Following cytotoxic nanoconjugates from injection to halting the cell cycle machinery and its therapeutic implications in oral cancer

Abstract

Background: The concept of personalized therapy has been proven to be a promising approach. A popular technique is to utilize gold nanoparticles (AuNPs) as drug delivery vectors for cytotoxic drugs and small molecule inhibitors to target and eradicate oral cancer cells in vitro and in vivo. Both drug and nanocarrier designs play important roles in the treatment efficacy. In our study, we standardized the nanosystem regarding NPs type, size, surface ligands and coverage percentage leaving only the drugs mode of action as the confounding variable. We propose that similarly constructed nanoparticles (NPs) can selectively leverage different conjugated drugs irrelevant to their original mode of action. If proven, AuNPs may have a secondary role beyond bypassing cancer cell membrane and delivering their loaded drugs.

Methods: We conjugated 5- fluorouracil (5Fu), camptothecin (CPT), and a fibroblast growth factor receptor1-inhibitor (FGFR1i) to gold nanospheres (AuNSs). We followed their trajectories in Syrian hamsters with chemically induced buccal carcinomas.

Results: Flow cytometry and cell cycle data shows that 5Fu- and CPT- induced a similar ratio of S-phase cell cycle arrest as nanoconjugates and in their free forms. On the other hand, FGFR1i-AuNSs induced significant sub-G1 cell population compared with its free form. Despite cell cycle dynamics variability, there was no significant difference in tumor cells' proliferation rate between CPT-, 5Fu- and FGFR1i- AuNSs treated groups. In our in vivo model, FGFR1i-AuNSs induced the highest tumor reduction rates followed by 5Fu- AuNSs. CPT-AuNSs induced significantly lower tumor reduction rates compared with the 5Fu- and FGFR1i- AuNSs despite showing similar proliferative rates in tumor cells.

Conclusions: Our data indicates that the cellular biological events do not predict the outcome seen in our in vivo model. Furthermore, our results suggest that AuNSs selectively enhance the therapeutic effect of small molecule inhibitors such as FGFR1i than potent anticancer drugs. Future studies are required to better understand the underlying mechanism.

Keywords: 5-flourouracil; Camptothecin; FGFR1 inhibitor, Oral cancer; Gold nanoparticles.

Fig. 1

Characterization of the synthesized AuNSs. a TEM image revealing a monodispersed spherical nanoparticle solution with an average size of 27.1 nm. b UV–VIS optical absorption spectra confirming the size of the AuNSs with the subsequent conjugations with a peak equal to 528 nm (scale bar 50 nm). It shows that no peak shift indicates that no clumping or aggregation of the nanoparticles occurred after each ligand conjugation. c UV-Vis spectra of CPT nanoconjugates at pH=5 with a peak at 340 nm which corresponds to free CPT being released from AuNSs at acidic pH

Fig. 2

Confocal images of tumor tissue sections showing intra-tumoral localization of FGFR1i- 5Fu- and CPT-AuNSs. a-c Untreated tumor tissue sections exhibited strong and diffuse fluorescent signal indicating strongly expressed FGFR1. d-f Tumor tissue sections treated with free-FGFR1i show decreased FGFR1 expression compared with control indicating successful tumor penetration and inhibition of FGFR1. g-i Tumor tissue section treated with FGFR1i-AuNSs shows total loss of FGFR1 expression as a function of enhanced FGFR1i-AuNSs tumor penetration and selective targeting. k Untreated tumor tissue section lacking any fluorescence signal. l, n, p, r) Tissue sections showing molecules of CPT and 5Fu localization on tumor cell plasma membranes rather than the nuclei. m, o, q, s Tissue sections treated with CPT-AuNSs or 5Fu-AuNSs showing drug localization within the nucleus, thus conjugating 5Fu and CPT to AuNSs enhanced tumor penetration and increased cellular uptake (CPT and 5Fu fluorescence emission at 405 nm and 495 nm, respectively). No nuclear fluorochrome was applied. Magnification × 63, Scale bar =25 μm except (M), which is 10 μm. j Percentage of nuclear localization denotes that 5Fu- and CPT- AuNSs significantly enhance nuclear accumulation by 2-fold and 3-fold, respectively; CPT-AuNSs shows marked intense intranuclear staining compared with 5Fu-AuNSs p< 0.001. (n=13)

Fig. 3

Cell cycle analysis in different study groups 4 weeks post-treatment compared with pre-treatment analysis. i Notice the G1 arrest in all study groups with a significant subG1 apoptotic population detected only in FGFR1i-AuNSs. (n=13)

Fig. 4

Immunohistochemical expression of PCNA and Ki67 in tumor tissue sections isolated from treated animals. a, b Untreated tumors cells expressing PCNA and Ki67 proteins indicating their cycling status, while PEG-AuNSs seems to induce a minimal inhibition to PCNA and Ki67 proteins. c-h FGFR1i- CPT- and 5Fu- AuNSs induced a significant inhibition to PCNA and Ki67 activity compared with the free form. i, j Bar chart plots of the average tissue section area showing positive immunoreaction for both PCNA and Ki67 normalized to the total area. The drugs and their corresponding nanoconjugates resulted in significant lack of proliferation of the tumor cells compared with untreated and PEG-AuNSs treated cells. There was no significance difference between the inhibitory effect induced by CPT, 5Fu and EGFRi (denoted by ns). Also, the same drug nanoconjugates appear to induce equivalent rates of cellular arrest. PCNA and Ki67 immunoreactions were significantly lower in tumor cells treated with FGFR1i- CPT- and 5Fu- AuNSs compared with the free forms. Findings were considered statistically significant when p< 0.001 and are denoted by asterisks. Scale bars=100 um (n= 13)

Fig. 5

Clinical follow up after different treatments. Tumor volume measurements were conducted at week 0 and week 4 post-treatments. Note lesions within the yellow dashed circles. a-d Intra and extra oral tumors at week 0 just before treatment with saline, FGFR1i-AuNSs, 5Fu-AuNSs and CPT-AuNSs, respectively. e A hamster treated with saline showed a significant increase in tumor volume, at 4 weeks post-treatment. Notice the formation of new intraoral exophytic polyps (yellow arrow). f A hamster treated with FGFR1i-AuNSs showed enhanced tumor shrinkage with decreased vasculature. g, h Animals treated with 5Fu-AuNSs and CPT-AuNSs also showed reduction in tumor size. i Tumor volume percentage change across the 8 groups showing enhanced tumor reduction rate related to FGFR1i-AuNSs treatment (− 63.09%, p< 0.001), followed by 5Fu-AuNSs (− 43.4%, p< 0.001). There was no significant difference between the 2 treatments indicating a favorable clinical effect (ns). j Kaplan Meier survival curve demonstrating significantly increased life span in animals treated with 5Fu-AuNSs, FGFR1i-AuNSs and CPT-AuNSs compared with control and free form of the drugs, denoted with asterisks (p< 0.001). Note the lack of significant difference among the nanoconjugates (ns). (n= 13)

Fig. 6

Off target different systemic effects. We conducted clinical, histopathologic and hemopoietic assessment 4 weeks post-treatment. a A hamster treated with CPT showing partial alopecia. b Normal liver section extracted from 5Fu-AuNSs conjugated group showing normal hepatocytes and liver parenchyma. The same images were obtained from the other 2 nanoconstructs. c Liver section showing focal necrosis near the portal vein obtained for both free cytotoxic drugs CPT and 5Fu. d, e Areas of glomerular shrinkage in hamsters receiving CPT and 5Fu. f Platelet count for the 8 groups across all time points; no significant difference was noted between the 3 nanoconjugates groups (ns) with only one exception seen for CPT-AuNSs at 48 h. A significant decrease in the platelets count was seen in both free 5Fu and CPT denoted with asterisks (p< 0.001). g RBCs count showing no significant difference between the 3 nanoconjugates groups (ns) (p< 0.001). h WBCs values also show no significant difference between the 3 nanoconjugates groups (ns) (p< 0.001), while a significant fluctuation was seen for WBCs count in both cytotoxic drugs denoted with asterisks (p< 0.001). i For haemoglobin, no significant difference was found across all groups except the unconjugated form of FGFR1i denoted with asterisks (p< 0.001). (n=13)