{'fraction_non_alphanumeric': 0.03146172697131325, 'fraction_numerical': 0.017767046273605303, 'mean_word_length': 5.070662874644498, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Mesoporous Silica Nanoparticles Decorated with Polycationic Dendrimers for Infection Treatment

This work aims to provide an effective and novel solution for the treatment of infection by using nanovehicles loaded with antibiotics capable of penetrating the bacterial wall, thus increasing the antimicrobial effectiveness. These nanosystems, named"nanoantibiotics", are composed of mesoporous silica nanoparticles (MSNs), which act as nanocarriers of an antimicrobial agent (levofloxacin, LEVO) localized inside the mesopores. To provide the nanosystem of bacterial membrane interaction capability, a polycationic dendrimer, concretely the poly(propyleneimine) dendrimer of third generation (G3), was covalently grafted to the external surface of the LEVO-loaded MSNs. After physicochemical characterization of this nanoantibiotic, the release kinetics of LEVO and the antimicrobial efficacy of each released dosage were evaluated. Besides, internalization studies of the MSNs functionalized with the G3 dendrimer were carried out, showing a high penetrability throughout Gram-negative bacterial membranes. This work evidences that the synergistic combination of polycationic dendrimers as bacterial membrane permeabilization agents with LEVO-loaded MSNs triggers an efficient antimicrobial effect on Gram-negative bacterial biofilm. These positive results open up very promising expectations for their potential application in new infection therapies.


Introduction
Over the last few years, the development of antimicrobial resistance and its therapeutic implications has become one of the main threats to global health, even talking about the emergence of a new "post-antibiotic era" in which antimicrobial treatment will be useless, given the multidrug-resistant nature of numerous microorganisms [1]. Although the majority of these microorganisms are Grampositive bacteria, infections caused by the Gram-negative microbes currently have rightly drawn attention, mainly due to the greater difficulty in dealing with them. This drawback lies basically in the presence of an outer membrane that serves as a highly impermeable barrier, as well as additional defense mechanisms that might be absent in Gram-positive bacteria [2][3][4]. Another added inconvenience is the formation of structures known as biofilms, which constitute a natural mechanism of defense of the microorganisms against external aggressions, including antibiotics, reason why their use even hinders the management of these infections [5]. The association of the phenomenon of multiresistance with the appearance of biofilm is results in almost intractable infections using the currently available antibiotic strategies [6].
Nowadays, there is an urgent need to combat biofilm-associated infections through the design of nanoplatforms able to prevent biofilm formation or to produce dispersion of preformed biofilms [7,8]. As well, one of the most important challenges in infection therapy is the design of nanocarriers able to selectively transport antimicrobial agents to the target site and, once there, release them in a controlled fashion [9][10][11][12]. Bacteria-targeted nanoparticles offer promising expectations to improve drug efficacy, compliance, and safety [13,14]. Both Gram-positive and Gram-negative bacteria possess different components in their outer membranes, which are easily deprotonated to produce negative charges, increasing their hydrophilicity [15]. Cationic polymers and moieties have been proposed as bacterial detection probes via electrostatic interaction [16] and they have been applied in the immobilization and detection of bacteria, mainly owing to the high toxicity of these components [15,17,18].
Dendrimers are single molecular weight, tree-like globular macromolecules with structural uniformity consisting of a central core, branching layers, and numerous end groups [19]. Their unique properties make them good nanoplatforms for different areas of biomedicine, used as protein mimics, drug delivery agents, imaging and antiviral and antibacterial agents [9,20,21]. Moreover, it has been reported that polycationic dendrimers, exhibiting high concentration of positively charged amine groups, are able to efficiently bind to negatively charged bacteria cell walls, increase bacterial membrane permeability and internalize inside the bacteria [10,22].
Regarding inorganic nanomaterials, mesoporous silica nanoparticles (MSNs) are excellent candidates to develop targeted drug delivery devices owing to their high biocompatibility, intrinsically large drug loading capacity and versatility in terms of chemical modification of its surface [23][24][25][26][27][28]. Herein, a new class of antimicrobial agent, named "nanoantibiotic", based on MSNs functionalized in the external surface with the polypropyleneimine (PPI) dendrimer of third generation (G3) has been developed. Since a generation dependent cytotoxicity has been observed when dealing with polycationic dendrimers, in this approach we have selected a low non-toxic third generation [29]. Moreover, to overcome the drawback of the limited surface charges of low generation dendrimers the attachment of several dendritic molecules to the same nanoparticle is accomplished [30][31][32]. The positive charge of this nanosystem behaves as internalization agent able to penetrate the negatively charged bacteria wall. The mesoporous structure allows the loading of levofloxacin (LEVO) as antimicrobial drug. The synergistic combination of both elements into a unique nanosystem produces an efficient antimicrobial effect on bacterial biofilm. The influence of the chemical nature of these G3 polycationic dendrimers on the performance of the nanoantibiotic has been investigated. For this purpose, this nanosystem was compared with MSNs externally functionalized with a model low molecular weight diamine. To date accurately mechanistic studies have been reported with star nanoparticles of organic nature (structurally nanoengineered antimicrobial peptide polymers), where membrane attachment and internalization in Gram-negative bacteria have been demonstrated [13]. However, to the best of our knowledge, this is the first study where internalization of inorganic MSNs in Gram-negative bacteria is reported.

