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Composite Porous Silicon-Silver Nanoparticles as Theranostic Antibacterial Agents Taeho Kim, Gary B Braun, Zhi-gang She, Sazid Hussain, Erkki Ruoslahti, and Michael J. Sailor ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b09518 • Publication Date (Web): 18 Oct 2016 Downloaded from http://pubs.acs.org on October 22, 2016

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ACS Applied Materials & Interfaces

Composite Porous Silicon-Silver Nanoparticles as Theranostic Antibacterial Agents** Taeho Kim,†,⊥ Gary B. Braun,‡,§,⊥ Zhi-gang She,‡ Sazid Hussain,‡ Erkki Ruoslahti,‡,§ and Michael J. Sailor,*,† †

Department of Chemistry and Biochemistry, University of California, San Diego

La Jolla, CA 92093, USA ‡

Cancer Research Center, Sanford Burnham Prebys Medical Discovery Institute

La Jolla, California 92037, USA §

Center for Nanomedicine and Department of Cell, Molecular and Developmental Biology,

University of California, Santa Barbara Santa Barbara, California 93106-9610, USA

Corresponding Author: *

E-mail: [email protected]

⊥

T.K. and G.B.B. contributed equally to this work.

Conflict of Interest: The authors declare no competing financial interest.

KEYWORDS: Plasmonic nanoparticles, electroless deposition, controlled release drug delivery, Pseudomonas

aeruginosa,

Staphylococcus

aureus,

time-gated

photoluminescence,

photoluminescence quenching, anti-bacterial

ACS Paragon Plus Environment

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ABSTRACT: A theranostic nanoparticle with biochemically triggered antibacterial activity is demonstrated. Metallic silver is deposited onto porous silicon nanoparticles (pSiNPs) by galvanic displacement. When aqueous diaminesilver ([Ag(NH3)2]+) is used as a silver source, the pSiNPs template the crystalline silver as small (mean diameter 13 nm) and well-dispersed nanoparticles embedded within and on the larger (100 nm) pSiNPs. The silver nanoparticles quench intrinsic photoluminescence (PL) from the porous silicon matrix. When exposed to an aqueous oxidant, the silver nanoparticles are preferentially etched, Ag+ is released into solution, and PL from the pSi carrier is recovered. The released Ag+ results in 90% killing of (Gram-negative) P. aeruginosa and (Gram-positive) S. aureus within 3h. When conjugated with the TAT peptide (sequence RKKRRQRRR), the pSi-Ag nanocomposite shows distinct targeting to S. aureus bacteria in vitro. Intravenously injected TAT-conjugated pSi-Ag nanoparticles accumulate in the liver, spleen, and lungs of mice, and in vivo release of Ag+ and recovery of PL from pSi is demonstrated by subsequent intraperitoneal administration of a hexacyanoferrate solution. The released Ag+ leads to a significant bacterial count reduction in liver tissue relative to the control. The data demonstrate the feasibility of targeted and triggered delivery of antibacterial Ag+ ion in vivo, using a self-reporting and non-toxic nanocarrier.


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INTRODUCTION Silver has long been known to display broad spectrum antibacterial activity and low toxicity to human tissues.1-2

Although various silver-based antimicrobials have been developed and

topically used in wound dressings, silver-based small molecule drugs are not practical in vivo due to the low bioavailability of free Ag+ ions.3 Metallic silver nanoparticles offer a promising alternative,4-5 though there is a need for systems that can effectively deliver the nanoparticles to infected tissues and convert them into therapeutically active silver ion in vivo. Porous silicon nanoparticles (pSiNPs) have recently received attention as a vehicle for in vitro and in vivo controlled drug delivery and theranostic applications.6 For controlled drug delivery, pSiNPs possess an open interior pore volume that can load and protect therapeutic agents,7,8 and the relatively large external surface allows conjugation with targeting moieties9-10 such as peptides11-12 and antibodies13 to allow selective homing to tissues. Furthermore, the porosity and surface chemistry can be readily tuned to enhance the solubility of loaded drugs, as demonstrated for the sustained release of chemotherapeutic agents14-15 or antibacterial compounds16 from pSi carriers. For theranostic17 applications, the intrinsic near-infrared photoluminescence of pSiNPs can be used to monitor targeting and degradation after the release of loaded therapeutics.

