canada, and assigned pKa values to its functional groups accordingly. Table 6.
Millipore water (Synergy UV); acetone (BDH, CAS#67-64-1, lot 010612B); HNO3 (Sigma Aldrich, CAS#7697-37-2, lot A0294591); ethylene glycol (Sigma Aldrich, CAS#107-21-1, grade 99+%, lot B0521395); 0.45 μm syringe filter (Thermo Scientific, nylon syringe filter 0.45 μm); magnetic stir bar (Fisher Scientific, 8-Agon stir bar 14-512-147).
HPL EbNP Synthesis.
The ξ-potential was measured with a Malvern disposable capillary cell DTS 1061. The following measuring settings were used: the solvent was H2O with 10% (v/v) acetone with an overall viscosity of 1.0684 cP. The effective voltage was 150 V.
IAT EbNP Synthesis.
The ξ-potential was measured with a Malvern disposable capillary cell DTS1061 in the size control and pH-stability studies. The following measuring settings were used: the solvent was H2O with 10% (v/v) ethylene glycol with an overall viscosity of 1.1932 cP. The effective voltage was 150 V. The ξ-potential was measured with a Malvern dip cell ZEN1002 in the ionic strength study. The dip cell allows ξ-potential analysis with low driving voltages. The following measuring settings were used: the solvent was H2O. The voltage was automatically adjusted by the equipment and chosen at values below 5.0 V for all measurements.
IAT EbNP Functionalization with Ag+ Ions.
Ag+ standard (Mettler Toledo, silver ISE standard 1000 ppm 51344770, lot ISEAG510L1); Ag+ ion selective electrode (Mettler Toledo, silver/sulfur electrode 51302822, reference filling solution C 51344752).
Reference Samples BPEI AgNPs and AgNO3 Solution.
Positively charged branched polyethylene imine (BPEI, Sigma Aldrich, Mw ˜25000 by LS, CAS#9002-98-6, lot MKB64206V) coated AgNPs with a z-average diameter of 20 nm were synthesized according to the literature.29 The molar ratio of the final AgNP solution was chosen to be 0.5 mM BPEI: 0.5 mM AgNO3 (Fisher Chemicals, CAS#7761-88-8, lot 016932): 0.1 mM HEPES buffer (Sigma Aldrich, CAS#7365-45-9, lot 98H5425). 10 ml of the mixture was exposed to UV light for 120 minutes to form BPEI capped AgNPs with 54 ppm silver equivalent. The z-average diameter was determined with DLS. The pH value of the final solution was 6.3. AgNO3 solutions were prepared from a 1000 ppm Ag+ reference standard. The target ppm concentrations for antimicrobial testing were reached by appropriately diluting the reference standard with Millipore water.
Media Used in Antimicrobial Testing.
PBS buffer (Sigma Aldrich, CAS#7778-77-0, lot 38H8503), LB ager (Fischer Chemicals, CAS#9002-18-0), LB broth (Acros 61187-5000, lot B012260G).
