Nanoparticles (NPs)‐Meditated LncRNA AFAP1‐AS1 Silencing to Block Wnt/β‐Catenin Signaling Pathway for Synergistic Reversal of Radioresistance and Effective Cancer Radiotherapy

Abstract Resistance to radiotherapy is frequently encountered in clinic, leading to poor prognosis of cancer patients. Long noncoding RNAs (lncRNAs) play important roles in the development of radioresistance due to their functions in regulating the expression of target genes at both transcriptional and posttranscriptional levels. Exploring key lncRNAs and elucidating the mechanisms contributing to radioresistance are crucial for the development of effective strategies to reverse radioresistance, which however remains challenging. Here, actin filament‐associated protein 1 antisense RNA1 (lncAFAP1‐AS1) is identified as a key factor in inducing radioresistance of triple‐negative breast cancer (TNBC) via activating the Wnt/β‐catenin signaling pathway. Considering the generation of a high concentration of reduction agent glutathione (GSH) under radiation, a reduction‐responsive nanoparticle (NP) platform is engineered for effective lncAFAP1‐AS1 siRNA (siAFAP1‐AS1) delivery. Systemic delivery of siAFAP1‐AS1 with the reduction‐responsive NPs can synergistically reverse radioresistance by silencing lncAFAP1‐AS1 expression and scavenging intracellular GSH, leading to a dramatically enhanced radiotherapy effect in both xenograft and metastatic TNBC tumor models. The findings indicate that lncAFAP1‐AS1 can be used to predict the outcome of TNBC radiotherapy and combination of systemic siAFAP1‐AS1 delivery with radiotherapy can be applied for the treatment of recurrent TNBC patients.


Screening of reduction-responsive NPs
The siLuc loaded NPs were prepared using the PDSA polymers with different chemical structure and molecular weight. Subsequently, Luc-expressing HeLa cells were seeded in 96well plates (5,000 cells per well) and incubated in 0.1 mL of RPMI 1640 medium) with 10% FBS for 24 h. Thereafter, the medium was replaced by fresh medium and siLuc loaded NPs were added. After 24 h incubation, the cells were washed with PBS buffer and allowed to incubate in fresh medium for another 48 h. The Luc expression was determined using Steady-Glo luciferase assay kits. Cytotoxicity was measured using AlamarBlue assay according to the manufacturer's protocol. The luminescence or fluorescence intensity was measured using a microplate reader, and the average value of five independent experiments was collected.
According to the above experiments, the PDSA8a nanoplatform with the best gene silencing efficacy was selected for the siAFAP1-AS1 delivery.

Evaluation of the reduction response of the siRNA loaded NPs
The si-1 loaded PDSA8a NPs (denoted NPs(si-1)) were prepared and then dispersed in 1 mL of PBS containing GSH with different concentrations. At a predetermined interval, the size of the NPs(si-1) was examined by dynamic light scattering (DLS), and the morphology of the NPs(si-1) was observed by transmission electron microscope (TEM). To examine the influence of reduction response on the siRNA release from the NPs, Cy5-labeled si-1 was encapsulated into the NPs (denoted NPs(Cy5-si-1)). Subsequently, the NPs were dispersed in 1 mL of PBS (pH 7.4) and then transferred to a Float-a-lyzer G2 dialysis device (MWCO 100 kDa, Spectrum) that was immersed in PBS buffer containing GSH at 37 o C. At a predetermined interval, 5 µL of the NP solution was withdrawn and mixed with 20-fold DMSO. The fluorescence intensity of Cy5-labeled si-1 was determined using a microplate reader.

Pharmacokinetics study
Healthy male BALB/c female mice were randomly divided into two groups (n = 3) and given an intravenous injection of either (i) NPs(Cy5-si-1) or (ii) naked Cy5-si-1 at a 1 nmol siRNA dose per mouse. At predetermined time intervals, orbital vein blood (20 µL) was withdrawn using a tube containing heparin, and the wound was pressed for several seconds to stop the bleeding. The fluorescence intensity of Cy5-labeled siRNA in the blood was determined by The blood circulation half-life (t 1/2 ) was calculated according to previous report [3].

Biodistribution
MDA-MB-231R xenograft tumor-bearing female nude mice were randomly divided into two groups (n = 3) and given an intravenous injection of either (i) NPs(Cy5-si-1) or (ii) naked Cy5-si-1 at a 1 nmol siRNA dose per mouse. Twenty-four hours after the injection, the mice were imaged using an IVIS Lumina Ⅲ imaging system. Organs and tumors were then harvested and imaged. To quantify the accumulation of NPs in tumors and organs, the fluorescence intensity of each tissue was quantified by Image-J.

Immune response
Healthy female BALB/c mice were randomly divided into six groups (n = 3) and given an intravenous injection of either (i) PBS, (ii) naked siCTL, (iii) naked si-1, (iv) PDSA NPs, (v) NPs(siCTL) or (vi) NPs(si-1) at a 1 nmol siRNA dose per mouse. Twenty-four hours after injection, blood was collected and serum isolated for representative cytokine analysis by enzyme-linked immunosorbent assay or ELISA (PBL Biomedical Laboratories and BD Biosciences) according to the manufacturer's instructions.

Blood and analysis
Healthy female BALB/c mice were randomly divided into six groups (n = 3) and given an