In particular, luminescent silicon nanoparticles18 provide a benign

alternative to quantum dots based on cadmium or indium,19-20 because the degradation byproducts of pSi are non-toxic and readily excreted.6-7 A distinguishing feature of pSi is that it is a good reducing agent. The skeleton of asprepared pSi consists of elemental Si with Si–H species on the surface, and both of these chemical species are competent reducing agents for a variety of metal salts and organic molecules.21 Redox-active drugs such as cisplatin22-23 and doxorubicin24 can be reduced by pSi,


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and metals such as nickel, copper, and silver can be coated onto pSi by electroless deposition.2528

This reduction reaction has been useful in preparing composite nanostructures with interesting

magnetic,29 catalytic,27 and plasmonic properties.30 In this work we harness the electroless deposition process to form silver nanoparticles within a pSi host matrix. The resulting pSi-Ag nanoparticles are colloidally stable, but under mild oxidizing conditions they release silver ion that is active against both gram-positive and gram-negative bacteria. A characteristic of the metallic silver nanoparticles is that they quench the intrinsic photoluminescence from pSi, which is recovered as the Ag nanoparticles dissolve. Capitalizing on this phenomenon, we demonstrate the potential for theranostic application of the pSi-Ag nanocomposites in a mouse infection model.

RESULTS AND DISCUSSION A Si-SiO2 core-shell formulation of pSiNPs was prepared using electrochemical etch of crystalline Si in an ethanolic HF electrolyte, lift-off of the pSi film, ultrasonication, and activation in an aqueous solution as previously described (see Supporting Information).31 During the activation step, a thin layer of silicon oxide grows on the hydrogen-terminated pSi surface. This SiO2 layer acts as an electronically passivating shell32 that enables relatively efficient photoluminescence from the quantum-confined Si domains in the pSiNPs.33 The as-formed pSiNPs had overall dimensions on the order of 100nm, with pores of ~15 nm running predominantly in the crystallographic direction (Figure 1a, Figure 2a, Figure S1). The oxide shell was sufficiently permeable such that exposure to aqueous diaminesilver ([Ag(NH3)2]+) resulted in electroless deposition of metallic Ag in the form of well-defined nanoparticles of size 5-20 nm within the pSiNP scaffold, with the smaller (~5 nm) AgNPs inside the pores and the


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larger (~15-20 nm) AgNPs on the exterior surface (Figure 1b, Figure 2b, Figure S1). The identity of the Ag nanoparticles was confirmed by energy-dispersive X-ray spectroscopy (EDS), inductively coupled plasma-optical emission spectroscopy (ICP-OES), X-ray diffraction (XRD), and by their characteristic plasmon band at 420nm in the UV-visible absorption spectrum (Figure 2). The X-ray diffraction spectrum also confirmed the presence of crystalline silicon in the pSiAg nanoparticle constructs (pSi-AgNPs) (Figure 2d). The maximal mass loading of Ag was 39.8% based on total pSi-AgNP mass (Figure S2). No free Ag NPs were detected in the TEM images, indicating that the Ag NPs formed predominantly at the surface or inside the pores of the pSiNPs. Fourier-transform infrared (FTIR) measurements on the isolated NPs revealed a decrease in surface Si-H species and an increase in silicon oxide (Figure S3), consistent with previous reports of electroless metal deposition in pSi.25

Figure 1. Schematic and representative TEM images of electroless deposition and oxidative stripping of silver nanoparticles within porous Si (pSi) nanoparticle carriers. Exposure of porous Si nanoparticles (a) to Ag+(aq) ions results in reductive deposition of isolated Ag nanoparticles and concomitant oxidation of the porous Si scaffold, generating a pSi-Ag composite (b). The plasmonic Ag nanoparticles effectively quench photoluminescence from the Si nanostructure,


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and the resulting pSi-Ag nanocomposite is not photoluminescent. Exposure of the material to the mild oxidant ferricyanide ion re-oxidizes the Ag nanoparticles to Ag+ ion, which is released from the pSiNPs into aqueous solution (c). At this point the pSiNPs regain photoluminescence.

