s2fieldsofstudy: Chemistry Biology

text:
Mitochondria-targeted half-sandwich iridium(iii)-Cp*-arylimidazophenanthroline complexes as antiproliferative and bioimaging agents against triple negative breast cancer cells MDA-MB-468

To reduce the side effects of marketed cancer drugs against triple negative breast cancer cells we have reported mitochondria targeting half-sandwich iridium(iii)-Cp*-arylimidazophenanthroline complexes for MDA-MB-468 cell therapy and diagnosis. Out of five Ir(iii) complexes (IrL1–IrL5), [iridium(iii)-Cp*-2-(naphthalen-1-yl)-1H-imidazo[4,5-f][1,10]phenanthroline]PF6 (IrL1) has exhibited the best cytoselectivity against MDA-MB-468 cells compared to normal HaCaT cells along with excellent binding efficacy with DNA as well as serum albumin. The subcellular localization study of the complex revealed the localization of the compound in cytoplasm thereby pointing to a possible mitochondrial localization and consequent mitochondrial dysfunction via MMP alteration and ROS generation. Moreover, the IrL1 complex facilitated a substantial G1 phase cell-cycle arrest of MDA-MB-468 cells at the highest tested concentration of 5 μM. The study verdicts support the prospective therapeutic potential of the IrL1 complex in the treatment and eradication of triple negative breast cancer cells. These results validate that these types of scaffolds will be fairly able to exert great potential for tumor diagnosis as well as therapy in the near future.

Introduction
Nowadays, breast cancer prevails over most of the deadly life threats towards women from all over the world. 11.7% of new cases of cancer diagnosed in 2020 were female breast cancer which is the highest number of cases among all types of cancer reported. 1 This disease is heterogenous at the molecular level but, as a consequence of diligent research over past decades, chances of healing have been increased by 70-80% in breast cancer patients when the cancer is non-metastatic and treated in the early stage. Over the years of research a few categorisations of tumours were rened based on the alterations at the molecular level. 2 Current clinical practices involve an alternate classication of 5 types that includes triple negative breast cancer (TNBC) without expression of ER, PR or HER2. TNBC is adenoid cystic and metaplastic in nature with a poor prognosis prole. 2 A thorough investigation of medical literature reveals that the term, "triple negative breast cancer", was rst mentioned in 2005 by Brenton and coworkers. 3 However, in the present situation 12-17% of all reported breast cancer cases have been recognised under this class.
It has been challenging to create an effective treatment regime for TNBC patients as this aggressive subtype does not respond to the HER2 targeting drugs or hormonal therapy. Consequently, chemotherapy remains as the main systemic treatment option even though TNBC develops resistance easily to existing targeted medicines like trastuzumab. Potential molecular targets for TNBC may include EGFR (a surface receptor), PARP1 (poly ADP-ribose polymerase 1) and DNA. The phenotypic similarity of TNBC to BRCA-1 associated malignancy helped researchers to develop few targeted cytotoxic agents currently which may lead to a new horizon of TNBC therapeutics. In 2020 FDA approved trodelvy (sacituzumab govitecanhziy) which is a topoisomerase inhibitor conjugate antibody directed to Trop-2 receptor for metastatic TNBC and for the rst-time improved progression of overall survival was witnessed. Triple negative breast cancer caused by BRCA1 and BRCA2 mutation can be a potential target of DNA damaging chemotherapeutics and a number of clinical data leads to the suggestion that involving platinum-based chemotherapeutics may be use in standard treatment regime of early stage as well as advanced TNBC. 4 As reported in earlier scientic research, platinum salts like cisplatin, carboplatin or oxaliplatin are able to initiate a platinum-DNA adduct formation followed by DNA damage in cancer cells leading towards apoptosis of the targeted cancer cells. BRCA1 decient breast cancer showed sensitivity towards platinum and gemcitabine neoadjuvant treatment but with poor chance of progression free survival. 5 A clinical study, reported by von Minckwitz in 2013 involving 315 early stage TNBC patients, let out the fact that a neoadjuvant treatment regime comprising doxorubicin and carboplatin achieved 59% pathologic complete response rate. 6 However, a combination therapy of gemcitabine/carboplatin given to advanced metastatic TNBC patients in a trial scored 34% objective response rate and progression free survival of 5.1 months. 7 Although cisplatin and its analogical platinum complexes showed slight prospect in TNBC treatment they have exerted some major drawbacks like toxicity, chemoresistance and narrow activity window. 8 This fact motivated broader area of investigation on efficacy of other metal complexes to target metastasis and defective cell proliferation. A handful of gold, iron, copper, ruthenium, rhodium and iridium complexes happened to be synthesized and tasted against breast cancer cell lines in vitro or in vivo and found to manifest excellent outcomes. 9 Of late, Ir(III) based metal complexes have been fascinating the mind of the investigators for being reconnoitered as highly active anticancer agents with their excellent photoluminescence property possessing high photostabilities, long-lived excited triplet states aer quick singlet to triplet intersystem crossing (ISC), larger Stokes' shi associated with high quantum yields. 10,11 Furthermore, the higher oxidation state of iridium metal, ability to display a wide range of ligand substitution, considerable redox properties, exible structural features and stability towards cancer cell environments has enabled them to be used in preparing anticancer metal complexes. 10,11 Instead, bioactivities of metal complexes are also reliant on the structure of the ligands. Arylimidazophenthroline compounds are well known as pertinent probes of DNA structure having the capability of disentangling the double stranded DNA through strong intercalative interaction. 12 Encouraged by our previous work with ruthenium and arylimidazophenthroline herein we have intended to design ve iridium(III)-Cp* N^N metal complexes with planar and p-extended arylimidazophenanthroline moiety as ligand having strong metal binding capacity (Fig. 1). [13][14][15][16][17][18] and evaluated their antiproliferative activity against MDA-MB-468 TNBC cells that are EGFR +ve, TGF alpha +ve, high in Ki67. These complexes exhibited dual properties like (i) killing of cancer cells via DNA damage (ii) mitochondrial dysfunction by ROS production. 19 The intrinsic phosphorescence property of these Ir(III) complexes are also helpful for cellular imaging and tracking of drug accumulation in subcellular organelles. 20