Reagents
All reactions for the synthesis of the silylated dendrimer and for the chemical modification of the silica surface were performed under an inert atmosphere by using standard Schlenk techniques.
Dichloromethane (CH 2 Cl 2 , Sigma-Aldrich) was dried by standard procedures over phosphorus (V) oxide and distilled immediately prior to use.
Deionized water was further purified by passage through a Milli-Q Advantage A-10 purification system (Millipore Corporation) to a final resistivity of 18.2 MΩ cm. All other chemicals (ammonium nitrate, absolute EtOH, dry toluene, NaOH, etc.) were of the highest quality commercially available and used as received.
The following analytical methods were used to characterize the synthesized compounds: solution state NMR spectroscopy; high resolution magic angle spinning (HR-MAS) NMR spectroscopy; solid state MAS NMR and cross polarization (CP) MAS NMR spectroscopy; Fourier transform infrared spectroscopy (FTIR); elemental chemical analyses; thermogravimetric and differential thermal analysis (TGA); low-angle powder X-ray diffraction (XRD); N 2 adsorption porosimetry; electrophoretic mobility measurements to calculate the values of zeta-potential (ζ); dynamic light scattering (DLS); scanning electron microscopy (SEM); energy dispersive X-ray spectroscopy (EDS); and transmission electron microscopy (TEM). The used equipment and conditions are described in the Supporting Information.

Synthesis of the silylated dendrimer G3-Si(OEt) 3
Under inert atmosphere, a solution of 3-isocyanotopropyltriethoxysilane (12 µL, 0.018 mmol) in dry CH 2 Cl 2 (20 mL) was added dropwise to a vigorous stirred solution of G3-PPI dendrimer (0.078 g, 0.018 mmol) in dry CH 2 Cl 2 (35 mL). The addition was carried out under an inert atmosphere of N 2 , vigorous stirring and with an addition time of about 2 h. The reaction mixture was stirred overnight at RT and the proper progress of reaction was checked by FTIR and 1 H NMR spectroscopies.
Subsequently, the solution was concentrated by partial removal of solvent under reduced pressure in the vacuum line and the product was immediately used for the covalent attachment to the silica surface.

Surface functionalization of the MSN materials
MSN materials were firstly dried under vacuum at 80 °C for 5 h and then re-dispersed in a dry solvent under N 2 , by using vortex shaking and ultrasound cycles for nearly 30 min or until an adequate suspension was attained.

MSN-DAMO:
A solution of 0.028 g of DAMO (57 µL, 5% excess) in 5 mL of dry toluene was added to a vigorously stirred suspension of 0.500 g of the CTAB-containing MSNs (34% wt, i.e., 0.329 g MSNs) in 100 mL of dry toluene and the mixture was heated to 110 °C overnight. The solid was isolated, exhaustively washed with toluene and acetone and then dried under vacuum at 30 °C.
Finally, the surfactant was removed by ion exchange as above described.