The mean hydrodynamic diameter (by dynamic light scattering, DLS) of the pSiNPs in aqueous solution was 93 nm, with a polydispersity index (PI) of 0.137 (Figure S4). After silver deposition the mean diameter and PI increased slightly (97 nm and 0.192, respectively), and the DLS measurement displayed a symmetrical distribution. The pSi-AgNPs showed excellent colloidal stability in aqueous media, maintaining hydrodynamic diameters of 90-110 nm for 1 month. Furthermore, pSi-AgNPs that were coated with polyethylene glycol (PEG) displayed stable mean diameter and PI values (177 nm, PDI: 0.278) when dispersed in phosphate-buffered saline (PBS) solutions (Figure S4d). By contrast, bare Ag nanoparticles are known to aggregate in the absence of surfactants or other stabilizing agents.34


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Figure 2. Characterization of silver deposited porous silicon nanoparticles (pSi-AgNPs). (a) Low-magnification TEM images of pSiNPs prior to silver deposition. (b) Low-magnification TEM images of silver-deposited pSiNPs (pSi-AgNPs). (c) UV-visible spectra of pSi-AgNPs prepared with increasing quantity of added [Ag(NH3)2]+: 0, 1, 2, 4, 8 and 16 µL of 0.1 M [Ag(NH3)2]+ solution, added to 0.4 mL of pSiNP in deionized water, stirred for 1h. Inset: Photograph of pSi-AgNP suspensions prepared with increasing concentration of silver ion. No distinct absorption band appears in the spectrum of pSiNPs under these conditions, but addition of [Ag(NH3)2]+ generates a broad band in the absorption spectrum at 420 nm, assigned to the surface plasmon resonance of Ag NPs. (d) XRD patterns of pSi-AgNPs, revealing the presence of crystalline silicon and silver. The XRD patterns for the cubic diamond phase of Si (JCPDS Card No. 27-1402, shown in red) and for the face-centered cubic (fcc) phase of Ag (JCPDS Card No. 4-783, shown in blue) are indicated as "ref-Si" and "ref-Ag", respectively.

The reaction of pSi with the nobler metals is known to form isolated metal deposits by galvanic displacement,25,

35

although the process can be difficult to control.36 In the present


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system, the extent of silver deposition was readily controllable by varying the concentration of [Ag(NH3)2]+ in the deposition solution (Figure 2c). For a fixed reaction time and concentration of pSiNPs, the intensity of the plasmon band of the silver nanoparticles at 420 nm increased monotonically with increasing [Ag(NH3)2]+. All the products contained silver nanoparticles of relatively uniform size, and these nanoparticles were uniformly dispersed and deposited in the pSiNPs (Figure 1b, 2b).

The identity of the silver precursor appears to be important in

determining the morphology of these composites; when AgNO3 was used as the silver source, Ag nanoparticles formed as larger heterogeneous aggregates, dispersed on the outer surface or existing as isolated AgNPs not associated with the pSiNPs (Figure S5). This is the metal morphology more typically seen with immersion-plated pSi.25, 35, 37

Si + 2H2O 4Ag+ + 4e-

SiO2 + 4H+ + 4 e-

(1)

4Ag

(2)

The difference in metal nanoparticle morphology observed with the two Ag+ sources (the amine complex vs. free silver ion) can be understood in terms of the local cell model of metal deposition in pSi, proposed by Ogata and coworkers.25 In this model, the silicon oxidation halfreaction (eq. 1) and the metal reduction half-reaction (eq. 2) occur at separate locations on the silicon nanostructure.

Silicon oxide formation supplies electrons, which travel through the

semiconducting silicon matrix to the region of growing metal islands, where additional metal ions are reduced. The metal surface is more favorable for metal deposition, and the result is a heterogeneous material with isolated metal deposits and patches of insulating silicon oxide containing no metal.

4Ag(NH3)2+ + 4eSiO 2 + 2H2O

4Ag + 8NH3

(3)

Si(OH) 4

(4)


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A distinct feature of the chemistry of the bis-amine adduct is that it releases ammonia upon reduction of the silver ion (eq. 3).

Whereas the Si oxidation half-reaction generates one

equivalent of acid (H+) for every electron released (eq. 1), the Ag(NH3)2+ reduction half-reaction generates two equivalents of base (NH3) for every electron consumed. The released ammonia will thus locally raise the pH, and this can be expected to solubilize silicon oxides formed during the redox reaction (eq. 4),38 limiting buildup of oxides at the surface.