As depicted in
The invention can be used as a platform technology for versatile synthesis of functionalized EbNPs, in achieving functionality and efficiency, in formulation, and in applications, and environmental safety and biodegradability at the same time. These EbNPs are systemized from natural materials available in abundance, potentially from waste or bio-products streams, by employing simple processes using green or no organic solvents. In addition to their safety, efficiency, and cost advantages, the benign nature of the new EbNPs opens multiple utilization opportunities in markets closed for persistent nanoparticles. The advantageous features of the invention include:
Functionalized EbNPs consisting of a core, one or more functionalizing agents, and one or more surface modifiers, whose details are outlined in the definition of terms, could be based on the following combination of materials. The EbNP core consists mainly of biopolymers or a combination of biopolymers from the group of modified lignin, modified cellulose, hemi or lingo cellulose, modified chitosan, modified chitin, prolamines, and gluten. Most preferable lignins are modified lignins extracted via Kraft pulping process including sulfonated (INDULIN C lignin) and unsulfonated lignin (INDULIN AT lignin) from MeadWestvaco and others, and extracted via organosolv process such as High Purified Lignin (HP-L™) from Lignol. Preferable modified lignins include Kraft pulping lignin extracted via Lignoboost process from Metso, and their derivatives. Most preferable modified celluloses include hydroxyl propyl methyl cellulose phthalate (e.g., HP-55 or HP-55S from Shin-Etsu), hypromellose acetate succinate (e.g., AS-HF from Shin-Etsu), and their derivatives. Other preferable modified celluloses include methyl cellulose, ethyl cellulose, cellulose acetate, hydroxyethyl cellulose, cellulose nitrate, cellulose propionate, and their derivatives. Most preferable modified chitosans include low molecular weight (Mw) chitosans, and their derivatives including chemically modified chitosan with amino groups in the backbone of chitin. Preferable chitosans include medium and high Mw chitosan and their derivatives. The active agent may be any biologically active component. This includes most preferably monovalent and divalent cationic metal ions including biocidal Ag+ and Cu2+, biocidal semiconductor compounds such as ZnO and TiO2, and natural and synthetic pesticides. Preferable active agents are amines, biopolymers with biocide activity including low Mw chitosan with amino groups, and synthetic polymers including negatively and positively charged polyelectrolytes. Other possible functionalization agents include the group of hydrophilic and hydrophobic pharmaceuticals, food additives, proteins, peptides, and others. Surface property modifiers are surfactants that include synthetic polymers and biopolymers as defined previously. Most preferably are positively charged biocidal amines such as polydiallyldimethylammonium chloride (PDADMAC) or poyallylamine hydrochloride (PAH), and oils and silicon based surfactants used as pesticide transfer and targeting agents. Other groups of surface modifiers include positively and negatively charged polyelectrolytes for surface charge modification, non-ionic surfactants, protein based surfactants, emulsifiers, and polysaccharides.
Negatively charged hydrophobic EbNPs are synthesized via one of the four suggested green synthesis routes with mean diameters most preferably in the range of 20 to 100 nm. Preferable EbNPs from synthesis may also result in bigger particles with mean diameters typically up to 500 nm, or more. The procedures include the water-water based pH-drop method, the solvent-water based pH-drop method, solvent-antisolvent method, and the polyelectrolyte-addition method. Taking lignin as input material, INDULIN AT lignin (IAT) and HP-L™ nanoparticles have been synthesized by the previously mentioned procedures. Table 7 compares the advantages and limitations of each method. In the water-water based pH-drop method, supersaturation and subsequent nanoparticle formation are achieved upon addition of acid to dissolved lignin in water at elevated pH to drop the pH into the range of pH 1.5 to 3.0. The pH stability can be increased upon adsorption of positively charged polyelectrolytes most preferably with PDADMAC, PAH, and others on the negatively charged EbNP surface. In the organic solvent-water based pH drop method, lignin is first dissolved in organic solvent such as ethylene glycol, toluene, or similar. Supersaturation is reached upon addition of acid precipitating out negatively charged hydrophobic EbNPs. The EbNPs formed in organic media may be transferred into water via dialysis or dilution. In the solvent-antisolvent method, biopolymer is dissolved in solvent such as acetone or ethanol. Supersaturation is reached upon rapid addition of antisolvent such as water precipitating out negatively charged hydrophobic EbNPs. In the polyelectrolyte-addition method, biopolymer is dissolved in solvent, typically water at adjusted pH. EbNPs are formed upon addition of positively charged polyelectrolytes. In comparison to prior art, each of the four green synthesis routes is performed at room temperature without crosslinking reaction. This differs significantly from the methods reported in patent literature in which chemically modified cross-linked lignin nanoparticles were synthesized at elevated temperatures32, 33. While chemically modified lignin nanoparticles may not biodegrade as easily, other advantages of the green synthesis methods described include utilization of inexpensive materials, low hazard potential, room temperature operations and therefore no need for external energy input or cooling, size control, scalability, and short synthesis times from prepared stock solutions to synthesized EbNPs in the minute range. According to the advantages outlined, the EbNP synthesis costs are low.
