Modular Drug-Loaded Nanocapsules with Metal Dome Layers as a Platform for Obtaining Synergistic Therapeutic Biological Activities

Multifunctional drug-loaded polymer–metal nanocapsules have attracted increasing attention in drug delivery due to their multifunctional potential endowed by drug activity and response to physicochemical stimuli. Current chemical synthesis methods of polymer/metal capsules require specific optimization of the different components to produce particles with precise properties, being particularly complex for Janus structures combining polymers and ferromagnetic and highly reactive metals. With the aim to generate tunable synergistic nanotherapeutic actuation with enhanced drug effects, here we demonstrate a versatile hybrid chemical/physical fabrication strategy to incorporate different functional metals with tailored magnetic, optical, or chemical properties on solid drug-loaded polymer nanoparticles. As archetypical examples, we present poly(lactic-co-glycolic acid) (PLGA) nanoparticles (diameters 100–150 nm) loaded with paclitaxel, indocyanine green, or erythromycin that are half-capped by either Fe, Au, or Cu layers, respectively, with application in three biomedical models. The Fe coating on paclitaxel-loaded nanocapsules permitted efficient magnetic enhancement of the cancer spheroid assembly, with 40% reduction of the cross-section area after 24 h, as well as a higher paclitaxel effect. In addition, the Fe-PLGA nanocapsules enabled external contactless manipulation of multicellular cancer spheroids with a speed of 150 μm/s. The Au-coated and indocyanine green-loaded nanocapsules demonstrated theranostic potential and enhanced anticancer activity in vitro and in vivo due to noninvasive fluorescence imaging with long penetration near-infrared (NIR) light and simultaneous photothermal–photodynamic actuation, showing a 3.5-fold reduction in the tumor volume growth with only 5 min of NIR illumination. Finally, the Cu-coated erythromycin-loaded nanocapsules exhibited enhanced antibacterial activity with a 2.5-fold reduction in the MIC50 concentration with respect to the free or encapsulated drug. Altogether, this technology can extend a nearly unlimited combination of metals, polymers, and drugs, thus enabling the integration of magnetic, optical, and electrochemical properties in drug-loaded nanoparticles to externally control and improve a wide range of biomedical applications.

The thin layer formation of the loaded NCs on the silicon wafer, and their removal after the metal evaporation were calibrated to set the conditions to yield as much metalcapped NCs as possible.PAH, PDDA and APTMS were used as positive electrolyte materials at 2% concentrations, in order to provide a positive counter charge to the surface, allowing the negative NCs to attach.The analysis of the SEM images showed that PDDA was the most adequate linker for this purpose (Figure S1).Detachment of the NCs from the surface of the silicon wafer to a 0.2% PSS solution was accomplished using a sonication bath for 5 minutes at 100% sonication power.The detachment conditions may vary with the use of different sonication baths; thus, the sonication time and the intensity of sonication must be calibrated accordingly.Figure S2 shows an example of an unsuccessful detachment, when the removal conditions were too aggressive, which resulted in metal domes without the core NP or with damaged wafers.

Morphology and composition characterization using SEM/EDS and DLS
The surface distribution of the particles on the wafer were examined using the environmental Scanning Electron Microscope (SEM) Quanta 200 (FEI Company, The Netherlands).The system includes an energy dispersive X-ray spectroscopy detector (EDS) (EDAX, TSL, AMETEK, Hillsboro, OR, USA) for NC surface elemental identification.Metal-PLGA-covered wafers were cut and fixed by a conductive adhesive tape and placed in a 35 ᵒ tilted platform.Chemical analysis was performed in frame mode, representing the sum of the elemental composition of the imaged NCs and the silicon substrate.
The EDS analysis of the samples confirmed the presence of the different materials in the diverse designed NCs (Figure S3A-C).Figure S3 also shows an example of metal evaporated NCs loaded on a silicon wafer before (Figure S3D) and after (Figure S3E) sonication using the above-mentioned settings, confirming a high NC recovery from the wafer of ~85%.Finally, the DLS measurements revealed the changes in size distribution of the half coated NCs after the addition of the metal cap, which probably derived from the known drawbacks of the instruments; non-spherical shape of the NCs, different refractive index for the different materials and, false peaks at 10 nm range for NPs larger than 40 nm, as seen previously with the Au-PLGA 1 .Red-whole NP, Blue-first layer and yellow-second layer.

UV/vis absorbance spectrum of NCs
Beside the morphology, composition, size and charge of the NCs, the UV/Vis absorption spectra were recorded using a spectrophotometer (FluoroMax4, HORIBA, USA).NCs were recovered from the wafers as depicted above and counted.Samples vials were photographed and 600 µl were loaded to a quartz vial for UV/vis absorbance spectrum measurement of each metal capped NCs, recorded in the range of 270-870 nm.

Magnetic properties and magnetic manipulation of the Fe-PLGA nanoparticles
Figure S6a shows the SQUID magnetometry magnetization loops of the Fe-PLGA nanoparticles.The magnetic properties of the nanocapsules were recorded using a SQUID magnetometer (MPMS-XL, Quantum Design).A magnetization curve at 300 K with a maximum applied field of 50 kOe was performed on tightly packed powdered sample after drying some drops of the nanodome suspension in water.
Note the loops show virtually zero magnetization in the absence of magnetic field.In fact, the constriction of the hysteresis loop at low fields is typical of a vortex magnetization reversal. 2Moreover, it is important to highlight the field required to saturate the magnetization is only about 2 kOe.

Photothermal heating efficiency measurement in Au-PLGA nanocapsules
The photothermal conversion efficiency of the NCs different nanocapsules was measured using a specially designed photothermal testing system.The system consisted of a NIR laser diode with emission wavelength of 808 nm (L808P500MM, Thorlabs) regulated by a laser diode controller (ITC4005, Thorlabs) and a power meter (PM100D, Thorlabs).Temperature monitored by an infrared camera (ETS320, FLIR).

Copper ion release from Cu-PLGA NCs
The release of copper ion was demonstrated using a spectrophotometer to measure the UV/Vis absorption spectra of Cu-PLGA NC solutions before and after 24 hour incubation at 37 ᵒC.The significant drop in absorbance, also visible with the naked eye where the solution became transparent, demonstrates the extensive copper ion release from the copper cap.To evaluate the extent of release, solutions were centrifuged at 14,000 rpm for 10 min and the supernatant and pellet were separated for ICP-OES measurements (Perking Elmer Optima 4300DV).The Cu ion release from the Cu-PLGA NCs to the medium was 77% from initial Cu content.

Figure S1 .
Figure S1.SEM images of the nanocapsules self-assembly on Si wafers precoated with positive

Figure S2 .
Figure S2.Optimization of metal coated nanocapsules detachment from the wafer.

Figure S4 .
Figure S4.Nanoparticle morphology.TEM micrographs and separation into layers of

Figure
Figure S6.a) SQUID magnetometry magnetization reversal loop of the Fe-PLGA