Results and discussion
Chemistry Synthesis and characterization. A series of imidazo [4,5-f] [1,10]phenanthroline ligands (L1-L5) was prepared by treating an equimolar mixture of 1,10-phenanthroline-5,6-dione and different aromatic carboxaldehydes (1)(2)(3)(4)(5) in the presence of ammonium acetate and glacial acetic acid, following the same procedure as mentioned in our previous communication. 21 Further to prepare Ir(III)-Cp*-imidazophenanthroline complexes (IrL1-IrL5), [(C 5 (CH 3 ) 5 IrCl 2 )] 2 was added to the prepared ligands (L1-L5) in a 1 : 2 ratio in methanol and stirred at room temperature for 2 h. Aer a change in colour from light yellow to orange, 2.5 equivalents of NH 4 PF 6 were added and stirred again for 90 min (Scheme 1). The complexes [Cp*Ir(L1-L5)Cl]PF 6 labelled as IrL1-IrL5 were obtained in good yield (92-95%). The structures of all the complexes (IrL1-IrL5) were analysed via 1 H, 13 C, 19 F and 31 P NMR, and mass spectroscopy. The complex IrL1 displayed a characteristic singlet peak at 1.74 ppm, corresponding to the ve methyl groups of pentamethylcyclopentadiene. The protons of complex IrL1 experienced a considerable downeld effect upon attachment to the iridium Cp* precursor. In the 13 C NMR spectrum, the ligand carbons appeared at around d 125.8-153.2 ppm. The aliphatic CH 3 carbons peaks were observed at d 8.7 ppm and aromatic CH carbons peaks were observed at d 89.6 ppm. In the 19 F NMR  Electronic absorption (UV-visible) and uorescence study. The absorption and emission spectra of all the complexes (IrL1-IrL5) at 298 K were recorded in a DMSO-water (1 : 1) solvent system, as shown in Fig. 2. The photophysical data is summarized in Table 1. The characteristic intraligand (p-p*) transitions (N^N ligands) appeared at 250-350 nm and metal to ligand charge transfer ( 1 MLCT) at 360-400 nm. 22,23 Among the complexes, we observed the maximum absorption in the 1 MLCT region for the benzothiazole derivative (IrL5). In the emission spectra, we observed the MLCT emission of all the complexes in the range of 350-525 nm (Fig. 2). Similar to the absorption spectra, the emission for the anthracene derivative is the most intense because of its strong p conjugation. Using the emission spectral data, the quantum yield of these complexes was calculated. These complexes didn't show remarkable quantum yield, complex IrL1 showed moderate quantum yield (0.002) though for the MLCT transition (Table 1).
Solubility, lipophilicity and conductivity study. Both hydrophilicity and lipophilicity studies were performed to determine the tumour-inhibiting potential of the metal complexes. These complexes were highly soluble in DMSO and moderately soluble in H 2 O, MeOH, EtOH and CH 3 CN. Furthermore, they were soluble in the range of 0.6-0.8 mg per mL of 10% DMSO in DMEM, 10 : 90 v/v (comparable to cell media) at 25 C ( Table 1). The lipophilicity of these complexes was determined by performing an n-octanol/water partition coefficient (log P o/w , where P o/w ¼ the octanol/water partition coefficient) study using the shake ask method (Table 1). 24 The experimental log P o/w values of these complexes were determined to be in the range of 0.27-0.34 (Table 1). Complex IrL2 exhibited the highest log P o/w due to the hydrophobic nature of its anthracene group. The lowest log P o/w value was observed for compound IrL3 because it's hydrophilic chromone group. The iridium complexes IrL1-IrL5 exhibited molar conductance values in the range of $7-9 S m 2 M À1 in pure DMSO. Furthermore, their molar conductance increased in 10% DMSO ($22-36 S m 2 M À1 , Table 1), suggesting Scheme 1 Synthesis of (h 5 -Cp*)iridium(III)-imidazophenanthroline complexes.  Stability study of the complexes by UV-vis spectroscopy. The stability studies of complex IrL1 were conducted in six different solvents, i.e. aqueous DMSO (H 2 O : DMSO ¼ 9 : 1) and aqueous GSH medium (Fig. 3) respectively in presence or absence of different concentrations of NaCl. It is essential that the complexes remain stable in the biological environments of cells, and thus the stability studies were performed.
The obvious change in absorbance ($15-30% decrease aer 24 h) with time in aqueous DMSO clearly revealed the moderate dissociation of the -Cl ligand from the Ir(III) complexes followed by aqua complex formation, which was also quantitatively determined based on the observed molar conductivity of the complexes (IrL1-IrL5) in aqueous DMSO and it favours DNA covalent binding as well. It has been reported that many cancer cells become resistant to various drugs by increasing their cellular glutathione level. 28 Hence, to determine the effect of GSH on the reported complexes, a stability study was performed in the presence of excess (10 eq.) glutathione (GSH) via time-dependent UV spectroscopy. Table 1 Photophysical characterization, solubility, lipophilicity and conductivity study of the complexes (IrL1-IrL5)  However, GSH didn't have much impact on the stability of metal complex and hence resistivity may not be induced against this complex in cancer cells containing high level of GSH. Cyclic voltammetry. The CV response of ten-continuous cycle shows a well-dened reversible redox peak at E 0 ¼ À0.485 AE 0.005 V versus Ag/AgCl with a peak separation potential of DE p value is 0.121 AE 0.005 (DE p ¼ E pa À E pc ). Peak current (i pa and i pc ) of the redox peak was found to increasing linearly which ensures the high stability and co-ordination of the metal-ligand complex (Fig. 4).