In addition, the

[Ag(NH3)2]+ ion is a weaker oxidizing agent than Ag+ ion, which will tend to slow the interfacial electron transfer rate at the metal nanoparticles. We suggest that both the elevated local pH and the slower electron transfer kinetics are responsible for the more uniform AgNP size and deposition morphology obtained with the [Ag(NH3)2]+ reactant.

Both of these factors are

expected to minimize the segregation between metal and silicon oxide domains that is a characteristic of the local cell mechanism driving formation of large, heterogeneous metal deposits.


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Figure 3. Photoluminescence properties of pSi-AgNPs. (a) Photoluminescence image (λex = 365 nm) of cuvettes containing aqueous dispersions (100 µg/mL, in deionized water) of (from left to right) empty porous Si nanoparticles ("pSi"), porous Si nanoparticles after incorporation of Ag nanoparticles ("pSi-Ag"), and pSi-Ag nanoparticles after addition of ferricyanide oxidant ("pSiAg(Etch)"). The ferricyanide oxidant selectively etches the Ag nanoparticle component of pSiAgNPs, regenerating empty pSiNPs and recovering the intrinsic photoluminescence from the quantum-confined Si domains in the nanostructures. (b) Steady state photoluminescence (PL) spectra (λex = 365 nm) collected from dispersions of pSi-AgNPs of increasing Ag content. The Si:Ag mass ratio for each sample is given in the legend. (c) Progression of PL spectra of a pSiAgNP sample with successive addition of the hexacyanoferrate (HCF) oxidant (calculated initial concentration of [Fe(CN)6]3− is 0.25, 0.5, 1, 2, and 4 mM, as indicated). Trace labeled "pSi" is the PL spectrum of the pSi nanoparticles prior to Ag deposition. (d) Normalized intensity decay of PL from the samples shown in (a) as a function of time: gate width 10 µs, gate step increase 10 µs, 20 accumulations, series length: 50.

Photoluminescence of the pSi matrix was strongly affected by the presence and quantity of the silver nanoparticles. As more Ag nanoparticles formed within the pSi matrix, the degree


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of PL quenching increased until it was essentially extinguished (Figure 3a,b). Although Ag metal absorbs light by virtue of its high optical density, metal-induced quenching of PL from pSi is generally too strong to be attributed to optical absorbance,39 and mechanisms involving energy transfer from the silicon excited state(s) to the metal plasmon, or charge transfer from pSi to metal acceptor states have been invoked.37,

40

In the present case we attribute the observed

quenching to photoinduced Si-to-metal electron transfer, because of the low spectral overlap between the silver plasmon and the pSiNP PL spectrum (Figure 2c). Silver nanoparticles are prone to oxidation due to their high surface energy and affinity for oxygen, releasing silver ions under ambient conditions in aqueous media.41-42 However, when contained within the pSi-AgNP constructs, the Ag nanoparticles were not readily oxidized to suppress premature release of Ag+. We attribute this stability to the presence of the pSi scaffold, which acts as a sacrificial anode to continuously supply electrons to silver at the interface. This interpretation is consistent with previously reported hybrid silver nanostructures such as pSi-Ag, Au-Ag and graphene-Ag, which also display a diminished tendency toward Ag oxidation.43-44 Although stable under ambient aqueous conditions, the silver nanoparticle payload could be induced to release silver ions in the presence of a mild oxidant. Here, we used ferricyanide ion, [Fe(CN)6]3−, which has been demonstrated to be a biocompatible,45 cell membraneimpermeable silver oxidation agent that has been used to quantify cellular internalization of nanoparticles both in vitro and in vivo.46 Exposure to a solution 4 mM in [Fe(CN)6]3− selectively etched the silver nanoparticles from the pSi-AgNPs within 5 min. Removal of Ag was apparent in the TEM images (Figure 1) and in the DLS measurement (Figure S6). The mean hydrodynamic diameter of the pSiNPs before Ag loading was ~90 nm and after Ag removal it


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had decreased to ~80 nm. The TEM images also indicated a slight decrease in size (Figure 1a and c), although the general morphology of the porous nanostructure was retained through the Ag deposition/removal cycle. The data are consistent with the expectation that the Si skeleton was partially consumed during Ag deposition (eqs 1 and 4). Further evidence of degradation and dissolution of the Si skeleton was observed in the PL spectrum, which derives from quantum confinement of electrons and holes in the thin crystalline features in the Si nanostructure. The PL spectrum of the pSiNPs prior to Ag deposition was centered at a lower energy than the spectrum obtained after Ag removal (Figure 3c).

This blue shift upon deposition/removal

indicates that the quantum-confined Si domains in the nanostructure were thinned during the process, consistent with the partial consumption of the Si skeleton. Up to 65% of the original PL quenched by Ag deposition was recovered upon oxidative Ag etching (Figure 3a,c). Timeresolved PL spectra indicated recovery of the long-lived (~60 µs) emission lifetime from the quantum-confined Si emitters in the pSiNPs, and an additional short-lived (nanoseconds) component, attributed to a silicon surface oxide defect emission,47 also became apparent (Figure 3d). Oxidative dissolution of the silver nanoparticles and recovery of PL from pSi could also be achieved with the biologically relevant reactive oxygen species (ROS) hydrogen peroxide (H2O2)48

and

superoxide/peroxynitrite,

the

latter

generated

by

addition

of

3-

morpholinosydnonimine (SIN-1) (Figure S7).49 ROS are commonly generated by host tissues in response to pathogenic infection,50 and the fact that PL from the pSi-AgNPs is recovered upon oxidation-triggered release of Ag+ suggests that PL recovery could be used as an indicator of the presence of bacterial infection and of the release of anti-bacterial Ag+. Thus the pSi-AgNP system could form a useful theranostic agent for simultaneous detection and treatment of bacterial infections.


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We tested the theranostic potential of the pSi-AgNP system in vitro, using cultures of gram-negative Pseudomonas aeruginosa (P. aeruginosa) and gram-positive Staphylococcus aureus (S. aureus). There is a great need for new therapeutics against these organisms, in particular for the strains that have evolved resistance to current antibiotics.51 Silver ion is active against these pathogens, and it shows relatively low toxicity to human tissues. Although the antimicrobial mechanisms of silver are not fully understood, silver cations (Ag+) are known to cause denaturation of microbial enzymes and other proteins, and to interfere with bacterial DNA replication.52 Silver cation-based drugs are not effective in vivo primarily because Ag+ interacts with chloride ion, thiols, and other biological compounds in the human body, which decreases the bioavailability of free Ag+ ions.3 Efforts to harness the antimicrobial activity of silver in vivo have employed various silver formulations,53 including organometallic complexes,54 metallic silver nanoparticles,4-5 and polymeric,55 dendrimeric,56 or inorganic nanoparticle57,58 hosts. Literature reports have noted the difficulty in generating high concentrations of bioactive Ag+ ion from AgNPs.3, 59 In the present system we found that addition of a selective etchant induced rapid and essentially complete release of silver ion from the pSiNPs (Figure 1c, Figure S6). When co-treated with the ferricyanide oxidant, pSi-AgNPs killed > 90% of S. aureus within 3h (50 µM Ag+ equivalent, Figure 4a-b). The level of killing was comparable to the positive control, in which an equivalent quantity of silver in the form of dissolved Ag+ ion (AgNO3) was administered.

On the other hand, empty pSiNPs or ferricyanide on its own exhibited no

significant bacterial killing and pSi-AgNPs introduced without the ferricyanide oxidant showed substantially less antimicrobial activity. The antimicrobial activity of the pSi-AgNP construct against P. aeruginosa (Figure 4c) was substantially greater than against S. aureus; in the presence of ferricyanide oxidant, pSi-AgNPs inhibited > 90% of P. aeruginosa growth within 3h


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at an equivalent Ag+ concentration of 15 µM (Figure 4c), three-fold lower compared to S. aureus. This result is consistent with the known antibacterial activity of free Ag+ ion, which is more potent against gram-negative bacteria such as P. aeruginosa than gram-positive bacteria such as S. aureus. As with S. aureus, pSi-AgNPs showed substantially lower activity when no oxidant was added, and the empty pSiNP or ferricyanide-alone controls showed no significant antimicrobial activity.

Figure 4. Antibacterial activity of pSi-AgNPs against S. aureus and P. aeruginosa. (a) Representative images of S. aureus colonies prepared by surface plating of 0.1 mL of S. aureus dilution on blood agar plates. S. aureus (2.6×107 CFU/mL in medium) was treated with 3 µL stock solutions of pSiNPs ("control"), pSi-AgNPs ("pSi-Ag", 50 µg Ag/mL), or pSi-AgNPs (50 µg Ag/mL) followed by 1 mM of hexacyanoferrate oxidant solution ("pSi-Ag(Etch)"), incubated at 37 ºC for 12 h, and plated. The presence of organisms is confirmed by the golden color of the colonies on the media. (b)-(c) Inhibition of growth of gram-positive S. aureus (b) and gramnegative P. aeruginosa (c), evaluated by optical density (OD) measurement at 600 nm after 3 h of incubation with the indicated concentrations of: saline ("control"); 1 mM of [Fe(CN)6]3−


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("Etchant only"); pSiNPs containing no Ag ("pSi"); pSi-AgNPs ("pSi-Ag"); pSi-AgNPs with added 1 mM of [Fe(CN)6]3− ("pSi-Ag(Etch)"); and AgNO3 solution ("AgNO3"). All experiments performed at 37 °C in LB media. Inhibition concentration values presented here represent the initial concentration of Ag+ in the nanoparticles determined by ICP-OES. Error bars represent standard deviations of the measurements (N = 3).

A substantial challenge for in vivo delivery of nano-therapeutics is the lack of target binding. In this work we evaluated targeted delivery of silver ion to bacteria using the HIV-1 TAT (CRKKRRQRRR) peptide as a targeting agent, attached to pSi-AgNPs. The TAT peptide is cationic, and it has been shown to be an efficient molecule for translocating nanoparticles into various mammalian cells and microorganisms.60-61 In particular, AgNPs conjugated with the peptide G3R6TAT (GGGRRRRRRYGRKKRRQRR) have recently been shown to display bactericidal activity with selectivity for S. aureus.62 We conjugated a cysteine-functionalized TAT construct (Rhodamine-C-C6-RKKRRQRRR-NH2) onto pSi-AgNPs via a maleimide-PEG moiety (TAT(Rhod)-pSi-AgNPs), and studied targeting efficiency to S. aureus. The fluorescent rhodamine tag was used to track the Ag-containing formulation, because the intrinsic luminescence from pSi was quenched by Ag in this construct (Figure S8). A transgenic strain of S. aureus expressing green fluorescent protein (GFP) was used to track the bacteria. The in vitro images clearly exhibited co-localization of the NPs with the targeted bacteria (Figure 5a), whereas control PEG-terminated pSi-AgNPs showed no co-localization with the bacteria (Figure S9). Furthermore, when administered to a GFP-expressing S. aureus biofilm and treated with ferricyanide oxidant, the TAT(Rhod)-pSi-AgNP formulation significantly reduced the biofilm mass, as assessed by a decrease in GFP intensity (Figure S10). Control experiments with silverfree pSiNPs or with ferricyanide alone (no pSi-AgNPs) showed no significant decrease.


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Figure 5. In vitro bacterial targeting and in vivo performance of pSi-AgNPs. (a) Fluorescence microscope images of S. aureus incubated with pSi-AgNPs conjugated to the TAT targeting peptide and a rhodamine indicator dye (TAT(Rhod)-pSi-AgNPs). Left panel: "TAT(Rhod)-pSiAgNPs" represents the red channel emission from the rhodamine dye attached to the NPs. Center panel: "GFP-S. aureus" is the green channel emission from GFP expressed by this strain of S. aureus. Right panel: Overlay of the two fluorescence channels, showing targeting of the TAT peptide-conjugated construct to gram-positive S. aureus. The NPs (0.05 mg) were incubated with the S. aureus culture (5 mL) for 30 min, and a drop of this solution was then placed on a glass microscope slide and washed prior to imaging. (b) Fluorescence microscope images of tissue histology sections for the indicated organs harvested from a healthy mouse, 2 hr after injection with TAT(Rhod)-pSi-AgNPs (5 mg NP/kg body mass). The nanoparticles were primarily accumulated in the liver and spleen, with some accumulation in the lungs. Red channel: rhodamine signal from the TAT(Rhod)-pSi-AgNPs; Blue channel: DAPI nuclear stain. (c) Representative time-gated (TG) photoluminescence images (λex= 365 nm, λem= 460 nm longpass filter; gate width: 400 µs, gate delay: 10 µs, 100 accumulations) of livers harvested from mice (N=3 for each group). Live mice were injected with TAT(Rhod)-conjugated pSiNPs


