Antiviral Properties of Polymeric Aziridine- and Biguanide-Modified

Feb 7, 2012 - Poly(hexamethylene biguanide) (PHMBG) (Arch UK Biocides Ltd., Manchester, U.K.) was treated and characterized as reported previously.(5)...
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Antiviral Properties of Polymeric Aziridine- and Biguanide-Modified Core−Shell Magnetic Nanoparticles Lev Bromberg,† Daniel J. Bromberg,† T. Alan Hatton,*,† Isabel Bandín,‡ Angel Concheiro,§ and Carmen Alvarez-Lorenzo*,§ †

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States Departamento de Microbiología y Parasitología, Instituto de Acuicultura, Unidad Ictiopatología-Patología Viral, §Departamento de Farmacia y Tecnología Farmacéutica, Facultad de Farmacia, Universidad de Santiago de Compostela, 15872-Santiago de Compostela, Spain

‡

S Supporting Information *

ABSTRACT: Polycationic superparamagnetic nanoparticles (∼150−250 nm) were evaluated as virucidal agents. The particles possess a core−shell structure, with cores consisting of magnetite clusters and shells of functional silica covalently bound to poly(hexamethylene biguanide) (PHMBG), polyethyleneimine (PEI), or PEI terminated with aziridine moieties. Aziridine was conjugated to the PEI shell through cationic ring-opening polymerization. The nanometric core− shell particles functionalized with biguanide or aziridine moieties are able to bind and inactivate bacteriophage MS2, herpes simplex virus HSV-1, nonenveloped infectious pancreatic necrosis virus (IPNV), and enveloped viral hemorrhagic septicaemia virus (VHSV). The virus−particle complexes can be efficiently removed from the aqueous milieu by simple magnetocollection.

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INTRODUCTION Magnetic nanoparticles (NP) that interact with living cells have emerged as one of the nanotechnology platforms in selective cell targeting, cell separation, and isolation in fermentation processes, drug delivery and diagnostics.1,2 Magnetic parameters of the magnetite NP, such as saturation magnetization, coercivity, and magnetocrystalline anisotropy, combined with benign nature and rich surface chemistry afford capability to selectively aim and manipulate NPs in magnetic fields, which can be uniquely important in their biological applications. We recently advanced a concept of nonspecific, polycationic magnetite NPs that are capable of capturing and killing a broad range of bacteria.3−5 Such magnetic NPs loaded with biocidal substances and colloidally stabilized in water by polycationic shells offer a green and cost-effective approach to the eradication of pathogenic microorganisms from contaminated water sources, aqueous media, and soil. In addition, since recovery of such polycationic NPs, along with any microbial nucleic acids or whole germs that were captured on the NP surface, is readily achievable by means of a magnet, these particles may enable not only in situ biodefense but also the monitoring of the presence of dangerous germs in aqueous habitats.3 We demonstrated that magnetite NPs, surfacemodified by poly(hexamethylene biguanide) (PHMBG) moieties, efficiently bind lipopolysaccharide and glycopeptide components of the bacterial membranes as well as nucleic acids and whole bacteria, and possess broad-range bactericidal © 2012 American Chemical Society

activity. Importantly, NPs based on PHMBG as a bactericide were manyfold less toxic to mammalian cells than to the bacteria.5 In the present work, we extended our studies to the effects of polycationic magnetic particles on viruses. Mechanistic studies of interactions between viruses and particle surfaces is a recent area of research, which can elucidate existing methods and usher in novel methods of disinfection or virus separation and vaccine preparation.6−8 From the practical perspective, we were interested in the potential of magnetic NPs in addressing viral diseases in aquaculture as well as eradication of human diseases. Noting that aziridines and ethyleneimines of low molecular weight have been widely utilized in virus inactivation in the vaccine development,9−15 as chemotherapeutic agents and chemosterilants,16,17 and broad-range virucides for red blood cell concentrates,18,19 we set out to develop magnetite NP with aziridine on its surface, which would be capable of electrostatically binding the virus and its DNA, but also of virus inactivation. Novel core−shell magnetite particles with a shell containing a functional silane, 3-glycidoxypropyltrimethoxysilane,5 were thus prepared by polymerization of aziridine on the NP surface. The ability of nanometric magnetite-silica core− shell particles functionalized with aziridine (A-M/SiO2), Received: December 28, 2011 Revised: February 6, 2012 Published: February 7, 2012 4548

