Light-Activated Controlled Release of Camptothecin by Engineering Porous Materials: The Ship in a Bottle Concept in Drug Delivery

Light-Activated Controlled Release of Camptothecin by Engineering Porous Materials: The Ship in a Bottle Concept in Drug Delivery Eva Rivero-Buceta,a Mirela E. Encheva,b Bradley Cech,c Eduardo Fernandez,b Germán Sastre,a Christopher C. Landry,c,* and Pablo Botella, a,* a Instituto de Tecnología Química, Universitat Politècnica de València-Consejo Superior de Investigaciones Científicas, Avenida de los Naranjos s/n, 46022 Valencia, Spain b Institute of Bioengineering, Universidad Miguel Hernández, Elche, Spain and Centre for Network Biomedical Research (CIBER-BBN), Avenida de la Universidad s/n 03202 Elche, Spain c Department of Chemistry, University of Vermont, 82 University Place, Burlington, VT 05405, USA

S4 previous work, 6 has also been tested, but our results indicate that the UFF, finally selected, reflects better the short range interactions in the azobenzene molecules. DL_POLY 2.20 7 was used for the molecular dynamic simulations, which were carried out including full flexibility and periodic boundary conditions (cubic box of 50 Å containing one molecule) for all the atoms of the molecule. The temperature chosen is 298 K within the NVT ensemble. We employed the Verlet-leapfrog integration algorithm and the Evans thermostat, with a time step of 1 fs. Each run comprised an equilibration stage of 1×10 4 steps followed by the necessary production stage so as to ensure sufficiently good statistics for the analysis of 3×10 5 steps (300 ps). The cut-off for the non-bonding forces was set to 9 Å, and the Ewald summation was employed for the Coulombic with a precision parameter set to 10 -4 . The configurations were saved every 10 time-steps (0.01 ps), giving a number of 30000, large enough to obtain good statistics and allowing a smooth visualisation of the dynamics.

Slide Preparation and STORM Imaging
Samples were prepared and imaged via STORM using a modified version of a previously published protocol. 8 Particles were resuspended in 500 µL phosphate-buffered saline (PBS, pH 7.4), briefly sonicated, and the resulting solution was added to a 35 mm MatTek glass slide with a 14 mm sample well. Particles were allowed to settle to the bottom of the glass slide for approximately 3-4 minutes.
During this time, fresh imaging buffer was prepared according to the protocol recommended by the microscope manufacturer (Nikon). The imaging buffer consisted of Tris buffer (690 µL, 50 mM Tris-HCl (pH 8.0), 10 mM NaCl, 10 % glucose), a GLOX solution (7 µL, glucose-oxidase (14 mg), catalase (17 mg/mL), 10 mM Tris-HCl (pH 8.0), 50 mM NaCl), and 2-mercaptoethanol (7 µL). Excess PBS was removed from the glass slide, sufficient imaging buffer was added to cover the well, and the sample was immediately imaged on a Nikon Eclipse Ti-E microscope.
The sample was excited using a 405 nm laser and a minimum of 7,500 frames were collected for each run. All molecules with a minimum peak height greater than 300 were considered for further analysis in NIS-Elements.

Data Analysis and Reconstruction
Several regions of interest (ROIs) were created to isolate data specific to individual spheres, from which Cartesian coordinates of the detected molecules were extracted to .txt files for data reconstruction.
The extracted Cartesian coordinates for each particle were used in a custom least-squares sphere of best fit algorithm written in Python, producing the calculated radius and center of sphere. This was done by minimizing the function: 9 For ease of calculation, the sphere of best fit along with the Cartesian coordinates were transposed to reflect the center of the sphere as the origin. Distances of each detected molecule from the center of the sphere were determined by the Pythagorean Theorem, again written in Python: Averaging the penetration depths for each respective sphere yielded the mean penetration depth. The total number of molecules within a sphere was found by counting the number of Cartesian coordinates detected by STORM.
Using the transformed Cartesian coordinates of each molecule and the sphere of best fit, a 3D model of each sphere was reconstructed using Blender modeling software. A Python code was written to import the molecule coordinates and construct a wire-frame sphere to reflect what was imaged via
Cells were seeded in 96-well plate at a density of 4500 (HeLa) or 8000 (U251, SH-SY5Y) cells per well and incubated in 5% CO 2 at 37 °C for 24h. Then, cells were treated with the CAABE-free LSN-1 nanoparticles, with final doses ranging from 100 to 0.005 µg mL−1 during 72 h. At the end of the incubation period, 15 µL of MTS solution was added into each well and incubated for another 4 h. Absorbance was measured with a Perkin Elmer Wallac 1420 VICTOR2 Multilabel HTS Counter (Northwolk, CT, USA) at the wavelength of 490 nm.
Three independent experiments (n=3) were performed for every sample and each experiment was carried out by in triplicate. The results are shown in Fig. S16.