MSN-G3:
For the covalent anchorage of the dendrimers onto the external surface of the MSN sample, the freshly obtained solution of silylated G3-PPI dendrimer, G3-Si(EtO) 3

Levofloxacin loading into MSN materials
LEVO loading in the inner pores of MSN materials was performed following an impregnation method [34]. MSN and MSN-DAMO materials were loaded by soaking 50 mg of the nanoparticles in 10 mL of a LEVO solution in ethanol (5 mg/mL) and the suspension was stirred at RT for 24 h preserved from light. Then, the materials were filtered, gently washed with absolute ethanol, dried under vacuum and denoted as MSN-L and MSN-DAMO-L samples. Taking into account the size of the third generation dendrimer, the loading process was performed during the same step of external functionalization of the MSN material with G3-Si(OEt) 3 , which is accomplished in dry CH 2 Cl 2 . In a first assay, MSN material was loaded by soaking 50 mg of the nanoparticles in 10 mL of a LEVO solution in CH 2 Cl 2 (5 mg/mL) and the suspension was stirred at RT for 16 h in dark conditions. Then, the sample was filtered, gently washed with CH 2 Cl 2 , dried under vacuum and characterized to assess the right loading of LEVO using this solvent. To afford MSN-G3-L, 250 mg of vacuum dried MSN were resuspended in 25 mL of a LEVO solution in dry CH 2 Cl 2 (10 mg/mL) and stirred at RT for 16 h in darkness conditions. To this suspension a solution of the freshly prepared G3-Si(OEt) 3 (amounts and conditions as described above for the functionalization of 250 mg of MSN) was added and stirring was maintained for another 16 h, at RT in the absence of light.
Then, the sample was filtered, gently washed with CH 2 Cl 2 and dried under vacuum. The amount of drug loaded in MSN materials was determined from the elemental chemical analyses and TGA.

"In vial" levofloxacin release assays from MSNs
The kinetic studies of LEVO release were carried out in phosphate buffered saline (PBS) at 37 °C and physiological pH of 7.4. A double-chamber cuvette with two different compartments (sample and analysis) separated by a dialysis membrane (12 kDa molecular weight cut-off) that only allows the LEVO diffusion was employed for the experiments. Briefly, 170 µL of a suspension of the loaded MSN material in PBS (8 mg/mL) was placed in the sample compartment and 3.6 mL of fresh PBS were located in the analysis one. This system was kept at 37 °C and magnetic stirring was applied to the analysis compartment. At every analysis time, the solution in the analysis compartment was removed for analysis and replaced by fresh medium. The amount of LEVO released was determined by fluorescence spectroscopy using a BiotekPowerwave XS spectrofluorimeter, version 1.00.14 of the Gen5 program, with λ ex = 292 nm and λ em = 494 nm. First, a calibration curve was established in a concentration range from 0.01 to 12 µg/mL.

Microbiological assays
Briefly, bacteria culture was carried out by inoculation in Todd Hewitt broth (THB; Sigma-Aldrich) and incubated overnight at 37 °C with orbital shaking at 200 rpm. After culture, bacteria were centrifuged for 10 min at 3,500 g at 22 °C. The supernatant was then discarded and the pellet was washed three times with sterile PBS. The bacteria were then suspended and diluted in PBS to obtain a concentration of 2 × 10 8 bacteria per mL. Bacteria concentration was determined by spectrophotometry using a visible spectrophotometer (Photoanalizer D-105, Dinko instruments). Gram-negative bacteria as Escherichia coli (E. coli ATCC25922 laboratory strain) were used.

Antimicrobial effect of levofloxacin dosage from MSNs
To determine the effectiveness of the LEVO dosages released from the different MSNs against bacteria growth, 100 µL of each dosage was inoculated in 900 µL of 10 8 bacteria per mL in PBS and incubated overnight. The presence or not of bacteria, as well as their quantification, was determined by counting the colony forming units (CFUs) in agar [35]. In this sense, 10 µL of this solution was seeded onto tryptic soy agar (TSA) and incubated at 37 °C overnight and subsequent counting.
Controls containing bacteria was also performed and the experiments were performed in triplicate. After incubation, the cover glasses were washed with HBSS and fixed with 2% wt/vol paraformaldehyde in PBS for 10 min at RT. The fixative was removed with HBSS twice and 0.5 mL of HBSS was added to each well before imaging. The samples were examined in an Olympus FV1200 confocal microscope. All images are representative of three independent experiments.