Biology
In vitro cytotoxicity study. The in vitro cytotoxicity of complexes IrL1-IrL5 and cisplatin were investigated using the typical 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay protocol against a panel of triple negative cancer cell line MDA-MB-468. The cells were initially incubated with the all test compounds at concentrations ranging from 0.5-50 mM for 48 h in vitro.
All the complexes exhibited signicant cytotoxicity with IC 50 values of 3.673 mM, 20.35 mM, 20.27 mM, 5.202 mM, 4.412 mM for IrL1, IrL2, IrL3, IrL4 and IrL5 respectively (Fig. 4). The dose dependent cytotoxic effects of these complexes were evident on MDA-MB-468 with IrL1 exhibiting the most potent effects thereby exhibiting the lowest IC 50 value of 3.67 mM (Fig. 5). Consequently, the IrL1 complex doses of 1 mM, 3 mM and 5 mM were selected for further experimental analysis. Furthermore, the possible cytotoxic effects of IrL1 on normal cell line were also examined on the immortalized human keratinocyte cell line HaCaT. The IC 50 value of IrL1 on HaCaT   is found to be 11.42 mM, which is signicantly $3 times higher than the IC 50 value of the complex on the breast cancer cell line MDA-MB-468, suggesting the low cytotoxic effects of the same on normal cells and safety implications to use the compound for selective therapeutics at lower concentrations (Fig. 4).

