Plasma-assisted synthesis of noble nanoparticles coated with hyaluronan: chemico-physical characterization and safety assessment

Nanosystems are rapidly gaining attention in the biomedical, chemical, material, computer and catalysis fields. Recent research has been focused on synthetic methods to decrease toxicity and side effects as compared to classic formulations. However, harmonization of the interpretative criteria of biological activity and strategies to improve the scalability of synthetic technologies are limited. The present work describes the use of atmospheric plasma as a promising strategy to produce size-controlled and safe noble nanoparticles, which did not show surface toxicity due to the absorption of chemicals during the synthesis reaction. Physisorption with Hyaluronic Acid was used to modulate nanoparticle aggregation kinetics and improve biological properties. Physico-chemical characterization was conducted using NMR spectroscopy, UV-visible and dynamic light scattering. Cytotoxicity on bacterial and Human Umbilical Vein Endothelial Cells was tested. The results demonstrated the efficiency of the plasma synthetic method to control nanoparticle size and toxicity selectively improving antibacterial activity against Gram-negative strain.

Physisorption with hyaluronic acid (HA) with low (200 kDa) molecular weight (Mw) modulates their aggregation kinetic, prevents secondary collateral phenomena such as coagulation and coalescence and plays a crucial role in morphology, size and biological activity [4][5][6][7][8]. AgNPs and AuNPs represent the most industrially produced and commercialized nanomaterials; their properties are unique and applications are found in different fields including material science, biosensing, organic chemistry, biotechnology, high molecular sensitivity detection, optics, biosensors, computers, medicine, microbiology, and catalysis [3,9].
Different approaches have been outlined to synthetize NPs, including chemicals, photo-and electro-chemical, extractive, and fermentative methods. The classical methods require high temperature, pressure and energy and involve the manipulation of hazardous chemicals with the need for complex purification steps, and the resulting products usually contain chemical contaminants. Moreover, extractive, and biological sources have a relatively slower rate [10][11][12][13][14].
Here we propose the use of atmospheric plasma as an environmentally sustainable, low energy consumption, affordable and highly efficient method to produce size-controlled and safe noble nanoparticles, which did not absorb toxic chemicals on their surface during the synthesis reaction [15][16][17][18][19]. Cold plasma obtained at atmospheric pressure and room temperature results as being rich with energetic electrons that are capable of dissociating, exciting and ionizing gas molecules [20][21][22]. Typical reactions include the formation of a Reactive Oxygen and Nitrogen Species (RONS) discharge that can diffuse/dissolve into water during exposure and react with the water molecules, resulting in a cocktail of chemical species whose generation depends on the release of hydrogen ions; moreover, neutral gas molecules subjected to cold plasma treatment are transformed into their oxides and their products can react with hydroxyl radicals, to produce hydrogen peroxide in plasma activated water (PAW) at the gas/liquid interface [23]. PAW was used as the reducing agent for Ag + or Au 3+ ions from Ag/Au salts to obtain AgNPs and AuNPs.
Physisorption with Hyaluronic Acid (HA) at a low molecular weight (200 kDa) and conjugation with the GHHPHGK peptide through an amide bond [24,25] were performed to improve pro-angiogenic activity [26][27][28]. The GHHPHGK derives from GHHPH, a 67 kDa peptide repeat present in histidine-proline rich glycoprotein (HPRG, Accession number: P04196, gene ID 3273) [29,30]. The amide linkage protected the polymer from enzymatic degradation [31,32]. Successively cold plasma technology as used to synthetize coated noble nanoparticles. Physico-chemical characterization was conducted using NMR spectroscopy, UVvisible and dynamic light scattering [33]. The safety assessment of polymeric nanosystems was evaluated on bacterial strains and HUVEC cell lines; moreover, their capacity to have no effect collagenase activity was tested for their application in wound healing. The resulting NPs can represent a promising technology because of their biological activity, an effective drug delivery system and suitable pharmacokinetic biomodulators [34,35]. volume ratio) and then rinsed with water before starting.

Synthesis of Hyaluronic Acid-GHHPHGK conjugate
The conjugation between hyaluronic acid (HA 200 kDa) and the peptide GHHPHGK (Glycine-Histidine-Histidine-Prolyne-Histidine linked to Glycine-Lysine-NH2) was obtained through an amide bond [25]. The amount of GHHPHGK peptide (p) conjugated with HA was determined from the ratio between the integration value of the signal of the acetyl groups of HA and the -CH2 Lysine signal or that of the H-2 or H-5 signals of the Histidine imidazole ring.

Wet chemical synthesis of AgNP and AuNP and HA conjugation
The bare silver nanoparticles (AgNPs) were synthetized following the method described by Mavani et al.
(2016) [36] Briefly, a solution of 1 mM AgNO3 was added with 2 mM NaBH4, see equation (1). The reaction was maintained in an ice bath and under stirring (200 rpm) for 45 minutes. Naked-eye detection was used to confirm the end of the reaction with the chromatic transition of the solution from colourless to yellow.
The bare gold nanoparticles (AuNPs) were synthetized starting from a 0.5 mM HAuCl4 aqueous solution by adding a 7 mM NaOH and 5 mM Glucose solution, see equation (2). The reaction at 25 °C occurred in 1 minute and its end was detected by the naked eye thanks to the chromatic transition from yellow to purple red.
In order to obtain the coated products, the AgNPs and AuNPs were synthetized solubilizing the AgNO3 and HAuCl4 salts in a 0.2 % w/v HA/pHA water solution.

Cold-plasma torch synthesis of AgNP and AuNP and HA conjugation
The plasma method to synthetize AgNPs and AuNPs used atmospheric pressure microplasma which can form the radical species • that acts as the reducing agent activator for the corresponding metal reduction [17].
Argon (Ar) at a flow rate of 25 sccm was injected from a pressurized stainless-steel capillary tube (0.6 mm inner diameter) whose tip was positioned approximately 2 mm above the plasma-liquid interface (Fig. 1). ii) growth, AuNPs size increases to 5.2 nm; iii) slow further growth, AuNPs size increases with a controlled growth mechanism where the limiting factor is the metal concentration within the solution; iv) fast final growth, a rapid increase in AuNPs size from 5-5.2 nm to 7.7 nm determined by an autocatalytic reduction on their surface [37]. In the absence of a stabilizing agent such as hyaluronic acid, which physioabsorbs on the surface of aggregating particles, the growth of nanoparticles continues even 24 h after the start of the reaction. The use of a stabilizing agent as a nucleation modulator is mandatory to regulate the size and kinetics of gold nanoparticle formation in aqueous media. The nucleation mechanisms have a substantial impact on gold nanoparticle bond length, causing it to decrease from 2.87 Å (calculated on the bulk) to the range of 2.55-2.70 Å. As the reaction progresses, the bond becomes shorter due to the increase in particle size and the decrement of the surface area per gold atom. A secondary mechanism also led to the formation of dimers (Au2Cl6) and trimers, with the occasional formation of gold clusters like Au13, the principal cause of coalescence and often considered as an undesired species in reaction mixtures [1]. b) Schematic representation of nanoparticle formation mechanism.
An electric charge of 3 mA (with a resistance value of 140 kΩ) was applied to a 1 mM solution of AgNO3 for 15 minutes at a temperature of < 5 °C, without stirring. Product formation was observed by the naked eye thanks to a chromatic transition from transparent to yellow brown.
An electric charge of 5 mA (with a resistance value of 140 kΩ) was applied to a 5 mM solution of HAuCl4 for 15 minutes at a temperature of < 5 °C, without stirring. Product formation was observed by the naked eye thanks to a chromatic transition from yellow to purple red.
For both reactions, a platinum (Pt) foil was immersed in the solution as a counter electrode to promote cathodic reduction reactions at the plasma-liquid interface.
In order to obtain the coated products, the AgNPs and AuNPs were synthetized solubilizing the AgNO3 and HAuCl4 salts in a 0.2 % w/v HA/pHA water solution.
As required by some experimental methods, after washing with ultrapure water, the concentrated NPs were obtained by centrifugation for 15-20 minutes at 15 °C and 8000-13000 rpm (Eppendorf Centrifuge 5417R, FA453011 Rotor, Italy). The NPs coated with HA were centrifuged for 4 minutes at 15 °C and 10000 rpm using a filter with a 30 kDa molecular weight cut-off (Merk Millipore, USA).