Antimicrobial effect of MSNs and LEVO-loaded MSNs in planktonic bacteria
To determine the antimicrobial effect of the different MSNs and LEVO-loaded-MSNs 10 µg/mL of each sample was inoculated in 900 µL of 10 8 bacteria per mL in PBS and incubated during 90, 180 and 1440 min. For comparison, the antimicrobial effect of free LEVO (1.88 µg/mL) was also evaluated. The presence or not of bacteria, as well as their quantification, was determined by CFUs in agar. Thus, 10 µL of this solution was seeded onto TSA, incubated at 37 °C overnight and subsequently the number of CFUs was counted. Controls containing bacteria was also performed and the experiments were performed in triplicate.

Antimicrobial effects against Gram-negative E. coli biofilm
Effectiveness of the LEVO loaded nanosystems against biofilm was also determined. For this purpose, E. coli biofilms were previously developed onto poly-D-lysine coated round cover glasses by suspended the round cover glasses in a bacteria suspension of 10 8 bacteria per mL during 48 h at 37 °C and orbital stirring at 100 rpm. In this case, the medium used was 66% THB + 0.2% glucose to promote a robust biofilm formation. After that, the round cover glasses containing the biofilm were placed onto 24 well culture plates (CULTEK) in 1.5 mL of new medium. Then, 0.5 mL of a suspension of MSN materials in PBS at final concentration of 5 and 10 µg/mL was added. After 90 min of incubation, the glass discs were washed three times with sterile PBS and 3 µL/mL of Live/Dead® Bacterial Viability Kit (Backlight TM ) was added. Also, 5 µL/mL of calcofluor solution was added to stain the mucopolysaccharides of the biofilm (extracellular matrix) in blue to specifically determine the biofilm formation. Both reactants were incubated for 15 min at RT.
Controls containing biofilm bacteria were also performed. Biofilm formation was examined in an Olympus FV1200 confocal microscope and eight photographs (60× magnification) were taken of each sample [36,37]. The surface area covered with adhered bacteria was calculated using ImageJ software (National Institute of Health, Bethesda, MD). All images are representative of three independent experiments.

Statistics
Data are expressed as means ± standard deviations of a representative of three independent experiments carried out in triplicate. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) version 19 software. Statistical comparisons were made by analysis of variance (ANOVA). Scheffé test was used for post hoc evaluations of differences among groups.
In all of the statistical evaluations, p˂0.05 was considered as statistically significant.

MSN material synthesis
Fluorescent MSNs were synthetized by covalently linking the fluorescent dye to the silica network following a modified Stöber method [38][39][40]. SEM analysis of the obtained MSNs revealed a uniform spherical morphology with an average particle diameter of ca. 150 nm (Fig.S1 in Supporting Information). Low-angle XRD measurement showed a well-resolved pattern of 2D hexagonal MCM-41 materials, with a unit cell parameter of 4.55 nm. The well-ordered mesoporous structure was also confirmed by N 2 adsorption porosimetry, possessing an average pore diameter of 2.4 nm and 1090 m 2 /g of surface area.

External functionalization of the MSN material
The functionalization of MSN materials was intended only in the outer surface of the nanoparticles to keep free the inner mesoporous surface (Fig.1). The post-synthetic method via condensation of alkoxysilane derivatives with silanol groups of the silica surface, under water-free conditions, was used to covalently attach the amine moieties to the silica support [41]. The amount of the alkoxysilane was established from the surface area (S BET ) of the MSNs, of which the 25% was taken into account for the external surface, with an average surface concentration of Si-OH groups of 4.9 OH/nm 2 [42]. A molar ratio of three Si-OH groups with one R-Si(OEt) 3 moiety was considered as the stoichiometry of the condensation reaction.
To graft DAMO moieties to the outer surface of the MSNs (MSN-DAMO), the as-synthesized material containing the organic template filling the pores was used in the condensation reaction and its removal was performed afterwards [43]. By using this approach, a preferential functionalization of the external surface of MSNs is expected [43]. A 100% nominal degree of the external surface functionalization was considered to attain a maximum coverage.
However, for the preparation of the MSN-G3 material, we kept in mind the possibility that the size of third generation dendrimers would prevent the functionalization of the interior of the MCM-41 pores [40]. In addition, the expected steric hindrance between third generation dendrimers at the silica surface prevents from a total silanol functionalization [33]. Thus, for the preparation of the MSN-G3 material, we calculated the amount of dendrimer required to functionalize a quarter of the external surface of the MSNs.

Materials characterization
The organic content of the MSN materials was quantified from TGA and elemental chemical analyses to follow the incorporation of the alkoxysilane derivatives ( Table 1) 4) and (5) Table 1). In MSN-DAMO most of the silanol groups (-Si-OH) over the nanoparticle surface have reacted during the functionalization, therefore not contributing to the -Si-O − formation in water (see also 29 Si MAS NMR discussion below). On the other hand, the pK a values of amine moieties in G3 are slightly lower than pK a values of amines in DAMO, mainly due to a polyelectrolyte behavior in the macromolecule that reflects the effect of electrostatic repulsion between different charged sites. This effect entails the decrease of the pK a value with the increase of the protonation degree. Therefore, under the same conditions in the water media protonation would be more favorable for MSN-DAMO than for MSN-G3 material. The same behavior was found in PBS medium; however, ζ-potential values measured in THB were found to be negative for bare  The presence of the G3 dendrimer on the surface of the nanoparticles is supported by the chemical shifts of the hybrid material, which closely match those of the respective functional groups in the solution NMR spectrum of the free compound [33]. The broadening of the signals with respect to the solution NMR spectrum is a normal fact due to the immovilization of the organic matter onto the solid nanoparticles. A sligth downfield shift of the signals can be adscribed to the use of different deuterated solvents as well as techniques and equipments [44,49]. Concerning the structural characterization of MSNs before and after functionalization, XRD confirms the 2D hexagonal mesoporous arrangement, showing three well-defined reflections that can be indexed as 10, 11 and 20 of a p6mm plain group for all the materials (Fig. 3A). These results agree with TEM studies (Fig. 3B-D), which show spherical shaped nanoparticles with an average diameter of ca. 120 nm. Regarding the mesoporous structure, TEM images evidence the presence of a honeycomb-like mesoporous arrangement typical of MCM-41 structure. These results reveal that neither the morphology nor the mesoporous order are affected by the used functionalization procedures. Fig. 3E shows the N 2 adsorption isotherms corresponding to pristine MSN, MSN-DAMO and MSN-G3 materials, respectively. All isotherms are IV type according to the IUPAC classification, which are characteristic of mesoporous materials exhibiting parallel cylindrical pores [50]. The appropriated treatment of the N 2 adsorption-desorption data allows to determine the main textural parameters (Table 3). Concerning the S BET , it experiences a decrease in both functionalized samples compared to pristine MSN, because of the post-grafting of organic moieties [51]. However, the pore volume (V P ) remains unaltered in MSN-G3 because, in good agreement with NMR results, due to the high volume of G3 this macromolecule is not able to functionalize the inner mesoporous cavities [33]. However, V P undergoes a remarkable drop in MSN-DAMO. This fact could be ascribed to the smaller size of DAMO, which may partially block the pore entrances. These findings are in good agreement with the calculated pore diameter (D P ) and wall thickness (t wall ) values, which remain constant in MSN-G3 and experience a significant variation (D P decrease and t wall increase) in MSN-DAMO, compared to unmodified MSN material.

"In vial" levofloxacin release performance
LEVO was loaded into the different materials by forming well-dispersed suspensions of nanoparticles in a solution of LEVO in ethanol or CH 2 Cl 2 , i.e., following an impregnation method (for further details see Experimental Section) [34]. The amount of LEVO loaded into the samples was 3.2%, 5.0% and 7.8% (wt.) for MSN-L, MSN-DAMO-L, MSN-G3-L, respectively. The effective drug loading was also evaluated by the reduction in the V P and pore diameter measured by N 2 adsorption (see Table S1 in Supporting Information). As above explained, the G3 macromolecule is not able to functionalize the inner mesoporous cavities and therefore the V P remains almost unaltered in MSN-G3 compared to pristine MSN, being available more pore volume accessible for drug loading than in MSN-DAMO. In addition, the impregnation method followed for LEVO loading in MSN-G3 is performed before the anchorage of the alkoxysilane on the external surface so the accessibility of LEVO to the inner pores is not impeded. To

