Plasmon-Enhanced Controlled Drug Release from Ag-PMA Capsules

Silver (Ag)-grafted PMA (poly-methacrylic acid, sodium salt) nanocomposite loaded with sorafenib tosylate (SFT), an anticancer drug, showed good capability as a drug carrier allowing on-demand control of the dose, timing and duration of the drug release by laser irradiation stimuli. In this study, the preparation of Ag-PMA capsules loaded with SFT by using sacrificial silica microparticles as templates was reported. A high drug loading (DL%) of ∼13% and encapsulation efficiency (EE%) of about 76% were obtained. The photo-release profiles were regulated via the adjustment of light wavelength and power intensity. A significant improvement of SFT release (14% vs. 21%) by comparing SFT-Ag-PMA capsules with Ag-PMA colloids under the same experimental conditions was observed. Moreover, an increase of drug release by up to 35% was reached by tuning the laser irradiation wavelength near to Ag nanoparticles’ surface plasmon resonance (SPR). These experimental results together with more economical use of the active component suggest the potentiality of SFT-Ag-PMA capsules as a smart drug delivery system.

SFT was loaded in SiO 2 with slight modifications with respect to the procedure reported in Ref. [2]. 1 mg of hydrophobic drug was dissolved in 10 mL of dichloromethane, then 5 mg of SiO 2 microparticles were added. The system was stirred for 6 h at r.t. To remove the unloaded drug, the mixture was centrifugated at 3000 rpm for 2 min, the precipitate was collected, washed twice and dried in a vacuum desiccator to remove the dichloromethane. The amount of drug in the supernatant was evaluated by UV absorption spectroscopy following the absorbance signal of SFT at the 265 nm. Then 13% drug loading (DL%) and 76% encapsulation efficiency (EE%) values in SiO 2 microparticles were computed by using the following equations [3]: Preparation of Ag-PMA colloid solution. To obtain Ag-PMA colloid solution, a two-step photoinduced reduction strategy under UV irradiation was just developed by some of us [4]. Briefly, a PMA 30 wt% (in Milli-Q water) solution was prepared and AgNO 3 powder was added in 10:1 AgNO 3 /PMA molar ratio. The mixture was then stirred 5 h at r.t. In order to optically reduce the silver ions, the mixture was irradiated at a UV light density of 470 nW/cm 2 . Afterwards, the mixture was also irradiated with a UV light radiation density of 378 µW/cm 2 for 5 hours. The system was stable up to one month.
Synthesis of Ag-PMASH. The synthesis was performed as reported in literature [5]. Ag-PMA solution (190 mg of 30 wt% solution) was prepared and diluted in 3 mL of PB solution (pH 7.4, 50 mM). Then, EDC (43 mg, 0.22 mmol) was added to the solution and the system was left under stirring for 30 min at r.t. Afterward, PDA-HCl (29 mg, 0.13 mmol) was added and the mixture was stirred for 24 h at r.t. In order to purify Ag-PMA-PDA compound, the mixture was dialyzed against Milli-Q water for 3 days and then lyophilized to give Ag-PMA-PDA like a powder. To obtain free thiol groups, the 2-mercaptopyridine moieties must be cleaved from Ag-PMA-PDA. In detail, Ag-PMA-PDA was solubilized in 0.5 M DTT solution in MOPS buffer (20 mM, pH 8.0) to reach a final concentration of 100 g/L and the solution was stirred 30 min at 37 • C. Then, the obtained Ag-PMASH was diluted up to 100 g/L by using NaOAc buffer solution (pH 4.0, 50 mM).
Determination of thiol group content. The thiol content was spectrophotometrically evaluated by using Ellman's reagent. In details, two different solutions, 50 mM DTNB (Ellman's reagent) in sodium acetate and 1 M TRIS solution at pH 8.0 were prepared. In the meantime, SH standard calibration curve by using acetyl Cysteine, staring from 10 mM concentration, was obtained. 10 µL of sample solution were added into 990 µL of DTNB solution and the mixture was incubated for 5 min at r.t. Finally, the optical absorbance at 412 nm was measured. 350±4.5 µg/mL SH content was estimated.
Preparation of SFT-Ag-PMA capsules. SFT loaded-SiO 2 microparticles were dispersed in 50 µL of NaOAc buffer solution, by vortex and sonication treatments. 5 µL of PVPON solution 4 g/L in 50 mM NaOAc buffer was added into SFT loaded-SiO 2 particles suspension and incubated overnight. Then, the PVPON-SiO 2 system was washed and resuspended in NaOAc buffer, by using the same method previously reported for SiO 2 microparticles. Finally, PVPON-SiO 2 system was resuspended in 50 µL of NaOAc buffer solution. At the same time, 50 µL of Ag-PMASH suspension (4 g/L in 50 mM NaOAc buffer) were added into PVPON-SiO 2 suspension and incubated overnight. The obtained system was washed and resuspended in NaOAc buffer three times and finally in 50 µL of NaOAc buffer solution.
Then, the interconnection of infiltrated polymer was allowed by cross-linkage processes via thiol oxidation by using CaT 2mM solution in 10mM MES buffer, the system was stirred 15 min. To release PVPON, the capsule were repeatedly washed with PB solution (10 mM, pH 7.2) [6]. Thus SFT-Ag-PMA SiO 2 capsules were obtained. In order to form the SFT-Ag-PMA capsules the silica template was removed by using HF 5 mM solution for 5 min, followed by three centrifugation/washing cycles in PB solution at 2000 rpm for 12 min [7].

