PF 06700841 br biodegradation and their less hydrophobic cha
biodegradation and their less hydrophobic character. Our work also demonstrates the potential of targeting non-canonical targets with non-viral combination nanoparticles. Our combination approach may also be able to be extended to polymeric nanoparticles that contain DNA  and other RNAs in addition to siRNA  in order to have a multimodal cancer pathway reprogramming eﬀect. r> 4. Conclusions
This study describes an advanced therapeutic platform the delivery of combination siRNA molecules intracellularly to the cytosol of tumor cells. Bioreducible PBAE-based nanoparticles are used to deliver five anti-GBM genes (Robo1, YAP1, NKCC1, EGFR, and survivin) in com-bination. Although the dose of each siRNA was relatively low (24 nM), significant functional eﬀects were observed in human glioblastoma PF 06700841 including inhibition of cancer cell growth and migration. The nano-medicine-induced knockdown was found to be specific to GBM while sparing healthy cells. In animal models, tumor burden is reduced over time by treatment with the bioreducible polymeric nanoparticles en-capsulating siRNA. This multimodal nucleic-acid based therapy is pro-mising for the treatment of glioblastoma and other solid tumors.
5. Experimental section
Chemicals used to synthesize the bioreducible PBAE monomer BR6 were purchased from Sigma-Aldrich (St. Louis, MO) and used as re-ceived. All other PBAE monomers were purchased from Alfa Aesar (Ward Hill, MA). AllStars Human Cell Death siRNA (siDeath) and a scrambled negative control siRNA (scRNA) were purchased from Qiagen; all other siRNA oligos were purchased from Origene Technologies (Rockville, MD). Antibodies against Yes-associated pro-tein 1 (YAP1), sodium-potassium-chloride cotransporter 1 (NKCC1), survivin, roundabout homolog 1 (Robo1), endothelial growth factor receptor (EGFR), and β-actin for use in Western blotting were pur-chased from the vendors listed in Supplementary Table S2. Cannulas used to implant orthotopic tumors and administer nanoparticles were purchased from Plastics One, Inc. (Roanoke, VA).
5.2. Bioreducible polymer synthesis
Monomer BR6 was synthesized as previously described . Briefly, bis(2-hydroxyethyl) disulfide was acrylated with acryloyl chloride under anhydrous conditions in the presence of triethylamine (TEA). The TEA HCl precipitate was removed via filtration, and other impurities were removed by washing with Na2CO3 and then with water. The final product was purified from organic solvents using rotary evaporation. Synthesis of the bioreducible polymer, referred to here-after as R646, was carried out in a method similar to that described previously . Briefly, the BR6 backbone monomer was mixed with the side chain monomer 4-amino-1-butanol (S4) at a 1.01:1 M ratio of BR6 to S4 in anhydrous tetrahydrofuran (THF). The monomer con-centration at the start of the reaction was 500 mg/mL. The poly-merization reaction was carried out for 24 h while stirring at 60 °C. Then, a ten-fold molar excess of the end-cap 2-((3-aminopropyl)amino) ethanol (E6) was added to the mixture and allowed to react with stir-ring at room temperature for 1 h. This end-capped polymer was twice precipitated and centrifuged with diethyl ether to remove unreacted monomers. Residual ether was removed by evaporation under vacuum for 48 h. Finally, the polymer R646 was dissolved in anhydrous DMSO at 100 mg/mL stored with desiccant at −20 °C. The structure of R646 is shown in Fig. 1 along with the physicochemical characterization of R646/siRNA nanoparticles. The structure of R646 was previously characterized via H1-NMR, as well as the polymer molecular weight and polydispersity via gel permeation chromatography (MW = 3978 Da and Biomaterials 209 (2019) 79–87
5.3. PBAE/siRNA nanoparticle characterization
All physicochemical characterization of PBAE/siRNA nanoparticles was carried out using scRNA. For sizing and zeta potential measure-ments, scRNA was diluted to 1.44 μM in 25 mM sodium acetate buﬀer (pH 5, NaAc), and R646 polymer was diluted separately in NaAc to 3.24 mg/mL. The R646 and scRNA solutions were mixed 1:1, vortexed briefly, and allowed to form nanoparticles over 10 min. The nano-particle suspension was then diluted in 1 × PBS to an appropriate concentration range for hydrodynamic size measurement by nano-particle tracking analysis (NTA, NanoSight NS300, Malvern Instruments, Worcestershire, UK) or zeta potential measurement (Zetasizer Nano, Malvern Instruments).
siRNA release from the PBAE/siRNA complexes was measured using a gel retention assay. Particles were made at higher concentration and then diluted 10-fold in either artificial cerebral spinal fluid (aCSF) or 1 × PBS with 5 mM glutathione (PBS/GSH) to simulate the extra-cellular and intracellular environments, respectively. The diluted par-ticles were incubated at 37 °C. At predetermined time points, some of the diluted particles were removed, added to sucrose as a lyoprotectant (final concentration 30 mg/mL sucrose), and lyophilized for 48 h. After all time points were collected, samples were rehydrated simultaneously and analyzed by gel electrophoresis using a 1% agarose gel (Ultrapure™ agarose, Life Technologies) at 100 V. To minimize any binding inter-ference, 30% glycerol without any other dyes was used as a loading buﬀer. The siRNA was visualized by ethidium bromide staining.