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Magnetite-Silica Core−Shell Particles Functionalized with Poly(Hexamethylene Biguanide) (PHMBG-M/SiO2). These were synthesized as described in our previous work.5 Elemental analysis, found (%): C, 27.8; H, 5.83; Fe,18.3; N, 19.7. Magnetite-Silica Core−Shell Particles Functionalized with Epoxy Groups. Synthetic route toward core−shell particles surface-modified with PEI-2 or aziridine groups involved prior synthesis of core−shell particles functionalized with epoxy groups. First, magnetite particles were prepared, which were well-dispersed in water with the aid of tetramethylammonium hydroxide (TMAOH). In the second step, the magnetic particles were encapsulated by a functional shell comprising tetraethyl orthosilicate (TEOS) and epoxy-functional 3-glycidoxypropyltrimethoxysilane (GPTMS).5 The third step comprised either attachment of PEI-2 or growth of aziridine chains off the particle surface-bound epoxy groups by boron trifluoride etherate (BF3·Et2O)catalyzed polymerization in chloroform. Thus, FeCl3·6H20 (7.58 g, 28 mmol) and FeCl2·4H2O (2.78 g, 14 mmol) were dissolved in 25 mL DI water and the solution was brought to 80 °C under nitrogen purge within ∼30 min. The solution was poured into 25 mL of 30% NH4OH and the resultant black precipitate was stirred and kept at 80 °C for 1 h. The resulting particle suspension was sonicated for 1 min and separated from supernatant by magnetocollection. The particles were then placed into a tube containing 30 mL of 0.33 M aqueous solution of TMAOH. The suspension was observed to be stable. The suspension was separated by magnetocollection and washed by 50 mL of deionized water twice. The resulting TMAOH-stabilized magnetite suspension (∼25 mL) was diluted by 40 mL ethanol. To the resulting suspension, 3.6 mL (16 mmol) of TEOS were added and the suspension was sonicated for 5 min, followed by addition of 4.6 mL (20 mmol) of GPTMS. The suspension was kept under vigorous shaking at room temperature for 2 days and the particles were separated using magnetocollection, dialyzed against excess of deionized water (MWCO 12−14 kDa) overnight, snap-frozen, and lyophilized. The resulting epoxy-modified particles designated as M/ SiO2 were characterized by FTIR5 and TGA. Elemental analysis, found (%): C, 17.0; Fe, 24.1; N, 0.04. The M/SiO2 particles were stored at −20 °C prior to the use. Core−Shell Particle Modified with PEI-2 (PEI-M/SiO2). This was synthesized as follows. To the TMAOH-stabilized magnetite suspension (∼25 mL) prepared as described above, 40 mL of absolute ethanol was added and the diluted suspension was sonicated for 1 min. To the resulting suspension, 3.6 mL (16 mmol) of TEOS were added and the suspension was sonicated for 5 min, followed by addition of 4.6 mL (20 mmol) of GPTMS. The suspension was shaken (200 rpm) at room temperature for 1 h and aqueous solution of PEI-2 (5 g in 100 mL water) was added and the resulting mixture was shaken at r.t. for 1 h, kept at 80 °C for 1 h, and then shaken at r.t. for 16 h. The suspension was then dialyzed against excess deionized water (membrane MWCO, 12−14 kDa). The resulting suspension did not exhibit any visible sedimentation of particles for several days at rest. The resulting PEI-M/SiO2 particles were separated by magnetocollection, snap-frozen, and lyophilized. Elemental analysis, found (%): C, 43.5; Fe, 7.92; N, 21.1. A-M/SiO2 Particles. These were synthesized by suspending 110 mg of the epoxy-modified particles (M/SiO2) in 5 mL chloroform by a brief sonication, followed by addition of 5 mL aziridine solution in chloroform (aziridine: 190 μL; 3.66 mmol). The reaction vessel was brought to 0 °C and 10 μL (0.08 mmol) of boron trifluoride etherate was added. The reaction vessel was sealed, briefly vortexed, and shaken at room temperature for 48 h. Then the reaction was quenched by addition of TMAOH (20 μL); the products were immediately separated by magnetocollection, rinsed by methanol and acetone, chilled in liquid nitrogen, and dried under vacuum. Elemental analysis, found (%): C, 36.0; Fe, 12.1; N, 15.9. A-M/SiO2 particles were stored at −80 °C prior to the use. The formation of poly(ethylene imine) species on the particles was confirmed by dissolution of a small fraction of the particles in 1 M trifluoroacetic acid. The particle digestion was followed by MALDI-TOF analysis of 1.5 wt % aqueous solution of the dissolved species with an acceleration voltage optimized in the range from 5 to 20 kV.

PHMBG (PHMBG-M/SiO2), and/or PEI (PEI-M/SiO2) to remove and inactivate the bacteriophage MS2, the herpes simplex virus HSV-1, the nonenveloped infectious pancreatic necrosis virus (IPNV), and the enveloped viral hemorrhagic septicaemia virus (VHSV) was discovered.

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EXPERIMENTAL SECTION

Materials. Branched poly(ethylene imine) (PEI-1; nominal average molecular weight, 25 kDa) with a molar ratio of primary to secondary to tertiary amino groups of 1:2:1 was obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO). After dialysis in water (MWCO 12−14 kDa) and removal of lower molecular weight fractions, the Mw was 38 kDa and Mw/Mn = 1.55. Lower molecular weight, branched poly(ethylene imine) (PEI-2, Lupasol G20, nominal molecular weight, 1300 Da) was obtained from BASF (Ludwigshafen, Germany) as a 20 wt % aqueous solution. The solution was snap-frozen and lyophilized prior to the use. Poly(hexamethylene biguanide) (PHMBG) (Arch UK Biocides Ltd., Manchester, U.K.) was treated and characterized as reported previously.5 Monomeric ethylene imine (aziridine, ≥99.8%) was obtained from ChemService Inc. (West Chester, PA) and was stored at −20 °C prior to the use. FeCl3·6H2O (98%), FeCl2·4H2O (99%), tetraethyl orthosilicate (99%, TEOS), boron trifluoride diethyl etherate (98%), tetramethylammonium hydroxide (TMAOH, 25 wt % in methanol), 2,5-dihydroxybenzoic acid (DHB, ≥99.5%), 4-(4nitrobenzyl)pyridine (≥98.0%), 3,5-dimethoxy-4-hydroxycinnamic (sinapinic) acid (99%), and 3-glycidoxypropyl trimethoxysilane (97%, GPTMS) were all purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Other chemicals and solvents were obtained from commercial sources and were of the highest purity available. General Methods. Bruker Avance 400 spectrometer was used for NMR measurements, including DEPT experiments to establish the structure of the model conjugates. Dynamic light scattering experiments were performed with a Brookhaven BI-200SM light scattering system (Brookhaven Instruments Corporation, Austin, TX) at a measurement angle of 90°. Particles dispersed in aqueous media (pH adjusted by 1 M NaOH or HCl) were filtered with a 0.45 μm syringe filter prior to the DLS tests. The particles were dispersed with sonication in 10 mM KCl aqueous solution at approximately 0.05 wt % concentration, and the pH of the nanoparticle suspensions was adjusted by adding 1 M HCl or NaOH aqueous solutions. All ζpotential measurements with magnetic NPs were performed using a Brookhaven ZetaPALS ζ-potential analyzer. The reported ζ-potential values are averages of five measurements, each of which was obtained over 25 electrode cycles. ζ-Potential measurements with MS2 virus suspensions were conducted using a Zetasizer Nano Series and disposable ζ-cells equipped with electrodes and a folded capillary (Malvern Instruments Ltd., UK). Particle or MS2 suspensions in 10 mM KCl with pH adjusted to a desired value were sterile-filtered directly into the ζ-cells. Thermogravimetric analysis (TGA) was conducted using a Q5000IR thermogravimetric analyzer (TA Instruments, Inc.; New Castle, DE). Samples were subjected to heating scans (20 °C/min) in a temperature ramp mode. Saturation magnetization of samples weighing 3−4 mg was measured at 300 K over a 0 to 50 kOe range using a Magnetic Property Measurement System model MPMS-5S (Quantum Design, San Diego, CA). Elemental analysis measurements were performed using an Agilent 7500a Series ICP-MS. Mass spectrometry was performed with a Microflex LRF20 MALDI-TOF mass spectrometer (Bruker Daltonics, Billerica, MA) equipped with a MicroScout ion source and a 337 nm nitrogen laser. Magnetocollection experiments were conducted throughout this work using a NdFeB, 2 in. × 1/2 in. Ni-plated disk magnet (surface field: 3309 G; grade N52; pull force, 130 lbs; K&J Magnetics, Inc., Jamison, PA). Particle Synthesis and Characterization. Poly(hexamethylene biguanide)- and Polyethyleneimine-Modified Magnetite (PHMBG-PEI-M). These particles were synthesized using PEI-1 as described in detail previously.4 Elemental analysis, found (%), PHMBG-PEI-M: C, 29.0; H, 6.04; Fe, 36.6; N, 14.3. 4549

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Aziridine Content Measurement. The aziridine group contents on the surface of A-M/SiO2 and analogous PEI-M/SiO2 particles were assayed using the well-known capability of aziridine to react with 4-(4nitrobenzyl)pyridine, forming purple, orange, or red-colored alkylated products.20,21 UV/vis calibration curves (λmax, 600 nm) were developed in 0−10 μM aqueous solutions of aziridine (pH 4 to 5) as described by Epstein et al.20 Although aqueous suspensions of A-M/ SiO2 and PEI-M/SiO2 particles possessed insignificant electronic absorption at λ > 500 nm, the absorbance readings of the particles (affected by the particle scattering) prior to the aziridine assay reaction were used as blanks in aziridine concentration measurements. The PEI-M/SiO2 particles did not form any detectable colored products in the aziridine assay, whereas A-M/SiO2 particles did. Bacteriophage MS2 Suspension and Enumeration.22 The bacteriophage MS2 (ATCC 15597-B1) was propagated in Escherichia coli (ATCC 12435). A high-titer stock of MS2 bacteriophage was made by pipetting 5 mL of SM buffer (NaCl, 100 mM; MgSO4·7H2O, 8 mM; Tris-Cl, 50 mM; gelatin, 0.002 w/v%) onto a plaque assay plate showing confluent lysis. After the plate was slowly rocked for 40 min at 4 °C, the buffer was transferred to a centrifuge tube containing 100 μL of chloroform to inactivate residual bacteria. After the tube was centrifuged at 4000 g for 10 min, the supernatant phage suspension was removed, filtered through a 0.22 μm membrane filter, 50 μL of chloroform was added, and this stock suspension (approximately 1011 PFU/mL) was stored at 4 °C prior to the use. To enumerate the phage stock, a plaque assay was performed. Samples were serially diluted in SM buffer, mixed with host E. coli suspensions and finally plated onto prepoured LB agar plates (Sigma-Aldrich Chemical Co.). In the assay, the plaques, formed due to the inoculation of E. coli with MS2 at 37 °C for 16 h, were counted. Only the plates that exhibited from 20 to 300 plaques were used for calculation of MS2 concentration. Suspension tests were performed five times in duplicate. The reduction factor was calculated as RF = log(PFU/ mL)(initial inoculum) − log(PFU/mL)(after exposure), where PFU stands for plaque-forming units. Removal of MS2 Phage by Magnetic Nanoparticles.23 A suspension of nanoparticles of known concentration in 10 mM NaH2PO4 (20 mL, pH 7.3) was sterilized by autoclaving at 115 °C for 15 min. Dilution of the particles was performed from a freshly dispersed stock suspension that was sonicated for 30 s using a Branson 450 Sonifier (Branson Ultrasonics Corporation, Danbury, CT). Upon equilibration at 25 °C, the pH was measured and readjusted by adding minute quantities of sterile solutions of NaOH or HCl. Virus was then added to the suspensions resulting in a titer of 2 × 106 or 2 × 107 PFU/mL, a sample was taken for virus titration and vigorous rotational shaking (∼250 rpm) commenced. Separate series of experiments were conducted at 37 °C, but otherwise identically. After 30 min, shaking was stopped, the particles were held to the bottom by magnetocollection, and samples were taken for titration of unadsorbed virus. Experiments were conducted in duplicate. The removal of the virions was characterized by the value Removed (%) = 100 × (Titer in the initial sample − Average titer after exposure)/(Titer in the initial sample) as well as by the reduction factor (RF) as defined above. Bacteriophage MS2 Inactivation. Inactivation of the MS2 virus by PHMBG solutions and PHMBG-M/SiO2 and PHMBG-PEI-M particle suspensions was tested as follows. The PHMBG and NP suspensions were prepared in aqueous 10 mM NaH2PO4, pH 7.3, with brief sonication, autoclaved at 115 °C for 15 min, and upon cooling to ambient temperature, the pH was adjusted to 7.3 with sterile 10 mM NaOH and HCl. Phage suspension (1 mL, 2 × 106 PFU/mL) was added to 9 mL of the tested solution or suspension and the mixture was briefly sonicated and then rocked at 200 rpm for 30 min at 25 °C. Magnetic particles were separated by magnetocollection. Then, 1 mL of the supernatant mixture was carefully withdrawn and added to 9 mL of neutralizer (6 w/v % Tween 80; 0.45 w/v % lecithin, and 0.1 w/v % L-histidine). After 2 min contact with the neutralizer, a sample was taken, serially diluted, and active bacteriophages enumerated with the plaque assay as above. In the control, PHMBG was replaced with sterile deionized water. The neutralizer has been previously shown to be nontoxic toward MS2 virus and its E. coli host, yet its use is