Experimental procedures used to prepare SFT-PMA capsules
Preparation of SFT-PMA capsules. These capsules were prepared by following the same method adopted for SFT-Ag-PMA capsules. In this case, PMA was not decorated with Ag NPs.

Experimental procedures used to prepare Ag-PMA capsules
Preparation of Ag-PMA capsules. These capsules were prepared by following the same method adopted for SFT-Ag-PMA capsules, but without the drug loading step.

Experimental procedures used to prepare PMA capsules
Preparation of PMA capsules. These capsules were prepared by following the same method adopted for Ag-PMA capsules. In this case, PMA was not decorated with Ag NPs.

Experimental procedures used to prepare Ag-PMA SFT* colloid solution
Here we briefly report the Ag-PMA SFT* colloid formulation strategy [4].

Preparation of Ag-PMA colloid solution.
To obtain Ag-PMA colloid solution, we performed a two-step photoinduced reduction strategy under UV irradiation. Briefly, a PMA 30 wt% (in Milli-Q water) solution was prepared and AgNO 3 powder was added in 10:1 AgNO 3 /PMA molar ratio. The mixture was stirred for 5 h at r.t. In order to optically reduce the silver ions, the mixture was irradiated at a UV light density of 470 nW/cm 2 . Afterwards, the mixture was also irradiated with a UV light radiation with density of 378 µW/cm 2 for 5 hours. The system was stable up to one month.

Characterization techniques
A Spectrum 100 Perkin-Elmer spectrometer working in ATR configuration was used to carry out FTIR measurements in the range 900-4000 cm −1 . Raman scattering response of the SiO 2 capsules was investigated after the deposition of some drops of the solution on a CaF 2 substrate. Micro-Raman spectra were excited by the 532 nm radiation of a 30 mW diode laser, for an integration time of 60 s. The backscattered radiation, collected by an Olympus BX40 microscope optics using a 100X objective lens, was analyzed by an XploRA 1800 cm −1 monochromator equipped with a Peltier CCD sensor. A Zeiss-Gemini 2 electron microscope, operating at 150 kV, is the apparatus used for SEM analyses. When the measurements were carried out in the transmission mode (STEM), the accelerating voltage was 30 kV. SEM apparatus is coupled with a Quantax EDX spectrometer to carry out energy dispersive X-ray (EDX) analysis. The EDX detected pear-shaped dimension is about 0.7 µm. A Horiba NanoParticle Analyzer SZ-100 was used to evaluate the Zeta potential using a laser Doppler method, based on the principle of electrophoretic mobility under an electric field. The thermal degradation of the samples was studied by thermogravimetric analysis (TGA) under air flow, also to estimate the percentage of Ag loaded into the nanocomposite. A Mettler Toledo TGA 851 apparatus (horizontal balance mechanism) was used. The sample weight is 3 mg. The thermogravimetric weight loss curve was recorded as a function of temperature. The balance sensitivity was 0.5 µg. The weight loss was calculated by the difference between the weights at r.t. and at 450 • C.

Stability and size distribution of Ag-PMA colloid solution
In order to investigate the colloidal stability of the Ag-PMA system, the colloids were irradiated with the 6W UV lamp for 1 hour and they were re-exposed to the UV light (radiation density of 378 µW/cm 2 ) by using a 25W UV lamp for 5 hours. No appreciable change in terms of intensity and lineshape of the Ag surface plasmon resonance (SPR) peak was observed after the second step of irradiation and also after a month from the formulation (Fig S1a). This finding proved that: i) during the second UV irradiation step, the polymer reticulate limits any further Ag-NP nucleation, growth and aggregation; ii) colloids are fairly stable. Moreover, an average size lower than 10 nm was estimated for the 80% of the Ag NPs, while the diameter of the remaining ones does not exceed 25 nm (Fig. S1b-d) [4].

Abbreviations
The following abbreviations are used in this Supplemental: