PD-l1 conjugated all-trans retinoic acid nanoparticles for targeted treatment of oral dysplasia and oral squamous cell carcinoma

Abstract

Objectives The present study conjugated all-trans retinoic acid (ATRA) with poly(lactic acid-co-glycolic acid)-block-poly(ethylene glycol) (PLGA-PEG) nanocarriers, and combined with anti–programmed death-ligand 1 (PD-L1) antibody to achieve targeted therapy for oral squamous cell carcinoma (OSCC) and oral dysplasia. Study Design The molecular mechanism of ATRA and PD-L1 was studied by using Cell Counting Kit-8 (CCK8), flow cytometry, Western blot, and immunohistochemistry. The effect of ATRA on OSCC and oral dysplasia cell proliferation inhibition, apoptosis induction, and PD-L1 downregulation was significantly enhanced after signal transducer and activator of transcription 3 (STAT3) signaling blockade. PD-L1 was highly expressed in patients with oral leukoplakia and OSCC compared with normal controls. By the measurement of dynamic light scattering, the average particle size and zeta potential of ATRA–PLGA–PEG and ATRA–PLGA-PEG–PD-L1 was 97 nm and –3.6 mV, 105 nm and –5.7 mV, respectively. The release of ATRA in nanoparticles reached 55% within 36 hours. The maximum encapsulation efficiency and loading capacity of ATRA into the nanoparticles reached 76.29% and 12.18%, respectively. The PEG-PLGA nanocarrier had almost no cytotoxicity, and the nanomedicine significantly inhibited cell proliferation of DOK and CAL27 cells on the in vitro cytotoxicity test. After 1 hour of incubation, the Nile red group showed few fluorescence intensity, whereas the 2 nanoparticle groups were readily detectable under confocal microscopy. Moreover, nanoparticles enhanced anticancer activity and reduced side effects compared with free ATRA in C3 H tumor-bearing mice. Furthermore, ATRA–PLGA-PEG–PD-L1 nanoparticles could target the tumor more effectively compared with the control group, as shown by whole-animal fluorescence imaging. Multicolor immunohistochemistry revealed effective regulation of expression of the related proteins after nanoparticle treatment. Results The mechanism of ATRA and PD-L1 downregulation was closely associated with STAT3 signaling inhibition in both OSCC and oral dysplasia. PD-L1, which was highly expressed in human oral leukoplakia and OSCC tissues, proved to be a specific target site for cancer therapy. The ATRA–PLGA-PEG–PD-L1 nanoparticles had lower toxicity and higher biocompatibility and targeted the tumor more specifically compare with free ATRA, both in vitro and in vivo. Conclusions These findings suggested that ATRA induced antitumor effects via STAT3 signaling inhibition in oral dysplasia and OSCC. The use of ATRA–PLGA-PEG–PD-L1 nanomedicine was an effective and nontoxic targeted therapy for oral dysplasia and OSCC, both in vitro and in vivo.