DNA binding studies
UV absorption method. To design effective chemotherapeutic drugs, it is essential to explore the interactions of metal complexes with DNA. Complex IrL1 displayed strong absorption band at 297 nm. DNA base pairs such as purine (adenine and guanine) and pyrimidine (cytosine and thymine) analogues are responsible for electronic transitions. Upon the addition of CT-DNA in increasing concentration from 5 mM to 60 mM, we observed a hyperchromic shi in p-p* region and hypochromic shi at the MLCT region resulting in the appearance of an isosbestic point, which indicates the prevalence of covalent interaction of the complex with DNA together with the intercalative mode of interaction (Fig. 6).
EtBr quenching study. The competitive binding of compounds IrL1 to CT-DNA was studied via uorescence spectroscopy using ethidium bromide (EtBr) as the uorophore. We clearly observed a gradual decrease in the uorescence intensity of the EtBr bound DNA in the presence of the complexes since they displaced EtBr from DNA, and consequently got bound between the base pairs of the DNA, suggesting the intercalative binding mode of action, as observed in Fig. 7. Intrinsic binding constant (K b ), Stern-Volmer quenching constant (K sv ) and apparent binding constant (K app ) were highlighted in Table 2.
The higher value of binding constant and lower values of Stern-Volmer's quenching constant and apparent binding constant is evidence of the fact that IrL1 complex interacts with DNA via covalent bond and not via intercalation mode.
BSA binding study. Upon excitation at 295 nm, the emission intensity of BSA at l em ¼ 350 nm decreased gradually on increasing the complex concentration, which conrmed that the interaction between complex IrL1 with BSA had occurred, as observed in Fig. 8. The Stern-Volmer quenching constant of these complexes with BSA (K BSA ) was calculated using the Stern-Volmer equation and the corresponding Stern-Volmer plots (Fig. 8, inset). The binding affinity (K) of the complexes was calculated from Scatchard plot analysis (Fig. 8, inset).
The complexes showed strong binding propensity with BSA, which is required for the transport of protein-bound complexes in biological systems. K BSA for complex IrL1 was found to be 5 Â 10 4 M À1 the K value was 6.3 Â 10 6 M À1 . The value of bimolecular quenching constant (k q ) calculated from K SV and s 0 (1 Â 10 À8 s) was observed to be 5 Â 10 12 M À1 S À1 . These values are higher than the maximum possible value for dynamic quenching (2.0 Â 10 10 L mol À1 s À1 ), 29,30 suggesting the involvement of static quenching mechanism by the present iridium(III) complexes.   The non-linearity of the Stern-Volmer plot of IrL1 is due to the formation of a ground state complex between the Ir(III) complex and BSA.
Co-localization study. To identify the subcellular localization of the IrL1 complex, the cells were treated with the complex and stained with Hoechst and further explored using uorescence microscopy. The complex exhibited cytoplasmic localization at 3 mM concentration with a green uorescence emission (Fig. 9). Mitochondria are one of the key targets for several anticancer drugs. Consequently, further experimental validation involved mitochondrial membrane potential (MMP) analysis by JC-1 staining and detection of ROS generation by DCFDA staining.
Mitochondrial membrane dysfunction study. The IrL1 complex demonstrated mitochondrial depolarization of MDA-MB-468 cells in a dose dependent manner. The cytoplasmic localization of the IrL1 complex as evidenced by sub-cellular localization study impelled us to explore the effect of the complex on cytoplasmic mitochondria. Consequently, the alterations in mitochondrial membrane potential (MMP, DJ m ) and associated mitochondrial dysfunction was demonstrated by JC-1, a cationic carbocyanine dye which exhibits potentialdependent accumulation in mitochondria. The cells treated with the mitochondrial uncoupler CCCP (carbonyl cyanide mchlorophenylhydrazone), which mediates the dissipation of mitochondrial membrane potential, served as positive control for the detection of mitochondrial dysfunction.
The ow cytometric quantication of JC-1-stained cells revealed a normal mitochondrial function in control cells devoid of any treatment exhibiting $94% JC-1 aggregates (red+ green uorescing healthy mitochondria). Impaired mitochondrial function was observed in only 3.30% of control cells with JC-1 monomers indicating green uorescence (Fig. 10B). Unstained control represented in Fig. 10A has been used for gating purposes. Conversely, the positive control with CCCP treatment, displayed a high percentage of cells ($79%) with damaged mitochondria indicating the presence of green uorescing JC-1 monomers against $20% cells retaining healthy mitochondria with red + green uorescing JC-1 aggregates (Fig. 10C).
Treatment of the MDA-MB-468 cells with IrL1 has ensued a dose dependent increase in JC-1 monomer expressing/green uorescing cells indicating damaged mitochondria (Fig. 10D-F). IrL1 complex at 1 mM concentration resulted in damaged mitochondria in 27.86% cells (Fig. 10D). However, the higher concentrations of the IrL1 complex at 3 and 5 mM induced alterations of mitochondrial membrane potential and associated damage in high percentage $47.52% and 67.02% respectively in MDA-MB-468 breast cancer cells (Fig. 10E and F). The proportion of green uorescing JC-1 monomers induced by high concentrations of the complex at 3 and 5 mM thereby indicates alteration in the MMP and mitochondrial depolarization and associated mitochondrial dysfunction leading to a dose dependent possible cell death meditated by the IrL1 complex comparable to the CCCP treated positive control cells (Fig. 10C).
ROS generation studies. The IrL1 complex exhibited a dose dependent reactive oxygen species (ROS) generation in the MDA-MB-468 breast cancer cells. The MDA-MB-468 breast cancer cells exhibited ROS generation suggestive of cellular stress leading towards cell death at elevated ROS levels.
Our results indicated a dose dependent increase in ROS production in the MDA-MB-468 cells treated with IrL1 concentrations 1 mM and 3 mM. The extent of ROS production is indicated by the 2 0 ,7 0 -dichlorouorescein (DCF) formed by the deacetylation and subsequent oxidation of 2 0 ,7 0 -dichloro-uorescin diacetate (DCFDA) through cellular esterases and ROS. 1 mM concentration of the complex has resulted in 55.01% DCF positive cells which has increased up to 70.67% DCF positive cells at 3 mM concentrations. ROS production was absent in untreated control. However, in the positive control, treated with H 2 O 2 , ROS production was high which is $99% (Fig. 11).
Cell cycle analysis. The IrL1 complex exerted a G 0 /G 1 cell cycle arrest at the highest tested concentration of 5 mM. The   16%), followed by a G 2 /M phase with $33.18% cells, and 31.66% of G 0 /G 1 cells (Fig. 12). Conversely, the treatment of IrL1 complex resulted in a decrease of G 2 /M phase cells upto $18.37% and a decrease of S phase cells upto $28.32% with the highest concentration of the complex i.e., 5 mM. Remarkably, 5 mm concentration considerably increased G 0 / G 1 phase cells (53.32%), indicative of a substantial G 1 arrest of MDA-MB-468 breast cancer cells mediated by the IrL1 complex.
Annexin FITC/PI assay. The Annexin FITC/PI assay has demonstrated signicant apoptotic initiation of IrL1 treated MDA-MB-468 breast cancer cells. At 3 mM concentration of  the complex, $34.89% of cells were found to be displaying early apoptotic phase and $9.49% cells exhibited late apoptotic phase (Fig. 13). The viable cell population constituted $51.37% which supports the IC 50 concentration of 3.673 mM demonstrated by IrL1 on MDA-MB-468 cells thereby corroborating the IC 50 data at this particular concentration.

Materials and methods
In all the experiments, the reagents and solvents used were of the highest grade and best commercial quality. All organic solvents used throughout the chemical synthesis and chromatography procedures were of analytical grade and used without  further purication as received from E. Merck (India). Pentamethylcyclopentadienyl iridium(III) chloride dimer, 1,10phenanthroline-5,6-dione, a-naphthaldehyde, 9-anthraldehyde, chromone-3-carboxaldehyde, indole-3-carboxaldehyde and benzothiazole-2-carboxaldehyde were procured from Sigma Aldrich Chemical Ltd, Merck and Spectrochem. Thin layer chromatography was performed on pre-coated silica gel 60 F 254 aluminium sheets (E. Merck, Germany) and the solvent system was an ethyl-acetate-methanol mixture. 1 2 , was dissolved in about 10 ml of methanol in a 50 ml round bottomed ask and was stirred continuously for 5-10 min to dissolve the reactant. To the completely dissolved solution, 2.1 equivalents of the previously synthesized ligands (L1-L5) were added and stirred at room temperature. Aer 90 min, 2.5 equivalents of ammonium hexauorophosphate (NH 4 PF 6 ) (13.3 mg, 0.082 mmol) was added as ligand exchange salt in order to increase crystallinity and hence, purity of the product and again the reaction mixture was stirred for 90 min more at room temperature. The progress of reaction was conrmed by TLC. Aer complete conversion of the starting materials to the desired product, the solvent was evaporated under reduced pressure. The crude product was washed with hexane and further recrystallized from diethyl ether/methanol (1 : 1) solvent system. Finally, the complexes (IrL1-IrL5) were obtained as light brown crystals with high yield (90-92%).

Cytotoxicity studies
Cytotoxicity study of all the synthesized compounds against triple negative breast cancer cells MDA-MB-468 and immortalized human keratinocyte cell line HaCaT were done with a drug incubation period of 48 h by following standard procedure that were mentioned in ESI. † Preliminary studies were done to understand the mode of action of the complex to induce cytotoxicity using ow cytometric methods.

Mitochondrial membrane dysfunction assay
Alterations in mitochondrial membrane potential (MMP, DJ m ) and associated mitochondrial dysfunction was demonstrated by JC-1, a cationic carbocyanine dye. The cells were treated with the mitochondrial uncoupler CCCP (carbonyl cyanide m-chlorophenylhydrazone), which mediates the dissipation of mitochondrial membrane potential, served as positive control for the detection of mitochondrial dysfunction. The ow cytometric quantication of JC-1 stained cells revealed a normal mitochondrial function in control cells and the results obtained from the quantied JC-1 stained cells among IrL1 treated cells were compared with the same of the control.

Imaging studies
Colocalization study and ROS generation studies were performed using live cell lines in their log phase.

Statistical analysis
As the study had more than one group, one way ANOVA was used for statistical analysis. The p value <0.05 was considered as signicant.

Conclusions
Five Ir(III)-imidazophenanthroline complexes were synthesized and characterized successfully. Our study has demonstrated the cytotoxic potential of IrL1 complex on MDA-MB-468 breast cancer cells in a dose dependent manner. The subcellular localization study of the complex revealed the localization of the compound in cytoplasm thereby pointing to a possible mitochondrial localization and consequent mitochondrial dysfunction. Subsequent analysis to demonstrate the alterations in MMP and ROS generation mediated by the IrL1 complex has revealed a signicant increase in mitochondrial dysfunction and a resultant increase in ROS production suggestive of the mitochondrial targeting potential of the IrL1 complex. The Annexin V-FITC/PI assay has validated the cytotoxic potential, along with the IC 50 dose of the complex on MDA-MB-468 breast cancer cells by initiating apoptotic pathway probably due to the cellular energetic stress triggered by elevated ROS levels. Furthermore, the IrL1 complex mediated a substantial G 1 phase cell-cycle arrest of MDA-MB-468 cells at the highest tested concentration of 5 mM. The study ndings support the prospective therapeutic potential of the IrL1 complex in the treatment and eradication of triple negative breast cancer cells.

Conflicts of interest
There are no conicts to declare.


Rheological Properties of Cholesteric Liquid Crystal with Visible Reflection from an Etherified Hydroxypropyl Cellulose Derivative

Optical properties of hydroxypropyl cellulose (HPC) derivatives have been widely investigated for their ability to exhibit cholesteric liquid crystal (CLC) phase. However, there are only a limited number of studies on their rheological properties even though they are quite important for the applications of such HPC derivatives to the versatile CLC photonic devices. In this article, we report on the optical and rheological properties of an HPC derivative possessing pentyl ether groups in the side-chains. The etherified HPC derivative exhibited thermotropic CLC phase with light reflection in the temperature range between 25 °C and 120 °C. After the HPC derivative was heated once at isotropic phase, followed by being cooled to the CLC phase, the reflection peak could not be observed, even at the CLC phase. At this stage, the HPC derivative exhibited solid-like rheological responses compared to that of sheared at a constant shear rate of 1.0 s−1. Such differences in the optical and rheological properties of the HPC derivative can be ascribed to the difference in CLC orientation state. From the rheological results, the etherified HPC derivative showed liquid-like behavior rather than the esterified HPC derivatives. This evidence provides a promising clue for fabricating high-quality CLC devices by the facile CLC orientation.