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("pSi"), TAT(Rhod)-conjugated pSi-AgNPs ("pSi-Ag"), or phosphate buffered-saline control ("PBS") via intravenous (tail vein) injection. The animal whose liver is denoted "pSi-Ag(Etch)" was treated with hexacyanoferrate-thiosulfate (etchant) solution by intraperitoneal injection prior to sacrifice, which selectively etched the Ag nanoparticles and re-activated PL from the silicon nanoparticles. (d) Signal-to-noise ratio (SNR) calculated for photoluminescence from pSiNPs accumulated in the liver for each group. ** p < 0.01; n.s., not significant, from two-tailed Student’s t-test. Data are presented as means ± SD (N = 3). (e) Bacterial counts in liver tissue showing bactericidal action of the TAT(Rhod)-pSi-AgNPs(Etch) group treated 2 h after intravenous inoculation of S. aureus (7.8×108 CFU/mL in medium). Nanoparticles (5 mg NP/kg body mass) and the etchant solution was administered 2 h after infection, and the livers were harvested 4 h later (6 h after infection). Viable bacteria in the liver tissue were quantified by multiplying colony counts by the dilution ratio. Mean ± SD (N = 3 for each) of S. aureus were calculated as log10 CFU/g in homogenized liver tissues. Statistical significance was calculated with the Student’s t-test; * p < 0.05 vs the untreated control (etchant injection only).

Finally, we assessed the compatibility and theranostic potential of the targeted pSi-AgNP system with pilot in vivo studies. The TAT(Rhod)-pSi-AgNP formulation was injected through intravenous (iv) route and allowed to circulate for 1 h prior to perfusion and harvesting of the organs. The tissue histology sections revealed that the NPs had mostly accumulated in the liver (27.9 ± 2.4% injected dose/g tissue), spleen (16.7 ± 3.1%), and lungs (2.6 ± 0.8%) of the animals (Figure 5b and Figure S11). In a separate experiment, one group of animals was treated with the etchant hexacyanoferrate-thiosulfate (HCF-TS) by intraperitoneal (ip) injection 1h after administration of the TAT(Rhod)-pSi-AgNPs. After an additional 1h of circulation, the animals were perfused and the organs harvested. This experiment was performed to assess if in vivo release of Ag+ ion from the pSiNP carrier could be detected using the recovery of intrinsic Si PL. The study focused on the liver of the animals, where nanoparticle accumulation was greatest. Microsecond time-gated (TG) photoluminescence imaging was employed, due to its ability to discriminate the longer-lived excited state of pSiNPs from exogenous dyes, media, and intrinsic tissue autofluorescence (Figure S12).11 The TG images revealed that PL from the pSiNPs appeared only after in vivo introduction of the ferricyanide etchant; the liver from the group of


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animals injected with TAT(Rhod)-pSi-AgNPs but no etchant, displayed baseline signals comparable to the saline controls (Figure 5c,d). This result is consistent with the bacterial inhibition experiments, where oxidation-induced release of Ag+ ion correlated with an increase in the intrinsic, NIR PL signal from the pSiNP carrier (Figure S12a). Also consistent with the in vitro experiments (Figure 3c), the liver from the control animal group injected with silver-free TAT(Rhod)-pSiNPs displayed the brightest signal in the TG images. We conclude that the in vivo oxidant selectively etched the Ag nanoparticles and re-activated PL from the silicon NPs, which shows the feasibility of using the long-lived excited state of silicon to monitor silver ion release in this theranostic system. To demonstrate therapeutic efficacy of pSi-AgNPs against infected tissues in vivo, mice were inoculated with S. aureus and 2 h later the TAT(Rhod)-pSi-AgNP formulation was administered (via intravenous injection) along with the ferricyanide etchant solution (via intraperitoneal injection). The animals were perfused 4 h later, and bacterial counts in the liver tissues were quantified. Bacteria in the treated group of mice (pSi-Ag(Etch)) were reduced by 10-fold (log10 CFU/g = 6.7 vs 7.6) when compared to the untreated control (Figure 5e). These results indicate that the nanosystem is capable of exhibiting effective bactericidal activity in vivo and they are correlated with the nanosystem's in vitro performance (> 90% S. aureus growth inhibition within 3h). Furthermore, when administered to healthy mice, histopathology revealed no significant toxicity of this pSi-Ag(Etch) system one day post-administration (Figure S13). As indicated by the observed antibacterial activity in vitro (Figure 4) and in vivo (Figure 5), by the TEM data (Figures 1, S6, and S7), by the optical density data (Figures S6 and S7), and by the recovery of photoluminescence from the porous Si nanoparticles both in vitro (Figure 3) and in vivo (Figure 5), the silver nanoparticles dissolve and are released from the pSi-AgNPs in