necessary to quench the activity of the dissolved PHMBG, to perform the plaque assay properly.22,24 The reduction factor after inactivation was calculated as RFi = log(PFU/mL)(after exposure) − log(PFU/ mL)(after phage separation and particle or PHMBG neutralization). In the RFi calculation, the titer values were adjusted for dilution. Kinetics of the MS2 virus inactivation by A-M/SiO2 and PEI-M/ SiO2 particle suspensions was tested as described previously.25 Namely, freshly prepared suspension of Aziridine-M/SiO2 particles in 0.15 M NaCl was diluted with the 0.2 M sodium 3-[Nmorpholino]propanesulfonate (MOPS) buffer (pH 7.0) to the required concentration and the calculated volume of the phage suspension with the original titer of 1011 PFU per mL was added. The mixture was then incubated at room temperature or at 37 °C for the desired time. Aliquots of the mixture were withdrawn intermittently; the withdrawn samples were immediately diluted 10-to-100-fold with 0.15 M NaCl. Then serial dilutions were prepared and the infectivity titer of the inactivated suspension was determined. The enumeration of viable MS2 bacteriophage counts was conducted in SM buffer as described above. Standard deviations of the rate constants were calculated from the results of three independent experiments. Analysis of Particle-Adsorbed MS2 Protein via Acid Digestion.26 A suspension of bacteriophage MS2 (initial titer, 1 × 1011 PFU/mL) and 1 mg/mL A-M/SiO2 was prepared and 7−8 logs of virus inactivation by exposure to A-M/SiO2 was confirmed. A 300 μL aliquot of the suspension was mixed with 300 μL of 50% acetic acid (AA), 10 μL of trifluoroacetic acid, and 50 μL of 1% Triton X-100 in a 10 mL Pyrex glass sample holder and briefly sonicated. Then, 1 μL of saturated sinapinic acid in 50%/50% (v/v) acetonitrile/deionized water containing 1% trifluoroacetic acid was applied to the sample plate and allowed to air-dry. Then, 1.0 μL of sample mixture was placed onto an MSP AnchorChip 600/96 target plate (Bruker Daltonics) and allowed to air-dry. In a final step, 1.0 μL of saturated sinapinic acid solution was added and allowed to air-dry. Acceleration voltage was optimized within varying m/z ranges. Spectra were acquired in the linear mode for positive ions and calibrated externally using the molecular ions of insulin ([M + H]+av m/z = 5735), cytochrome c ([M + H]+av m/z = 12 362), and apomyoglobin ([M + H]+av m/z = 16 952). In the control experiments with 1 mg/mL A-M/ SiO2 particles using DLS, it was shown that under the acidic conditions (50% acetic/3% trifluoroacetic acid/1% Triton X-100), over 90% of the particles dissolved, with the weight-average hydrodynamic diameter declining from approximately 210 nm prior to the digestion to ∼12 nm after the digestion. Simplex Herpes Deactivation.27 Monolayers of African monkey kidney cells (Vero, ATCC CCL-26) were grown in polymer tissue culture flasks (Corning, NY) in RPMI-1640 complete medium (Sigma-Aldrich Chemical Co.) supplemented with 10% heatinactivated fetal bovine serum, L-glutamine (2 mM), HEPES buffer (10 mM) and MEM sodium pyruvate (1 mM, Invitrogen Co.). Penicillin G sodium (100 U/mL), and streptomycin sulfate (100 mg/ mL) were added. Cells were incubated at 37 °C in a humidified atmosphere with 5% CO2. Vero cells were inoculated with 1 × 106/mL of HSV-1 (herpes simplex virus, MacIntyre strain, ATCC VR-539). The Vero cells were allowed to adsorb with HSV-1 for 1 h at 37 °C with gentle shaking and the supernatant from the flasks was discarded. HSV-1 adsorbed cells were then covered with fresh RPMI-1640 medium at 5% CO2 and incubated at 37 °C for 5 days and examined for plaque formation. The flasks were then freeze−thawed 3 times to disrupt the cells and centrifuged at 300 g for 15 min. The supernatant was collected and stored in 1 mL aliquots at −80 °C. Subsequently, a virus titration assay was performed from these stock cultures as follows. Monolayers of Vero cells on 6.0 × 1.5 cm Petri dishes, in triplicate, were overlaid with 1 mL of the HSV-1 suspensions to be titrated. A Petri dish with monolayer of uninfected Vero cells was included as a negative control. The cells were allowed to adsorb with HSV-1 for 1 h at 37 °C with gentle rocking. The supernatant fluids were aspirated from the dishes. The plates were overlaid with RPMI1640 buffer containing 1% methyl cellulose (MW 88 kDa, SigmaAldrich Chemical Co.). The plates were incubated undisturbed at 37 °C with 5% CO2 and examined daily for 5 days. Supernatant fluids 4550

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was multiplied by 0.7. The reduction factor was calculated as RF = log(PFU/mL)(initial inoculum) − log(PFU/mL)(after exposure), where PFU stands for plaque-forming units. The RF(1) and RF(2) denote RF values obtained in the method involving dilution into the medium containing magnetic particles and the method involving first separating the NP and the virions following supernatant dilution, respectively.

were aspirated from the plates at the end of 5 days and plaques were counted as clear zones within the monolayer of cells. The cytotoxicity of NPs and PHMBG solutions on Vero cells was measured by MTT assay.28 Particle suspensions or PHMBG solutions of known concentrations in RPMI-1640 medium (50 μL) were syringed upon a 3-day-old monolayer of Vero cells grown in 24 well microwell plate. After 1 h of incubation, the supernatant was replaced with fresh RPMI-1640 buffer. Then, 10 μL (per 100 μL of cells) of aqueous solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye (MTT, 5 g/L), was added to the cells and the plates were incubated for 4 h. The plates were read on a plate reader and the mean readout value for each set of test conditions was compared as a percentage of the untreated control cells, which represented 100% metabolic activity. NP suspensions and PHMBG solutions of known concentrations in RPMI-1640 medium were tested for antiviral activity as follows. The suspension or solution to be tested (50 μL) was mixed with HSV-1 suspensions (50 μL, 3 × 105 virus/mL). Each mixture was incubated for 30 s at room temperature and diluted with RPMI-1640 medium (900 μL). The test samples of the suspension (briefly sonicated just prior to the use) or solution (1 mL) were added upon 3-day-old monolayers of Vero cells on 6.0 × 1.5 cm Petri dishes in triplicate. The Petri dishes were shaken gently at 60 rpm and the HSV-1 was allowed to adsorb onto the Vero cells for 1 h at 37 °C and 5% CO2. The supernatant was then carefully aspirated and the Petri dishes were overlaid with 5 mL of 1% methyl cellulose in RPMI-1640 CM and incubated for 5 days at 37 °C with 5% CO2 and above 100% humidity. The development of plaques was observed after 5 days. A minimum suspension or solution concentration that completely suppressed formation of plaques (minimum suppression concentration) was thus determined. Inhibitory Effect of Magnetic NP on Fish Viral Pathogens. Infectious pancreatic necrosis virus (IPNV), C2 strain, a nonenveloped, dsRNA-containing aquabirnavirus, was obtained from the American Type Culture Collection. Hemorrhagic septicaemia virus (VHSV), strain Fr07/71, a ssRNA-containing, enveloped rhabdovirus was isolated from trout in France. IPNV and VHSV were propagated in Chinook salmon embryo cells (CHSE-214) and in epithelioma papulosum cyprinid (EPC) cells, respectively, both of which were obtained from the European Collection of Cell Cultures (ECACC, Health Protection Agency, UK). Each virus inoculum (200 μL) was mixed with an equal volume of NP suspension at varying concentrations (A-M/SiO2, at 0.1 mg/mL, PHMBG-PEI-M, 0.2 mg/ mL or 0.4 mg/mL, and PHMBG-M/SiO2, at 0.4 mg/mL). The following two experimental approaches to evaluation of the inhibitory effects of NP were adapted. In the first set of experiments, following incubation of the virus and NP suspensions for 5 min at room temperature, the mixture was subjected to serial 10-fold dilutions in a medium containing magnetic particles at the concentrations cited above. Then, the magnetic particles were removed by centrifugation and magnetocollection and the viral dilutions were inoculated onto semiconfluent monolayers of CHSE-214 (for IPNV) or EPC (for VHSV) in 48-well plates (100 μL per well). Adsorption of the virus was allowed for 1 h at room temperature with gentle shaking, followed by supernatant removal and addition of fresh cell growth medium. The inoculated monolayers were incubated for 1 week at 15 °C and visualized daily for development of cytopathic effect (CPE). In a second set of experiments, the incubated virus and NP suspensions were first separated by magnetocollection, and then the virus-containing supernatant was serially diluted in the cell growth medium. The initial concentration of PHMBG-PEI-M NP was set at 0.4 mg/mL, and incubation times of 0.5, 1, 2, and 3 h were tested. Inoculation in cell cultures was performed as described above. Viral titers were determined using the tissue culture infectious dose (TCID50 per mL) method, calculated according to Reed and Müench,29 and expressed as the highest dilution of the mixture which produced CPE in 50% of the inoculated wells. Untreated virus suspensions were used as controls. To express the mean virus concentration value number of PFU/mL, the TCID50 per mL value