The second part of the invention includes nanoparticle functionalization in order to infuse the matrix with an active ingredient or otherwise create the desired usage characteristics. Functionalization methods of the EbNP carrier include infusion, and absorption and/or physical and chemical adsorption of active agent. Both weak and strong binding of the active agent are possible mechanisms involved in the functionalization. This binding of the agent can occur because of electrostatic interaction, hydrophobic or hydrophilic interactions, reduction processes, chemical linking, kinetic and entropy driven capture of the functional molecules. In comparison to persistent nanoparticle systems compromised of the active agent alone, the functionalized EbNP technology suggests higher efficiency in terms of optimized smaller amount of active agent used to deliver the same functionality ultimately minimizing risks and hazards stemming from excess active agent.
The adjustment and customization of the surface properties is used to replicate and enhance the particle properties needed for its functionality, and can be achieved by introducing one or more modifiers on the EbNP surface. Depending on the surface modifier chosen, the binding strength and the adsorption processes can vary accordingly. Surface properties that are controlled on this stage include surface charge, pH stability, hydrophobicity, biocidal activity, and others. Changes in surface properties can specifically be performed for better particle targeting, to increase the shelf life of the system, to alter the colloidal stability, to modify the interaction potential with the environment or a specific target, to protect the active agent, to customize depletion and transport effects of active agent, and others.
Applications:
The EbNP systems suggested in the present invention can find applications in different areas of technology and industrial products. The key new element is that the functionalized EbNPs may be designed to exhibit locally confined and temporarily limited bioactivity, by delivering the same desired activity as permanent nanoparticles currently employed in various applications, but only during the time of their application. Since functionalized EbNPs can be engineered to have complete functional equivalency to a variety of permanent hazardous nanoparticles, EbNPs may therefore replace a wide range of metal or semiconductor nanoparticles employed at moderate temperatures.
Besides these multimillion dollar applications, the benign nature of the invention opens opportunities for its use in markets presently closed for persistent nanoparticles. New additional applications may be found in the multibillion pesticide, food, and drug industries. Applications of functionalized EbNPs are sectioned in immediate applications, new applications, and new applications with FDA approved EbNP matrix.
Immediate applications of these new particles include/invention can be employed as:
New applications include:
New therapeutic or nutraceutical applications (possibly for FDA-approved or other regulatory approval) of EbNPs described herein include:
Executive Summary.
One of the most widely used classes of nanomaterials today is the silver nanoparticles (AgNPs), which exhibit general antimicrobial, antisporal and antifungal activity, while being of low toxicity to humans. The application of Ag nanoparticles, however, has met a number of serious problems, due to their relatively large cost and the rapidly growing concerns about the environmental and human dangers by the persistent nanoparticles released post application. We have pioneered a set of new ideas that resulted in the demonstration of a novel class of functionalized, environmentally-benign, nanoparticles (EbNPs) as highly efficient microbicidal substitutes of the AgNPs (
Background.
Engineered nanoparticles are in use or being considered for use across a wide range of fields. However, lingering concerns about the potential health hazards of nanoparticles and their accumulation in the environment have limited the expansion of nanoparticle technology into many medical, environmental, and military applications. Silver nanoparticles (AgNPs) have emerged as the most widely employed persistent nanoparticles (PNPs), as their broad-spectrum antimicrobial properties allow them to combat bacteria strains exhibiting antibiotic resistance,11 which are reported in human pathogens including Escherichia coli (E. coli)12, Pseudomonas aeruginosa (P. aeruginosa)13, and others. Furthermore, AgNPs are effective agents against viruses (e.g. herpes), spores (e.g., anthrax), and fungi (e.g. Candida albicans)34. Such broad-spectrum biocidal agents can minimize the potential for nosocomial infections15 in applications including antimicrobial wound dressings, textiles,20 and water filters.21 However, toxicity studies on AgNPs in contact with skin, specifically in commercially available wound dressings, indicate that AgNP containing solutions exhibit stronger cytotoxic effects toward keratinocytes than do PNP free counterparts.23 In addition, in vitro studies on mammalian fibroblasts have revealed that AgNP can induce apoptosis.24, 25 In this context, AgNP may potentially affect human health.26
The environmentally benign nanoparticles (EbNPs) that we have developed address these safety concerns associated with nanosystems without sacrificing the powerful nano-scale functionality. Biopolymers such as lignin serve as suitable matrix for benign nanoparticle systems. Lignin is the most abundant aromatic polymer in nature,4 has an amorphous structure, and is biodegradable. Matrixes of INDULIN AT lignin (TAT), depicted in
We further proved that by infusing IAT EbNPs with functional metal ions, such as antimicrobial silver ions, and additional surface modification, such as switching the surface charge from negative to positive, it is possible to synthesize degradable nanoparticles that match the nanoparticle functionality of their respective PNPs, while increasing utilization and post-utilization safety. The schematic in
Antimicrobial Testing and Comparison of AgNPs with Silver-Infused EbNPs.
We compared the antimicrobial activity of Ag-EbNPs-PDADMAC with the one of positively charged branched polyethylene imine AgNPs (BPEI AgNPs) and AgNO3 solutions, We performed quantitative antimicrobial tests on Gram-negative E. coli BL21 (DE3), a common human pathogen, and qualitative tests on Gram-negative P. aeruginosa, a human pathogen not susceptible to antimicrobial amines such as BPEI and PDADMAC. Therefore, any antimicrobial activity in the P. aeruginosa tests will predominantly stem from silver. The testing procedure are reported in Section 1.
The quantitative antimicrobial efficiencies of each active agent in the E. coli tests are compared in
As mentioned above, the qualitative antimicrobial test on P. aeruginosa can distinguish the antimicrobial effect of PDADMAC from the effect of silver (
presents a comparison of the properties of common AgNPs with our novel silver-infused EbNPs.
Hypothesis of the Antibacterial Mechanism of Ag-EbNPs-PDADMAC.
The antibacterial effect of Ag-EbNPs-PDADMAC, is a combinatorial effect of the antimicrobial properties of Ag+ ions and of the quarternized amine PDADMAC. For the bacteria not susceptible for bactericidal amines such as P. aeruginosa,38, 39 the antimicrobial effect is based on the bactericidal activity of silver-ions. We suggest that the Ag+ ions are weakly bound to the EbNP binding sites and locally concentrated on the EbNP surface. These Ag+ ions may be surface active and could be released upon contact with a bacteria cell membrane. A possible mechanism of the antimicrobial Ag-EbNPs-PDADMAC activity is illustrated in
Additional data, examples and embodiments may be found in Appendix A attached to the specification of U.S. provisional patent application No. 61/776,274, filed Mar. 11, 2014, the benefit of which application is claimed by the present application, and the contents of which application are incorporated by reference herein in its entirety.
The article “a” and “an” are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object(s) of the article. By way of example, “an element” means one or more elements.
Throughout the specification the word “comprising,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. The present invention may suitably “comprise”, “consist of”, or “consist essentially of”, the steps, elements, and/or reagents described in the claims.
The following Examples further illustrate the invention and are not intended to limit the scope of the invention.
It is to be understood that, while the invention has been described in conjunction with the detailed description, thereof, the foregoing description is intended to illustrate and not limit the scope of the invention. Other aspects, advantages, and modifications of the invention are within the scope of the claims set forth below. All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.
This invention was made with government support under Grant No. 554,871 awarded by the U.S. Environmental Protection Agency. The government has certain rights in the invention.
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| Number | Date | Country | |
|---|---|---|---|
| 20140256545 A1 | Sep 2014 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61776274 | Mar 2013 | US |