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the form of silver ions. Prior work has shown silver ion to have low background toxicity to cells in vitro and to mice in vivo while displaying high bactericidal activity in a mouse infection model.63 The in vivo toxicity of the present system also appears to be directed primarily toward bacteria, combining the high affinity of TAT and the greater toxicity of silver ions to bacteria relative to mammalian cells. Recent examples of nanoparticle-enabled in vivo delivery of silver have shown good efficacy against bacterial infections when administered directly to the infected tissues. For example, shell cross-linked "knedel-like" polymeric nanoparticles (SCK NPs) loaded with a silver carbene complex (SCC) have been administered as aerosolized therapeutics in a mouse pneumonia model of P. aeruginosa.55 Additionally, mesoporous silica nanoparticles (IBN-4) loaded with silver in the form of a pH-triggered silver-indole-3 acetic acid hydrazide complexes were effective in treating intraperitoneal infections of E. coli in a mouse model via direct intraperitoneal (ip) administration of the nano-therapeutic.57 The current nanoparticle system differs from these studies in that it shows effective delivery of an antibacterial silver nanoparticle construct via intravenous (iv) injection.

There is a significant challenge in delivering

therapeutics to diseased tissues by iv injection, and targeted systems hold promise to improve efficacy of delivery to the intended site while reducing overall systemic toxicity.64 Prior studies have shown effective targeting of silver-containing nanoparticle systems to bacteria when conjugated to antibodies65 or antimicrobial peptides66 that selectively bind to bacterial cell membranes, and the present work demonstrates that active targeting of silver can also be applied in vivo. Silver has been shown to act in synergy with some antibiotics.63 In this regard, the ability of pSi to simultaneously accommodate disparate payloads such as small molecules and nanoparticles is relevant.67-69


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CONCLUSION In summary, the in vitro and in vivo data presented here demonstrate a new non-toxic nanocarrier capable of targeted and triggered delivery of Ag+ ion in vivo. This work shows that the unique redox chemistry of porous silicon can be harnessed to load, protect, and release anti-bacterial silver, and the [Ag(NH3)2]+ chemistry is important in driving the relatively narrow size distribution of small Ag nanoparticles in the pSiNP host. The surface chemistry of these NP constructs is compatible with targeting peptides, and the TAT peptide was shown to selectively target the NPs to bacterial cells. The release of Ag+ in the vicinity of bacterial cells was triggered by mild oxidants, generating >90% killing of bacteria in vitro and in vivo. Exploring the potential for theranostic applications, we found that silver loading and release could be tracked using the intrinsic photoluminescence from silicon "quantum dot" NPs.

The

photoluminescence from the Si quantum dots was easily distinguished from tissue autofluorescence using time-gated photoluminescence imaging, and the delivery of therapeutic Ag+ resulted in a turn-on type of sensor. The released Ag+ effectively decreased the bacterial burdens in the targeted tissues with no obvious in vivo toxicity.

Acknowledgements: This work supported by the Defense Advanced Research Projects Agency (DARPA) under Cooperative Agreement HR0011-13-2-0017), by the National Science Foundation under Grant No. CBET-1603177, and by internal research funds supplied by the Sanford Burnham Prebys Medical Discovery Institute. The content of the information within this document does not necessarily reflect the position or the policy of the Government.

Supporting Information: Experimental methods and Figures S1−S14. The Supporting Information is available free of charge on the ACS Publications website at DOI:


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