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RESULTS AND DISCUSSION Particle Characterization. In this work, we present functionalized, polycationic, and paramagnetic nanoparticles as a means of manipulation and inactivation of the virus in aqueous milieu. Polycationic particles modified with PEI and/ or PHMBG, including core−shell particles structured as ∼150 nm clusters of primary magnetite particles (∼5 nm each) fused together and encapsulated with silica shells have been described in our previous works3−5 and are used herein to test their interactions with virions. The magnetite-containing NPs were superparamagnetic, i.e., exhibited no residual magnetism upon removal of the magnetic field. For details of the synthesis and characterization, see the Experimental Section. The structures of these particles are depicted schematically in Figure 1, and the size, polymer content, and saturation magnetization of all particles species are collected in Table 1.

Figure 1. Schematic of functionalized paramagnetic particles of the present study.3−5

Core−shell particles modified by aziridine (A-M/SiO2) are introduced herein for the first time, and thus, their design, synthesis, and characterization are described in detail. The AM/SiO2 particles are compared with their PEI-M/SiO 2 counterparts, which are modified with branched PEI-2 and are of a very similar structure overall, except being devoid of aziridine groups. To obtain the core−shell particles surfacemodified by aziridine group, we used a synthetic route with two principal steps. First, magnetic particles were obtained with a 4551

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Table 1. Properties of the Functionalized NP of the Present Study particle species PHMBGPEI-M PHMBGM/SiO2 PEI-M/ SiO2 A-M/SiO2

number-average hydrodynamic diameter (nm)

polymer content (wt %)

saturation magnetization (emu/g of magnetite)

120 ± 24

55−63

40−65

160 ± 11

55−60

60−69

240 ± 16

57−60

80−90

190 ± 12

59

90

Figure 3. MALDI-TOF spectrum of the polyethyleneimine species obtained by dissolution of A-M/SiO2 particles in 1 M trifluoroacetic acid followed by dilution with water to 1.5 wt % concentration. For the sample preparation, 1 μL of the analyte solution was mixed with 1 μL of 10 mg/mL methanolic solution of 2,5-dihydroxybenzoic acid (DHB); 1 μL of the resulting mixture was applied to the target using the dried-droplet method.

shell consisting of TEOS and epoxy-functional GPTMS that underwent a polycondensation reaction using a modified Stö ber method,5 and then the aziridine monomer was polymerized off the glycidoxy groups on the particle surface in the presence of boron trifluoride etherate (Figure 2).

TOF analytic techniques optimal for PEI have been described recently.31 Series of individual signals were ordered in a Gaussian-like curve characteristic of polymers. The spectra were well-resolved, showing singly charged ions with m/z signals corresponding to protonated oligoethyleneimine species separated by one CH2−CH2−NH repeat unit (43 Da). The main ion series appears at m/z = 43n + 18, wherein the term n represents the number of repeat units. The distribution of the observed oligomers stretches from the trimer ion at m/z = 147 to the 30-mer ion at m/z 1308. This series consists of protonated PEI oligomers [H2N−(CH2CH2NH)n−H+H]+ or [PEI+H]+, with NH2 end groups. This result is analogous to the MALDI of branched PEI (MW∼800 Da) by acid-catalyzed polymerization of aziridine.31,32 Doubly charged ions with the composition [H2N−(CH2CH2NH)n−H+2H]2+ appearing at every m/z ∼ 43n/2 are also quite apparent in the MALDI spectrum, giving a prominent peak for the ethyleneimine hexamer (m/z = 258) (Figure 3). A low abundance ion series, probably resulting from the fragmentation of the main distribution (m/z = 43n + 18) is observed at m/z = 43n + 1. Fully and partially hydrolyzed products of GPTMS acid hydrolysis, corresponding to [M+H] + ions of (3hydroxypropyl)silanetriol (m/z = 138) and (3-hydroxypropyl)(methoxy) silanediol (m/z = 154), respectively, were also found. Since the superparamagnetic nature of our particles prohibits their study by conventional NMR, we conducted a model reaction between GPTMS and aziridine (S-1) without paramagnetic materials, with the purpose of elucidating the structure of the GPTMS-PEI-aziridine conjugates, as follows. GPTMS (118 mg, 0.5 mmol) and aziridine (86 mg, 2 mmol) were dissolved in 3 mL CDCl3 and the reaction mixture was chilled to 0 °C. Then, 5 μL of boron trifluoride etherate was added and the reaction vessel was gently shaken sealed at room temperature for 24 h. NMR spectra of the reaction mixture (S2) confirmed (i) the presence of residual aziridine groups in the resulting conjugate and (ii) the presence of the aminopropanol links between the GPTMS residues and the PEI chains forming via polymerization of aziridine (signals at 3.58 and 3.74 ppm). In summary, we obtained core−shell paramagnetic particles with a significant content of aziridine groups covalently attached to the PEI termini tethered to the silica shell of the core−shell paramagnetic particles. The A-M/SiO2 particles kept in water at pH 7.4 at room temperature maintained their saturation magnetization (∼90 emu/g of magnetite) for at least

Figure 2. Modification of GPTMS-functionalized core−shell particles (M/SiO2) by polymerization of aziridine.

Cationic ring-opening polymerization of the aziridines catalyzed by Lewis acids such as boron trifluoride etherate is wellknown.30 The reaction was terminated by the addition of tetramethylammonium hydroxide, leaving reactive aziridine groups on the particle surface. Beyond measuring the composition of the A-M/SiO2 particles by elemental analysis and TGA, we analyzed their aziridine content, which was found to be 0.7 mmol/g of dry, freshly made material. The assay was repeated on 2 mg/mL aqueous particle suspensions stored at −20, 0, and 25 °C. The particles retained 100% of the initial aziridine content for approximately 3 days, 10 days, and 1 year at 25, 0, and −20 °C, respectively. Such retention of the aziridine groups is sufficient for potential applications of the particles such as eradication of wild poliovirus. While biguanide-based NPs did not show toxicity to mammalian cells in our experiments (see below), the aziridine-modified particles may be toxic. However, decay in the aziridine group concentration at ambient temperature within days (over 60% within 12 days) can be advantageous if the particles were to remain in the environment. In the virus deactivation studies herein, we used freshly prepared particles. The polymeric species (PEI) resulting from the polymerization of aziridine on the surface of the A-M/SiO2 NP was further characterized by dissolving the particles with the aid of trifluoroacetic acid followed by MALDI mass spectroscopy (Figure 3). 2,5-Dihydroxybenzoic acid (DHB) without salt addition proved to be the most suitable matrix for the PEI species that was found on the A-M/SiO2 NPs. Similar MALDI4552

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one year. It should be also noted that the saturation magnetization did not change as a result of the autoclaving. We have also tested that the particles are stable for hours in ocean water at ambient temperature. Interactions between Virions and Paramagnetic Particles. The presence of lipids and the virion size are key parameters for the virucidal efficiency of a biocide.33,34 The lipid envelope in a virus is associated with a high degree of susceptibility to interactions with disinfectants; the absence of lipid and small size are responsible for the resistance to lipophilic chemical agents. Consequently, interactions of several viruses, both enveloped and nonenveloped, were investigated herein. The present work focused on the binding and capture of several representative viruses with four types of polycationic magnetic particles, which contained biguanide, amine, and/or aziridine groups on their surfaces. Namely, herpes simplex virus type 1 (HSV-1) and MS2, a RNA bacteriophage belonging to the Leviviridae family, were studied along with testing susceptibility of the viruses relevant for aquaculture. Bacteriophage MS2 was examined extensively in the present work, as it is often used as a model virus for studies of virus−substrate interactions due to its availability in high titer in a purified form; the ease, speed, and accuracy with which viable virus can be titrated; and because of its similarity in size, structure, and chemical composition to the human-pathogenic enteroviruses such as poliovirus.22,24,35,36 The MS virus particle is sized 22− 29 nm and possesses a pI of 3.1.35 The full virion consists of a ∼27 nm protein capsid surrounding an RNA genome of 3569 nucleotides.37 The second virus of the present study, HSV-1 virion, is a large (hydrodynamic diameter, ∼180 nm)38 enveloped virus. Herpes viruses infect a wide spectrum of species ranging from oysters to man, with a trait of establishing a latent infection within their hosts. The HSV-1 possesses 120− 300 kbp DNA genome enclosed by an icosahedral capsid that is surrounded by a tegument layer and a lipid-bilayer envelope.8 For infection, HSV-1 fuses its lipid membrane with the lipid membrane of the cell. There are approximately 12 different glycoproteins embedded in the lipid bilayer of the envelope. Some of the glycoproteins bind to the cell-surface receptors to initiate the infection of the cell with the HSV.39,40 Acidic heparan sulfate glycosaminoglycans mediate the attachment of the virion to cells. Our choice of HSV-1 as one of the model viruses to study was motivated by the possibility of cationic nanoparticles competing with the cellular surface for binding of this virus, leading to a lower cell-virus infection probability, analogous to the effect of polylysine- and PAMAM-based dendrimers.41,42 We have demonstrated previously a strong binding of biguanide-modified particles with glycoproteins and lipopolysaccharides,4,5 which, albeit belonging to the bacterial membranes, are not dissimilar to the glycoproteins of the virion’s envelope. ζ-Potential of the magnetic particles and MS2 virions of the present study as a function of pH is shown in Figure 4. The observed negative charge on MS2 virions corresponds well with the published data.35 It is expected that electrostatic attraction between the negatively charged virus proteins and positively charged particles will dominate the interactions of the virus and paramagnetic NPs. At pH < 4, the MS2 virus particles aggregated and sedimented, and thus we present no ζ-potential measurements at acidic pH. The PHMBG-PEI-M particles that is the highest charge density species (contains up to 1.45 × 1021 total positive charges per 1 g of dry particles)4 showed

Figure 4. Effect of pH on ζ-potential of MS2 virions and magnetic nanoparticles surface-modified with amine (PEI-M/SiO2), biguanide (PHMBG-PEI-M and PHMBG-M/SiO2), and aziridine (A-M/SiO2) groups. Measurements were conducted in 10 mM KCl at 25 °C.

somewhat higher ζ-potential at pH > 7.4 than other particles that contained 15% to 50% fewer charged groups. Nevertheless, all four types of magnetic particles studied were positively charged and colloidally stable in the pH range from 4 to 10, under conditions where they can realistically function in the environment. For the long-term chemical stability, core−shell particles with magnetite cores encapsulated by silica are preferred over the PHMBG-M-PEI particles that will tend to dissolve and lose paramagnetic properties when exposed to water over a month.5 Although the particle charges declined at pH > 7−8 due to the partial neutralization, the polymeric chains provided sufficient stability, so that no dramatic aggregation was observed within the pH range from 4 to 10. In subsequent experiments, we highlight the ion-exchange adsorption of the MS2 virus on the polycationic nanoparticles (Figure 5). As concentration of the polycationic PHMBG-M/

Figure 5. Effect of biguanide-, amine-, and aziridine-modified particle concentrations on the removal of MS2 bacteriophage from aqueous suspension at pH 7.3 and 25 °C by magnetocollection. Circles, squares, triangles, and diamonds show data points obtained with PHMBG-M/SiO2 (1), PHMBG-PEI-M (2), A-M/SiO2 (3), and PEIM/SiO2 (4) particles, respectively. Filled and open points show percent of MS2 virions removed and reduction factor (RF), respectively. For RF definition and experimental details, see Experimental Section.

SiO2, PHMBG-PEI-M, A-M/SiO2, and PEI-M/SiO2 particles increased above 0.05−0.2 mg/mL, over 99% of MS2 virions was removed from its aqueous suspensions, with the reduction factor approaching and exceeding 4 (Figure 5). Particle concentration-wise, the removal of the MS2 virus by cationic particles herein is 30-to 100-fold more efficient than in previously reported analogous experiments with unmodified magnetite particles at pH 4, where the net surface charge of the magnetite and MS2 virus particles is of the opposite sign.23 The 4553

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negative charge at the N-7 position of guanine. This binding opens the imidazole ring structure of the guanine molecule and becomes a stop signal for DNA and RNA polymerases. Thus, the altered nucleic acid can no longer serve as a template for transcription or replication, and this disrupts nucleic acid replication, which inactivates contaminating pathogens.18,44−46 The kinetics of the MS2 virus survival by A-M/SiO2 particles and their analogue, PEI-M/SiO2, are presented in Figure 6. A

drawback of the unmodified magnetite particles is that magnetite is an amphoteric solid, which can develop charges in the protonation (Fe−OH + H+ → Fe−OH2+) and deprotonation (Fe−OH → Fe−O− + H+) reactions of Fe− OH sites on the surface. The presence of the excess protons necessary for bare magnetite surface to be positively charged and bind the MS2 virus renders the virus-magnetite binding sensitive to the presence of ions as well as colloids that maintain equilibria with the magnetite surface. As a result, the electrostatic attraction between bare magnetite particles and MS2 virus in salt solutions or sewage effluents dramatically declines at pH > 5,23 despite the fact that the effective pKa of the magnetite surface in the absence of salts is 7.9.43 In the present work, proper design of the magnetite coating not only ensures the chemical and colloidal stability of the NPs, but also maximizes the particle−virion electrostatic attraction. In the case of PHMBG-modified particles, the presence of hydrophobic hexamethylene groups on the particle surface can enhance the complexation between the particle surface and amphiphilic proteins of the virion coat proteins. Exposure of the virions to the PHMBG strongly polycationic chains with multiple alkane, hydrophobic segments decreases the polarity of the MS2 virion particles, leading to their aggregation in aqueous media.22 In fact, the effect of the hydrophobic groups of PHMBG on viral infectivity due to the entropy-driven aggregation has been recently demonstrated.22 Analogous entropy-driven enhancement of the MS2 virion removal by PHMBG-M/SiO2 particles has been confirmed herein. Thus, the MS2 removal at 25 °C by PHMBG-M/SiO2 particles (Cparticle = 5 μg/mL) was measured to be 66.3%, but when the temperature was increased to 37 °C, the removal increased to 84.5%. In these experiments, the pH 7.3 was maintained. On the other hand, with PEI-M/SiO2 particles, which are cationic at pH 7.3, but lack any hydrophobic groups, no effect of the temperature increase from 25 to 37 °C on the MS removal was observed. Approximately 35% removal was observed at these temperatures at the effective particle concentration of 5 μg/mL. The inactivation factor (RFi), reflecting upon the ability of the virions remaining after the contact with the particles to infect the host cells, was measured with PHMBG solutions and suspensions of the particles of the present study. The RFi values were measured at 20 μg/mL particle or PHMBG concentrations (see Experimental Section). We were unable to measure the inactivation factor RFi at higher particle concentrations because of the insufficient concentration of the virus left in the samples. In all cases, except for A-M/SiO2 particles, the RFi values varied from 1 to 1.6, demonstrating that the amine- and biguanide-modified particle species of the present study lowered the ability of the virus to form plaques 10- to 40-fold. The RFi of 1.3 was measured for PHMBG, which corresponded well with the literature data.22 Much higher RFi values of approximately 4 were measured for A-M/ SiO2 particles, albeit the efficiency of the neutralizer on the aziridine groups potentially toxic to the host cells was not established. The efficiency of the A-M/SiO2 particles in the virus removal (Figure 5) and high RFi values indicated that the particles with aziridine groups present possess mechanisms of virus binding and inactivation that are additional to and more potent than the simple electrostatic binding. The mechanism of virus inactivation by aziridine groups has been investigated in some detail and is believed to be primarily via ionic binding to RNA or DNA by covalent interaction of the aziridine group with the

Figure 6. Survival kinetics of bacteriophage MS2 during treatment with A-M/SiO2 and PEI-M/SiO2 nanoparticles in 0.15 M NaCl at pH 7.0 and 25 °C. Effective aziridine group concentrations in suspensions of A-M/SiO2 particles were 1.0 and 0.5 mM, corresponding to 1.43 and 0.73 mg/mL particle concentrations, respectively. The effective concentration of PEI-M/SiO2 particles in the buffer was 0.73 mg/mL. The kinetics with 0.73 mg/mL A-M/SiO2 was conducted with and without magnetocollection of the particles. Data points obtained with magnetocollection are shown by filled squares, in blue. The moment of magnetocollection is shown by an arrow.

dramatic contrast between the effects of the particles with and without aziridine was observed. The kinetics with PEI-M/SiO2 particles, devoid of aziridine, showed somewhat rapid ∼100fold decline in the virion count after 30 min, and then the kinetics plateaued. The log(So/S) values in this case are close to the inactivation factor (RFi) with the PEI-M/SiO2 particle species, measured in 10 mM NaH2PO4 (pH 7.3). It is clear that the adsorption and complexation of the negatively charged virions with the positively charged surfaces of the PEI-M/SiO2 particles (compare with Figure 2), resulting in the virion removal from the medium, controlled the kinetics in this case. The adsorbed virions occupying the particle surface exhausted the availability of the positive charges for further virion adsorption, and thus the kinetics plateaued. On the other hand, the A-M/SiO2 particles caused exponential decline in the MS2 survival. When the A-M/SiO2 particles were quickly removed from the reaction medium by magnetocollection (Figure 6), the kinetics immediately plateaued, confirming that indeed the particles, rather than aziridine that dissociated from their surface, were the reaction agents. Due to the important feasibility of vaccine preparation by the aziridine treatment, the mechanisms of virus inactivation by aziridines have been studied in detail.14,15,25,47−50 It has been established that the viruses are inactivated predominantly through aminoalkylation of their RNA or DNA by aziridines, which results in the abortive termination of genomic replication of the nucleic acids, thereby terminating the virus reproduction. The presence of one modified nucleotide is sufficient for infectivity inactivation, and the kinetics of virus survival reflects the kinetics of alkylated genome components. If the modification rate is constant and the same for all virions present in the reaction medium, then 4554

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the survival kinetics can be described by a simple rate equation25,48 k = −2.3 log(St/S0)/Cat

where k is the effective rate constant of infectivity inactivation, Ca is the effective aziridine concentration in the suspension, and S0 and St are the infectivity titers of the phage suspension before inactivation and at time t, respectively. The rate constant k incorporates inactivating damages of any kind. The kinetics with A-M/SiO2 not interrupted by the particle removal were linear (R2 > 0.98), which afforded estimation of k values of 320 and 240 M−1 min−1 in the cases of Ca = 0.5 and 1.0 mM, respectively, from the slopes. It is interesting to observe that k values found with the A-M/SiO2 particles were well within the range of the rate constants found for MS2 phage with aqueous solutions of ethyleneimine trimers and tetramers under similar conditions.25 This is an indication that our nanoparticles sized ∼150 nm possessed an inactivation capability similar to that of the small water-soluble molecules. It can be hypothesized that the aziridine groups present at the termini of the polymeric PEI chains on the A-M/SiO2 surface are capable of alkylation of the 2.1−2.5-nm-thick A protein capsid51 of the MS2 virus. Protein modification by highly electrophilic aziridines through nucleophilic attack of amino acid residues such as cysteine, histidine, lysine, tryptophan, and so forth is well-documented.25,52 Such modification causes inhibition of viral cell entry or the release of the RNA genome. Altering the protein capsid can cause conformational changes in the protein shell necessary for the release of the genomic RNA attached to the inner surface of the virus coat during RNA ejection.51 Furthermore, because of the high degree of dynamics of the protein capsid in solution, it is possible that the aziridine groups attached to a relatively long and flexible arm of the PEI polymer could directly access and alkylate the viral genome.52 In order to obtain experimental proof of our AM/SiO2 particles alkylating the MS2 capsid’s proteins, we applied methodology of acid dissolution reported previously for bacteriophage MS2.22 The details of the particle dissolution are given in the Experimental Section. The bacteriophage MS2 coat protein is a 129-amino-acid protein, with a molecular mass of 13 728 Da. We observed two base peaks of the MS2 capsid protein detected at m/z = 6864 and 13 729, corresponding to [M+2H]2+ and [M+H]+, respectively (Figure 7).26,53−55 There were also three peaks at m/z = 13 989, 14 048, and 14 091, which corresponded to the [H2N−(CH2CH2NH)6−H+2H]2+ ethyleneimine hexameric ion (m/z = 259), heptamer (m/z = 319), and octamer [M+H]+ (m/z = 362) bound to the capsid protein. The above results indicate that the A-M/SiO2 NPs are capable of alkylating the phage’s capsid protein with the aziridine groups on the particle surface. It is plausible that such bonding of the particle to the capsid of the virus damages the capsid and leads to the viral RNA being available for modification. Compared to the virus capsid protein, it is much more difficult to ascertain the presence of chemically modified RNA on the particle surface. However, such virus genome modification is evident from the inactivation curves presented in Figure 6. Suppression of Herpes Virus. Herpes virus is an encapsulated virus with a DNA-containing, icosahedral capsid surrounded by a proteinaceous tegument, which in turn is encased in a lipid bilayer containing about a dozen different viral glycoproteins.56 Like many other bacteria and viruses with negatively charged, amphiphilic lipid constituents of the outer

Figure 7. MALDI spectra of bacteriophage MS2 capsid protein suspended in 50% acetic acid/Triton X-100 solution resulting from digestion of the phage bound to A-M/SiO2 nanoparticles: (a) area of [M+2H]2+protein ion; (b) area of [M+H]+ protein ion. The intensity is normalized to the intensity at m/z = 13 729. Products of the protein alkylation by aziridine-terminated PEI of the particles are found at m/z = 13 989, 14 048, and 14 091.

membrane that are efficiently bound to and thus their bilayers disrupted by strongly positively charged biguanides with hydrophobic segments, HSV-1 is inhibited in vitro by biguanides such as PHMBG used herein as well as by chlorhexidine, its small-molecular-weight antiseptic analogue.27,57−59 While exhibiting efficient inactivation, PHMBG shows no toxic effects on human cells when applied in formulations such as mouth rinses and antiseptic/disinfectant solutions, and thus, it was used herein as a standard to compare effects of functionalized core−shell particles (Table 2). It is Table 2. Minimum Suppression Concentration of Simplex Herpes Virus by PHMBG and PHMBG- and AziridineModified Magnetic Particles (HostAfrican Monkey Kidney Cells) species

minimum suppression concentration (mg/mL)

PHMBG PHMBG-PEI-M PHMBG-M/SiO2 A-M/SiO2

0.1 0.2 0.4 0.002a

a

A-M/SiO2 NPs are toxic to the host cells at 0.1 mg/mL concentrations.

interesting to observe that, when minimum suppression concentration was expressed per effective concentration of PHMBG in the particles (polymer content, ∼50 wt %), the PHMBG-PEI-M particles possessed approximately the same suppressing concentration as PHMBG, but PHMBG-M/SiO2 were ∼2-fold less efficient (Table 2). The difference in activity can be explained by the significantly higher charge density in the PHMBG-PEI-M particles, which contain branched polyethyleneimine, loosely bound to PHMBG by Schiff-base groups (Figure 1).3 4555

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We further investigated possible toxicity of the NPs on the Vero cells hosting HSV-1 by using MTT assay (see Experimental Section). The metabolic activity values for the PHMBG-M/SiO2 and PHMBG-M particle suspensions ≤2 mg/mL as well as PHMBG solutions at concentrations