UV-Visible nanoparticle characterization
UV-Visible characterization was conducted at 25 °C using a Lambda2S spectrophotometer (Perkin Elmer, USA). The measurements were performed using quartz cuvettes (1.0 or 0.1 cm path length). The absorbance and maximum wavelength were recorded. The assay was performed in triplicate twice and the reproducibility of the synthesis was evaluated as a percentage of relative standard deviation (% RSD), as given in equation (3): where ̅ is the average value of the measured variable, and is the standard deviation.
The theoretical size of NP diameters was calculated from equation (4) as described by Haiss et al. (2007) [38], considering each force involved in the system, including plasma frequency and collision frequency.
where is the cross section of a spherical particle with R as the radius embedded in a medium with dielectric function ∈ at a defined wavelength ; = �∈ ⁄ 2 is the wave vector; ( , ) and ( , ) are the scattering coefficients in terms of Ricatti-Bessel functions.

Dynamic light scattering nanoparticle characterization
The NPs hydrodynamic diameter size was investigated by Dynamic Light Scattering (DLS), measuring the particle size distribution at 25 °C with a 650 nm light source (laser diode, 5 mW and photo multiplier tube detector) by using DLS HORIBA LB-550, USA [33]. The measurements were performed using quartz cuvettes (1.0 cm path length). The assay was performed in triplicate twice.

ATR-FTIR nanoparticle characterization
The chemical structure of the functional groups was determined by Fourier-transform infrared spectroscopy (ATR-FTIR) following the method described by Sanfilippo et al. (2020) [39]. Briefly, the silicon wafers are cut to give a surface of 0.8 cm x 1 cm and washed with methanol and iso-octane. 100 µL of the sample (HA, NP or HA-NP) were put on the cleaned silicon surface and left to dry at room temperature. The ATR-FTIR spectra was recorded using a Spectrum Two™ FTIR Spectrometer (Perkin Elmer, USA).

Atomic Force Microscopy (AFM) nanoparticle characterization
The surface morphology and nanomechanical properties were studied using Atomic Force Microscopy (AFM),

Diameter size determination by transmission electron microscope (TEM)
The morphology, composition and size of the NPs were verified by means of a transmission-electron microscope (TEM) using a Philips CM20 microscope 200 kV operating with a tungsten electron gun, using the method described by Haiss et al. (2014) [38]. The Feret diameter of the particles was determined automatically using "Image Tool" software.

Agar diffusion assay
The putative inhibitory activity of the NPs was investigated determining the zone of inhibition by the agar diffusion assay, modifying the method described by CLSI M7-A7 for bacteria [41]. the negative control was sterile MH broth. The 96-well plates were incubated as described by CLSI M100-S23 [43]. The absence of growth was confirmed by spreading 100 µL from each well on MH agar in which bacterial growth seemed inhibited. The experiments were carried out twice in duplicate.

Cell lines and treatment culture and maintenance
Human

Determination of bare and HA/pHA coated AgNPs and AuNPs toxicity
To find a safe and non-toxic amount of bare and HA/pHA coated AgNPs and AuNPs the 3-(4, 5-dimethyl-2-

Effects of NPS on collagenase activity
The effect of tested nanoparticles on the enzymatic activity of collagenase was determined spectrophotometrically by quantifying the fragment produced by the enzymatic reaction against an external standard. The assay was performed by modifying the method described by Wunsch-Heidrich et al. acid. The organic solution was dehydrated using anhydrous Na2SO4 and the absorbance was determined spectrophotometrically (at 320 nm, using a UV-1200 spectrometer). The substrate solution mixed with the buffer solution was used as the blank. The enzymatic activity was quantified as follows: where standard concentration was 0.02 µmoles/mL, 50 was the sample dilution factor, 1000 was the conversion factor from µmoles to nmoles, 900 was the number of seconds in 15 minutes, df was the dilution factor of collagenase to obtain a solution with a final activity ranging from 0.35 to 1.2 nkat/mL.
The results are expressed as a relative percentage of collagenase activity (%A) calculated as follows: %A = Activity of treated sample Activity of untreated sample × 10 (6)

Chemical and spectroscopical characterization of HA conjugate (pHA)
The 1 H-NMR spectrum of Hyaluronic Acid-GHHPHGK-conjugate (pHA) is shown in Fig. 2 (Fig. 2). The amide bond formation between the free carboxylic group of HA residue and the amino group of the Lysine side chain was underlined by a resulting upfield shift (Δδ = -0.10 ppm) of ε-methylene protons (δ = 3.13 ppm) relative to the Lysine-NH2 moiety, as compared to the corresponding resonance of the non-conjugated peptide (δ = 3.03 ppm).
The conjugate shows all the expected signals from the protons of peptide and hyaluronic acid units; all proton resonance signals were completely assigned. The amount of GHHPHGK peptide conjugated with HA results as being ~ 15%.  The Absmax peak of AuNPs was right shifted with respect to those of AgNPs for both chemically and plasma synthetized ones. The Absmax peak of p AuNPs was left shifted with respect to the chemically synthetized ones.

UV-Visible and Dynamic light scattering nanoparticle characterization
For both syntheses it is possible to observe a red shift of the λmax of HA-AuNP compared to AuNP, relative to the increase in NP optical diameter (Fig. 3). This red shift is more intense for pHA-AuNP (1.1 nm for both chemically and plasma synthetized), a linear variation of the λmax was observed for c AuNP with respect to plasma synthetized ones, as a function of the HA presence. The broad plasmon band for pHA-c AuNP is evidence of the presence of several populations of various sizes. On the contrary, p AuNP and HA-p AuNP exhibited a smaller size than the p AgNPs. The pHA-p AuNP plasmon band points to the formation of a monodisperse solution of larger NPs. These differences could be due to the different charge of the respective metal precursors salts, monovalent silver and trivalent gold. The hydrodynamic diameter is higher than the optical size except for glucose-capped c AuNP and HA-c AuNP which showed quite similar values. The glucose used as reducing agent for synthesis of c AuNP could contribute to forming a dense and rigid shell around the nanoparticles which roughly corresponds to the hydration shell around the NPs.

Atomic Force Microscopy nanoparticle characterization
The atomic force microscopy (AFM) images of HA (200 kDa) show a layer of macromolecules < 0.5 nm thick, indicating an aggregation force between chains, as well as rather strong hydrophilic interactions between the mica substrate and HA, typical of the low molecular weight. The stiffness of the HA molecules is apparent and is due to dehydration (Fig. 5 a-c). HA-AgNP and HA-AuNP show the HA monolayer around the core of aggregated NPs, especially for HA-AuNPs with respect to HA-AgNP. The dimensions of the height images were 25 nm and 60 nm for HA-AgNPs obtained by chemical synthesis and 27 nm and 30 nm for plasma synthetized ones, with different microscopic characteristics typical of globular hybrid-polymer metal nanoparticle systems (Fig. 5).

Diameter size determination by transmission electron microscope (TEM)
The chemically and plasma synthetized AgNPs and AuNPs showed two different aging trends: AgNP aging

Biological properties 3.3.1 Antibacterial activity
The MIC values of all the tested NPs were lower than those of chloramphenicol (CHL); NPs were more active against Escherichia coli ATCC 9637 and no significant differences were observed for HA and pHA coated NPs. AuNP was less toxic with respect to AgNP.
With the agar diffusion method, the antibacterial activity of NPs was shown to be lower with respect to that observed using the broth microdilution method and AuNPs did not show any inhibitory activity. These results could be due to interactions between NPs and the agar mesh. All chemically synthetized AgNPs ( c AgNPs, HAc AgNPs, pHA-c AgNPs) showed a clear inhibition zone for both tested strains (Fig. 6 A).

Cytotoxic activity on Human Umbilical Vein Endothelial Cells (HUVEC)
The results of the MTT (tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay on Human Umbilical Vein Endothelial Cells (HUVEC) showed that cell viability is a function of NPs and of the final yield depending on the method used for the synthesis (Fig.6 B)

Effect on collagenase activity
After treatment with AgNPs, collagenase activity was reduced by 20% (Fig. 6 C). In the presence of plasma synthetized AgNPs, collagenase activity is higher respect to what determined in presence of chemically synthetized NPs (91% vs 84,57%). Nevertheless, collagenase activity stays at high levels when chemically synthetized AgNPs are coated with HA (HA-AgNP).

Conclusion
The results collected in this study lead to the consideration of cold plasma technology as an efficient method for the synthesis of gold and silver nanoparticles, which is also applicable in the industrial field. The nanoparticles obtained were stable and the physisorption with Hyaluronic Acid contributed to modulating their aggregation kinetic and stabilizing their biological properties, especially against Gram-negative bacteria, maintaining their safety profile when tested on HUVEC cells. Our research on the synthesis of nanoparticles composed of noble metals provides promising bioactive compounds with reduced side effects, using substances that could constitute a reference for the fight against bacterial resistance, the elusive mechanisms of onco-related pathologies and other ailments where these systems have been shown to be applicable.

Acknowledgements
The authors acknowledge to the European Union (M-ERA.NET 2 Cofound H2020 Joint transnational Call 2016 DM 593 MUR project n. SmartHyCAR 4274.