Pro-apoptotic and size-reducing effects of protein corona-modulating nano-architectures enclosing platinum prodrug in in vivo oral carcinoma

The selective and localized delivery of active agents to neoplasms is crucial to enhance the chemotherapeutic efficacy while reducing the associated side effects. The encapsulation of chemotherapeutics in nanoparticles decorated with targeting agents is a strategy of special interest to improve drug delivery. However, serum protein adsorption often compromises the in vivo efficiency of targeting agents, leading to protein corona formation that interferes with the targeting process. Here, the enhanced efficacy of hybrid nano-architectures enclosing a platinum prodrug and decorated with a customized peptide (NAs-cisPt-Tf2) is demonstrated by employing alternative in vivo models of oral carcinoma. The peptide binds to transferrin and modulates the protein corona formation on NAs-cisPt-Tf2, supporting the identification of its receptor. Optimized chorioallantoic membrane cancer models (CAMs) enabled a thorough assessment of the tumor-suppressing effect of NAs-cisPt-Tf2 as well as the quantitative evaluation of angiogenesis and cell cycle associated mechanisms. The treatment strategy resulted in a significant tumor volume reduction coupled with anti-angiogenic and pro-apoptotic effects inferred from the downregulation of the vascular endothelial growth factor gene and increased expression of cleaved caspase-3. Overall, this study provides a potentially effective tumor-targeted approach for a non-invasive management of oral carcinoma.


Note 1. Synthesis procedures
Synthesis of fluorophore-modified poly(L-lysine)  Poly(L-lysine) hydrobromide (PL; 15-30 kDa) was dissolved in milliQ water to a final concentration of 40 mg/mL. In a microtube, 75 μL of PL was mixed with 2 μL of AlexaFluor-647 NHS ester (10 mg/mL in dimethyl sulfoxide; Invitrogen A20006), and 100 μL of acetate buffer 0.3 M (pH 5.4). The mixture was kept in dark and incubated on a shaker (700 rpm) overnight at room temperature. The product PL-647 was used without further purification.

Synthesis of fluorophore-loaded (NAs-647) or prodrug-loaded nano-architectures (NAs-cisPt)
Gold ultrasmall nanoparticles (USNPs) were synthesized through rapid reduction of gold salt, beginning with 200 μL of aqueous solution of tetrachloroauric (III) acid (HAuCl 4 ; Alfa Aesar, ACS 99.99% metal basis; stock: 10 mg/mL) and 10 μL poly(sodium 4-styrene sulfonate) (PSS; 70 kDa; 30% aqueous solution) being added to 20 mL of milliQ water. Freshly prepared aqueous solution of sodium borohydride (200 μL of the 8 mg/mL stock) was quickly added to the vigorously stirring solution containing the gold salts. After 2 min of vigorous stirring, the solution was further aged for another 10 min before 177 μL of PL-647 (for NAs-647) or 165 μL of PL-cisPt (for NAs-cisPt) was added. Then, the solution was incubated for 20 min. The gold USNP polymeric arrays with the fluorophore or prodrug were collected by centrifugation at 14000 rpm for 5 min. After removing the supernatant, the product was resuspended in 2 mL of milliQ water.
The Stöber process was modified to construct silica shell on the periphery of gold polymeric arrays. Two 50-mL tubes were filled each with 35 mL ethanol and 1.2 mL ammonia solution (Merck, 32%). Once the gold polymeric arrays were ready, 20 μL tetraethyl orthosilicate and 1 mL of the gold arrays were added on each tube. The mixture was incubated for 3 h at room temperature under moderate shaking. Then, the resulting nano-architectures (NAs-647 or NAs-cisPt) were collected through a 30-minute centrifugation at 4000 rpm. After discarding the supernatant, the resulting product was added with, sonicated, and resuspended in ethanol. The suspension was spun at 14000 rpm for 3 minutes and the washing was discarded. The product was again added and sonicated with ethanol for another round of washing. After another centrifugation at 14000 rpm for 3 min, the washing was discarded and ethanol was again added and the product was sonicated. Then, short spin (15 s or until rotational speed reaches 14000 rpm) was done to remove larger nanoparticles. The supernatant was separated and spun at 14000 rpm for 3 min to collect the final NAs-647 or NAs-cisPt. After removing the supernatant, the final products (stable for at least 1-year) were resuspended and stored in 1 mL of ethanol.

Synthesis of surface functionalized nano-architecture (NAs-647-Tf2 or NAs-cisPt-Tf2)
The ethanolic suspension of the nanoparticles (1 mL) was mixed with the freshly prepared solution of the linker silane-poly(ethylene glycol)-maleimide dissolved in ethanol (1 mL of the stock 4 mg/mL). The volume was adjusted to 10 mL with ethanol and the mixture was stirred vigorously for 15 min. The nanoparticles were recovered through centrifugation at 14000 rpm for 3 min. After removing the supernatant, the particles were recollected and resuspended in 1.2 mL of N-(2hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES) buffer (20 mM, pH 7.2). Meanwhile, the Tf2 peptide was prepared as reported in Santi et al (2016) [2]. A stock Tf2 solution (2 mg/mL) was prepared by dissolving the lyophilized sample in degassed HEPES buffer. Then, 300 μL of the peptide solution was added to the buffered solution containing the nanoparticles, resulting in a Tf2 final concentration equal to 0.4 mg/mL. The mixture was stirred for 2 h, added with 500 μL Lglutathione (GSH; final concentration equal to 200 μg/mL in HEPES buffer), and stirred for another hour. GSH was added to react with the excess maleimide ends that did not react with the Tf2 peptide. After the cumulative 3-hour incubation, the mixture was spun at 14000 rpm for 3 min to recover the nanoparticles. Finally, the recovered NAs-647-Tf2 or NAs-cisPt-Tf2 was washed twice with ethanol, and stored in ethanol at -20°C.  Volume fold change were measured for each tumor and referred to the respective changes in volume post-treatment (EDD 12 or 14) with respect to pretreatment volume (EDD 10).

12)
This metric compares the change in volume after experimental treatment to the initial effect of the serum-free medium vehicle solution on the tumors treated on EDD 12. The "average volume fold change" refers to the values (metric #2) for each treatment type. The data are reported as mean ± standard error of the mean of pooled samples from two independent experiments, with N > 8 eggs per condition. The data are reported as mean ± standard deviation of two independent experiments, with N > 8 eggs per condition, per experiment.  using Aperio ImageScope software. The algorithm automatically generated a scoring system based on the intensity of positive staining, and classified them into weak, moderate, or strong (brown signal). The data are reported as mean + standard deviation of three areas of the same slide section. Statistical analysis was performed through one-way ANOVA *p-value < 0.05, **pvalue < 0.01 (right).