Evaluation of bacterial internalization
To directly observing the internalization capability on bacteria cells confocal microscope studies were conducted. in the positively charged amine functional groups of MSN-G3 that are exposed to the negatively charged bacterial wall [10]. Therefore, MSN-G3 not only binds to the negatively charged phospholipid membranes, but also provokes a slight change in membrane permeability, which finally leads to nanosystem internalization into the bacteria, as it has been reported elsewhere for other systems [13,55]. Further studies to analyze the effect of MSN-G3 internalization in the bacteria at longer times, as well as to elucidate the interaction and internalization between the nanosystem here proposed and bacteria cell membrane are being conducted for a better understanding of the mechanism against Gram-negative bacteria. Moreover, to evaluate the potential antimicrobial effect of the different samples (MSN, MSN-DAMO and MSN-G3), preliminary studies were carried out by incubation of 10µg/mL of each material in 10 8 bacteria/mL suspensions during 90, 180 and 1440 min, followed by plating and counting of CFUs (Fig. S3A in Supporting Information). The obtained results reveal no significant antimicrobial effect for all materials after 90 min of assay, in good agreement with confocal microscopy images (Fig. 5)

In vitro antimicrobial efficacy against E. coli biofilms
After evaluating the internalization effectiveness and antibiotic release profiles, in vitro antimicrobial assays can be performed to assess the efficacy of these nanosystems against E. coli biofilms. Fig. 6 shows showing vastly live cells (green), dispersed mucopolysaccharide (blue) fragments and scarcely dead cells (red). Finally, the best results are derived from MSN-G3-L assays, which is able to totally destroy the biofilm since no blue staining and almost negligible live cells are observed. It should be mentioned that even at nanoparticles concentrations of 5 µg/mL the biofilm is destroyed, existing dead bacteria (red) covering almost the entire biofilm surface (Fig. S2B in Supporting Information).
This finding agrees with the synergistic combination of the bacterial penetration capability of polycationic dendrimers present in MSN-G3 (Fig. 5) and the antimicrobial effect of LEVO released from this matrix (Fig. 4).
In addition, control experiments with G3 dendrimer, LEVO and a combination G3 and LEVO were carried out (Fig. S4 in Supporting Information). The concentration of each compound was that corresponding to the G3 grafted to MSNs (MSN-G3) and to the amount of LEVO released from MSN-G3-L after 90 min during the "in vial" assay. The results evidence not significant antimicrobial effect in any of the cases with respect to the control.

Conclusions
A new class of antimicrobial agent, named "nanoantibiotic", based on mesoporous silica nanoparticles (MSNs) decorated with polypropyleneimine dendrimers of third generation (G3) and loaded with levofloxacin (LEVO) as antibiotic has been developed. The covalently grafting of these G3 dendrimers to MSNs (MSN-G3) allows an effective internalization in Gram-negative bacteria.
This internalization can be ascribed to the high density of positive charges and flexibility on the surface of the MSN-G3, which produces electrostatic interactions with the negatively-charged bacteria walls and triggers their permeabilization. Furthermore, the LEVO antibiotic loaded into the mesoporous cavities is released in a sustained manner at effective antimicrobial dosages. These findings demonstrate that the synergistic combination of bacterial internalization and antimicrobial agents into a unique nanosystem provokes a remarkable antimicrobial effect against bacterial biofilm. This novel nanoantibiotic is envisioned as a promising alternative to the current available treatments for the management of infection.  carbons, i.e. they may be reversed. C) 29 Si MAS NMR spectra of the MSN materials synthetized in this work. For A) and B) signals due to ethoxy groups from the incomplete hydrolysis and condensation during the sol-gel synthesis of the MSNs are indicated with a # mark. Residual methoxy groups from DAMO are also contemplated.

Figure Captions
As well, the signals due to residual surfactant in the materials are indicated with a * mark in the 13 C spectrum.     Graphical abstract