Nanoparticles derived from insect exoskeleton modulates NLRP3 inflammasome complex activation in cervical cancer cell line model

Immune evasion is an important hallmark of cancer progression and tumourigenesis. Among the cancer types, cervical cancer has very high global prevalence, severely affecting female reproductive health. Its preponderance is also observed in the Indian health sector. The NLRP3 inflammasome, an intracellular complex regulates the innate immune activity and a variant gene of it has been significantly associated with cervical cancer development. We aimed to evaluate the potential role of our chitosan engineered nanoparticles (CSNP) and gallic acid conjugated chitosan (gCSNP), to modulate the NLRP3 inflammasome complex in cervical cancer cell lines to explore their novel physicochemical properties. The encapsulation of gallic acid (GA) with chitosan was performed using ion gelation method. The CSNP and gCSNP nanoparticles ranged between 155 and 181 nm as determined by zeta sizer. The conjugations were validated by FTIR and XRD analysis. In the cervical cell line model, CSNP suppressed NLRP3 inflammasome activation in contrast to gCSNP at higher doses. In contrast to gCSNP, the CSNP not only demonstrated inhibitory effect on the expression of genes coding for the NLRP3 inflammasome complex (signal 1—priming), but also decreased relative expression of gene involved in the activation of NLRP3 inflammasome complex (signal 2—activation). Conjugation of gallic acid reversed the immunosuppressor mimicking action of CSNP in cervical cancer cell line. Future research can reveal the immunomodulatory mechanism of CSNP may have its translational significance as potential treatment strategies targeting immune evasion as an important hallmark of cancer.


Background
Cervical malignancy is the fourth most prevalent neoplasm globally and second most common among Indian women (Arbyn et al. 2020;Mishra et al. 2016). Inflammation induced through microbial or danger signals affects all stages of tumor development. The pro-inflammatory cytokines, IL-1β and IL-6, are important mediators for inflammation-induced tumourigenesis (Moossavi et al. 2018). The NLRP3 inflammasome is a multimeric protein complex that regulates the innate immune activity through modulation of the production of pro-inflammatory cytokines. A variant NLRP3 gene has been significantly associated with cervical cancer development (Pontillo et al. 2016). However, the role of NLRP3 inflammasome activation in tumourigenesis, transformation and invasion, remains a conundrum (Moossavi et al. 2018). Activation of the NLRP3 inflammasome is a two-step process where microbial toxins or danger signals and ATP are necessary for inflammasome activation. There is a lack of adequate therapeutic strategies targeting key immune signaling pathways involving the inflammasome complex in cancer treatment.
Nanoparticles hold promise in offering a unique opportunity that increases the efficiency of cancer immunotherapy as well as ameliorates their toxic side-effects (Hamarsheh and Zeiser 2020;Park et al. 2018). We hypothesize that our engineered nanoparticles CSNP/gCSNP can prevent the outcome of the priming signal by inhibiting the expression of genes coding for the NLRP3 inflammasome complex proteins in human cervical epithelial adenocarcinoma cells. Furthermore, we aimed at studying the role of CSNP/gCSNP on the genes coding for the purinergic receptor (P2RX7) which functions as a major contributor in providing the second kick necessary for activating the NLRP3 inflammasome complex. Additionally, the study aimed at evaluating the effect of our engineered nanoparticles on the gene coding for pro-inflammatory cytokine IL1β and its release from stimulated cervical cancer cells.
A pilot study by one of our co-investigators indicated a positive effect of chitosanbased nanoparticles and oligosaccharides on the anti-inflammatory cytokines, in the in vivo study conducted on the cyclophosphamide-treated mice model (Mudgal et al. 2019). As an extension of these findings, this study was undertaken to investigate the effect of chitosan nanoparticles on the NLRP3 inflammasome complex to establish the relationship between innate immunity, inflammation and disease progression in cervical cancer cell line model.
Cancer immunotherapy has come a long way to significantly augment conventional cancer therapy in recent years (Kruger et al. 2019;Sun 2017). Although NLRP3 inflammasome has been implicated both in tumourigenesis and as a response to antitumour therapy, it provides a lucrative target for cancer immunotherapy (Hamarsheh and Zeiser 2020;Kantono and Guo 2017;Karki and Kanneganti 2019). Hence the present study further investigated the multipronged potential of CSNP/gCSNP as a robust immune modulator in cervical cancer cells, to address the molecular mechanisms behind the activation of inflammasomes. Here, we not only targeted the expression of critical genes in the canonical pathway, but also the activation mechanism of the NLRP3 inflammasome complex in cervical cancer cells. Moreover, the present study aimed at exploring the possibilities of pharmacological potential of the CSNP nanoparticles and its modified form as gCSNP.

Physicochemical characterization of engineered nanoparticles
The formulated nanoparticles (NP) were dissolved in an appropriate volume of HPLC-grade water and the average hydrodynamic diameter, polydispersity index (PDI) and zeta potential were estimated with ZetaSizer Nano ZS90 (Malvern Instruments Limited, UK) equipped with a 4.0 mW He-Ne laser. The detection angles of 90° and 120° were used for determination of: (a) size and PDI , and (b) the zeta potential of the synthesized nanoparticles, respectively. The FTIR spectra of CSNP, gCSNP and GA were recorded on a Jasco FT/IR-6300 Spectrometer equipped with a DR PRO410-M (Jasco, Japan) by scanning from 4000 to 500 cm −1 at a resolution of 4 cm −1 and analyzed using Microsoft Excel (Microsoft Office version 2016, USA). The crystallographic structures of CSNP and formulated gCSNP were acquired on a Rigaku Ultima MiniFlex 600 X-ray diffractometer (Rigaku, Germany) using 119 Cu Kα radiation operating at 40 kV and 15 mA.
In vitro studies to evaluate the effect of engineered nanoparticles on NLRP3

inflammasome-activated cervical adenocarcinoma cell line (HeLa 229) model
HeLa 229 cell line was procured from the National Centre for Cell Science, Pune and the passage No. 102 was used in the present study. Cells were maintained in DMEM (Gibco, India) supplemented with 10% fetal bovine serum (Gibco, India) and 1% penicillin-streptomycin (Merck, India) at 37 °C in 5% CO 2 humidified incubator (Thermo Fisher Scientific, India).

Cytotoxicity assay
MTT Cell Growth Assay (CT02, Merck, India) was used for assessing the 24-h cytotoxicity of CSNP and gCSNP on HeLa 229 and normal kidney cells (HEK293) cell lines. The cytotoxicity assay was used only as a reference for dose selection of the engineered nanoparticles since the selected doses were used for a time duration limited to 5 h as per the present study design.

Relative mRNA expression of genes coding for activated NLRP3 inflammasome complex in cervical cancer cell line
The total RNA was extracted from HeLa 229 cells homogenized in TRI-Reagent (T9424, Merck, India) following the manufacturer's protocol. The extracted RNA was carefully assessed for its quality, purity and integrity using the 260/280 ratio, 260/230 ratio obtained from BioSpectrometer basic, (6135000009, Eppendorf, India) and agarose gel electrophoresis (intact gel bands corresponding to 28S and 18S RNA), respectively. The extracted RNA from each group was diluted to 180 ng/μl using nuclease-free water to ensure a known amount of starting mRNA concentration from every group for cDNA synthesis. High-capacity reverse transcriptase cDNA synthesis kit (ThermoFisher Scientific India Pvt. Ltd., India) was used to prepare cDNA (final volume of 20 μl) from the extracted total RNA using thermocycler (Mastercycler X50, Eppendorf, India). Synthesis of cDNA involved the use of random primers from the cDNA synthesis kit with internal controls as + RT/-RT (with or without reverse transcriptase) to reduce experimental errors. TaqMan ™ 20X Assay probes (Table 1; Ther-moFisher Scientific, India) were mixed with Universal PCR Master Mix (4304437, Applied Biosystems ™ , ThermoFisher Scientific, India) for the qPCR experiments. Quantitative polymerase chain reaction (qPCR) was performed in CFX96 (BioRad, India) to assess the relative change in mRNA expression of genes coding for activated NLRP3 inflammasome complex proteins. The PCR conditions used were as follows: to achieve a maximum of the reaction, conditions used were optimized for 20 μl of the reaction volume with initial denaturation step (hold at 95 °C for 10 min) followed by 40 cycles of 95 °C for 15 s, 60 °C (TaqMan 20X assay specific) for 1 min followed by a final hold at 4 °C.
On completion, the plates were stored at − 20 °C and the mean cycle quantification (Cq) values were recorded (Bustin et al. 2009). Fluorescence signals were detected in the channel 1: Fam (CFX96, Bio-Rad, India). The efficiency of the gene assay mixes was determined using standard dilution method following MIQE guidelines (Bustin et al. 2009). The recorded Cq values from the test group was normalized against efficiency of assay 20X for each gene, sample mRNA amount, number of experimental repeats, number of qPCR repeats and with Cq values of three selected reference genes that were most stably expressed across all experiments. Relative expression of genes for the test groups was compared with that of control and represented by converting to their log 2 Cq value.

Statistical analysis
All experiments were performed in triplicates and statistical significance of variation was determined using One-way ANOVA with appropriate post hoc test using GraphPad Prism software version 8.04 for Windows (GraphPad Software, San Diego, California USA) and GeneX 7.0 (GenEx ™ , Sweden). A value of p < 0.05 was considered significant.

Preparation and characterization of nanoparticles
The CSNP and gCSNP were fabricated by ion gelation method using TPP as the crosslinker. The physicochemical characteristics were estimated initially with the zeta sizer to establish the protocol for the size distribution of the nanoparticles. Table 2 summarizes the zeta sizer profile of the nanoparticles prepared by the ion gelation protocol. The size of CSNP was 155.1 ± 5.7 nm in diameter while the size of gCSNP was 181.1 ± 6.9 nm in diameter. The polydispersity index of both the NPs was between 0.35 and 0.4 ± 0.03. The NPs were later subjected to FTIR and XRD detection protocols to ascertain the conjugation and the structure of the NP (Fig. 1). Since the FTIR spectra between 4000-2500 cm −1 and 1000-400 cm −1 for all the samples were identical, these regions were not plotted. In the FTIR, the spectra of gCSNP were compared with those of CSNP, chitosan (CS) and GA. The FTIR spectra of the CS and its CSNPs revealed a new trough peak indicating the appearance of PO stretching at 1256 cm −1 . The FTIR of CS was also compared with gCSNP. The characteristic transmittance trough of CS appeared at 1640, 1585, 1374 and 1150-1040 cm −1 . In addition, significant changes in the troughs of amide III group around 1374 cm −1 could be observed. The gCSNPs displayed a decrease in the peak at 1320 and 1380 cm −1 . Moreover, the intensity of (NH 2 ) trough band at 1628 cm −1 found in CS decreased dramatically and a new band at 1550 cm −1 appeared denoting the cross-linking with TPP during the nanoparticle formation. The FTIR of CS was also compared with gCSNP. The characteristic transmittance trough of CS appeared at 1640, 1585, 1374 and 1150-1040 cm −1 . In addition, significant changes in the troughs of amide III group around 1374 cm −1 could be observed. The gCSNP displayed a decrease in the peak at 1320 and 1380 cm −1 attributed to the NH bending observed in the glucosamine units and at 1420 cm −1 (the symmetric NH 3 + bending region) when compared with CSNPs. . In addition, in gCSNP as compared to the CSNP, some changes in the spectra were observed at 1730 and 1640 cm −1 representing C=O stretching in esters and C=O stretch of chitosan amide. The XRD spectrum of CS was compared with gCSNP ( Fig. 1B). We observed a shift towards the right side in the case of gCSNP signifying an increase in the amorphous nature of the nanoparticle as compared to the CS.
However, we observed that the conjugation of gallic acid reversed the immunosuppressor mimicking action of CSNP, as all three gCSNP doses significantly (p < 0.0001) increased IL1β protein secretion in the extracellular solution of treated HeLa 229 cells.

Discussion
Inflammasomes are the multi-protein platform in the innate immune system that induce procaspase-1 activation and inflammatory cytokines maturation such as IL1β. Chitosan-based nanoparticles are good immune enhancers by dint of being able to reverse cytokine changes in immunosuppression preclinical models (Mudgal et al. 2019). To explore the efficacy of the nanoparticles as immunomodulators in the HeLa 229 cell line, we fabricated CSNP and gCSNP. The fabrication of the chitosan nanoparticles and the conjugation of gallic were analyzed with the FTIR. The FTIR spectra of the CS and its CSNPs revealed a new trough peak indicating the appearance of PO stretching at 1256/ cm. The characteristic transmittance trough of CS appeared at 1640, 1585, 1374 and 1150-1040 cm −1 . This corresponds to amide I, amide II, amide III groups, and glycosidic linkage (C-O-C), respectively (Pawlak and Mucha 2003). Moreover, the intensity of (NH 2 ) trough band at 1628/cm found in CS decreased dramatically and a new band at 1550/cm appeared. This confirmed the cross-linking of the TPP with chitosan to form the nanoparticles. The FTIR of CS was also compared with gCSNP. The characteristic transmittance trough of CS appeared at 1640, 1585, 1374 and 1150-1040 cm −1 . In addition, significant changes in the troughs of amide III group around 1374 cm −1 could be observed (Wei and Gao 2016). Amide III bands arose from the C-N stretching vibration. This might be due to the conjugation of GA to the amino group in CS in gCSNP (Wei and Gao 2016). The gCSNP displayed a decrease in the peak at 1320 and 1380/cm attributed to the NH bending observed in the glucosamine units and at 1420/cm (the symmetric NH 3 + bending region) when compared with CSNPs. The conjugation of GA with CS was also confirmed by XRD which revealed an increase of amorphous characteristic due to a reduction of the inter-and intra-hydrogen bonds. This is in agreement with the results by other groups . The size of the engineered nanoparticles is another factor which determines the bio-nano interaction (Nel et al. 2006). The size effect of ENMs on NLRP3 inflammasome activation has been examined with the nanoparticles in the lower range showing pronounced effects on IL-1β production by several studies, though contradictory results were obtained (Sun et al. 2013). The effect of the size on the NLRP3 inflammasome regulation was studied with CSNP and gCSNP. The size of CSNP was found to be 155 nm while the encapsulation of GA increased the size by 26 nm to 181.1 nm. The encapsulation efficiency of GA was found to be 90.2% based on the already reported earlier (Lamarra et al. 2016). Activation of NLRP3 inflammasome involves coordinated processes between stimuli and cells. The nanoparticles were fabricated based on the hypothesis that CSNP due to improved surface area would act as antioxidant curbing the NLRP3 inflammasome and its activity would be further augmented by gallic acid conjugation. Three different models have been proposed for the NLRP3 inflammasome activation. In the present paper, we have tried to explore the K + efflux model (Tschopp and Schroder 2010). In the context of the present study, the CSNP induced downregulation of NLRP3 inflammasome complex genes in cervical cancer cells, might prove beneficial in ameliorating the adverse effects of inflammasome gene expression thus avoiding the ensuing adjacent normal tissue damage. Thus, this immunosuppressor mimicking activity of CSNP can potentially inhibit the infiltration of myeloid and myeloid derived cells, thereby preventing the development of inflammatory microenvironment around cancer cells to attenuate tumor progression (Mudgal et al. 2019).
Through our study, we infer that the conjugation of gallic acid reversed the immunosuppressor mimicking action of CSNP, indicated by the release of pro-inflammatory cytokine IL1β from HeLa 229 cells. It is known that gallic acid and CSNP work synergistically as is observed in our study where in contrast to CSNP alone, gCSNP upregulated the expression of genes coding for NLRP3 inflammasome pathway as part of the priming signal (Hamarsheh and Zeiser 2020;Mudgal et al. 2019;Zheng et al. 2018). Hence, we infer that CSNP alone at 155 nm size, downregulated the expression of genes coding for NLRP3 inflammasome complex even at higher doses. The present study demonstrated how a bifunctional role of CSNP can be elicited with the mere conjugation of gallic acid, by reversing its role in HeLa 229 cells. This allowed an unopposed priming signal for the inflammasome complex proteins. This effect on the priming signal by gCSNP could be due to the physicochemical property of the nanoparticle and its interaction with the biological system that is occurring at the nano-bio interface.
Furthermore, with respect of the activation signal, our study helps to infer that unlike gCSNP, CSNP was also effective in controlling the formation of NLRP3 inflammasome complex probably by inhibiting the P2RX7 gene even in the presence of external ATP as per the K + efflux model (Hamarsheh and Zeiser 2020;Kantono and Guo 2017;Karki and Kanneganti 2019). The significant increase in the IL1β secretion from HeLa 229 cells when treated with gCSNP indicate that conjugating gallic acid led to the reversal of drug action. To the best of our knowledge, we demonstrated for the first time that conjugation of gallic acid reverses the immunosuppressor mimicking property of CSNP in NLRP3 inflammasome-stimulated HeLa 229 cell line model. We can speculate within reason that the conjugation of gallic acid with CSNP could potentially activate the NLRP3 inflammasome as previously observed with other NPs by Jo et al. 2016. In the presence of higher doses of gCSNP, the increased IL1β release from cervical cancer cells accompanied with the upregulation of P2RX7 gene implicated the unopposed activation of NLRP3 inflammasome complex.

Conclusions
The present study demonstrated that the role reversal of CSNP can be achieved when conjugated with gallic acid at higher doses. Interestingly, given its wide range of safe limits of cytotoxicity, as observed in our study, alternative administration time can be tested to come to a conclusion in case of HeLa 229 cells. Additionally, on a larger perspective, it is an interesting finding to note that gallic acid conjugated nanoparticles

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