Introduction
Cellulose is the most abundant natural polymer on the Earth. Cellulose derivatives are attractive because of their safety and biocompatibility. Hydroxypropyl cellulose (HPC), as depicted in Figure 1, is a cellulose derivative that can be obtained by reacting natural cellulose with propylene oxide. Owing to its safety, HPC has been utilized not only as food and pharmaceutical additives, but also as thickeners and coating agents in interdisciplinary industrial fields [1][2][3][4].
Another interesting property of HPC is its ability to exhibit cholesteric liquid crystal (CLC) phase with light reflection [5]. Gray and Werbowyj succeeded for the first time in the preparation of CLCs using pristine HPC. When powdery HPC is dissolved in water at high concentration, the viscous solutions show lyotropic CLC phase with visible reflection characteristics depending on the concentration [6,7]. Following their archetypal demonstrations, numerous studies have shown that HPC derivatives also exhibit thermotropic CLC phase with visible reflection after the hydroxy groups in side-chains of HPC are chemically substituted with alkyl chains through ester [8,9] or ether bonds [10][11][12], that is, esterified or etherified HPC derivatives, respectively. At CLC mesophase, the rod-shaped molecules spontaneously form the periodic helicoidal structure owing to their chirality, thereby enabling the emergence of unique light reflection at a specific wavelength. Such a light-reflection phenomenon can be recognized as a kind of Bragg reflection [13,14]. The maximum reflection wavelength (λ) can be numerically expressed according to the following equation proposed by De Vries [15]: where n stands for the average refractive index and p is the helical pitch length of CLC.
In the case of thermotropic CLCs, the p value readily fluctuates with temperature. As a result, the reflection colors of CLCs are significantly dependent on the temperature, leading to their wide range of potential applications as temperature indicators, reflective color displays, full-color recording media, tunable lasers, and so forth [16].
eco-friendliness for the next-generation sustainable society. To date, several previous studies have dealt with the CLCs of esterified HPC derivatives possessing alkanoyl sidechains. For instance, our previous report has shown that the rheological properties of esterified HPC derivatives are greatly affected by the CLC orientation [23,24]. On the other hand, we have recently established a promising methodology to synthesize etherified HPC derivatives possessing alkoxy side-chains [11]. Such etherified HPC derivatives also exhibit thermotropic CLC phase in a manner similar to that of esterified HPC derivatives. Furthermore, it is predictable that the completely etherified HPC derivatives exhibit stable reflection colors under highly humid conditions because of the chemical stability of the ether linkage rather than the ester [11]. Such stability of the etherified HPC derivatives is highly attractive for the technological developments of CLC photonic devices when compared to the esterified HPC derivatives whose optical and rheological properties have been extensively studied. However, there has been no report to explore the rheological behavior of etherified HPC derivatives.  Figure 5, the previous results of an esterified HPC derivative possessing propionyl side-chains (HPC-PrEs) will be discussed in Section 3.4.
In this research, we investigated the optical and rheological properties of an HPC derivative possessing pentyl side-chains through ether bonds (HPC-PeEt), as shown in  Figure  5, the previous results of an esterified HPC derivative possessing propionyl side-chains (HPC-PrEs) will be discussed in Section 3.4. In this context, the rheological properties of CLCs have been attracting interest for decades [17,18]. To understand their rheological properties, which are distinct from those of other polymer solutions, aqueous solutions of cellulose derivatives have been one of the most frequently investigated motifs. This is because the cellulose derivatives generally form the rigid helical conformation in solution [19]. Based on this background, a few precedents have been made on the rheological behavior of lyotropic CLCs from mixtures of cellulose derivatives such as HPC or sulfonated cellulose nanocrystal with water [20,21].
In the case of thermotropic CLCs, it is quite interesting that sticky products can be obtained by the simple substitution of alkyl chains to the hydroxy groups of pristine HPC. This phenomenon can be observed when HPC is either esterified [22] or etherified [14]. In addition, it is of prime importance to understand the rheological behavior of CLCs from HPC derivatives because they are potentially applicable to CLC photonic devices with eco-friendliness for the next-generation sustainable society. To date, several previous studies have dealt with the CLCs of esterified HPC derivatives possessing alkanoyl sidechains. For instance, our previous report has shown that the rheological properties of esterified HPC derivatives are greatly affected by the CLC orientation [23,24]. On the other hand, we have recently established a promising methodology to synthesize etherified HPC derivatives possessing alkoxy side-chains [11]. Such etherified HPC derivatives also exhibit thermotropic CLC phase in a manner similar to that of esterified HPC derivatives. Furthermore, it is predictable that the completely etherified HPC derivatives exhibit stable reflection colors under highly humid conditions because of the chemical stability of the ether linkage rather than the ester [11]. Such stability of the etherified HPC derivatives is highly attractive for the technological developments of CLC photonic devices when compared to the esterified HPC derivatives whose optical and rheological properties have been extensively studied. However, there has been no report to explore the rheological behavior of etherified HPC derivatives.
In this research, we investigated the optical and rheological properties of an HPC derivative possessing pentyl side-chains through ether bonds (HPC-PeEt), as shown in Figure 1. The transmission spectral measurements revealed that a reflection peak of HPC-PeEt is reversibly shifted in the full visible-wavelength range by changing the temperature between 25 • C and 120 • C, arising from the thermotropic CLC feature. The rheological behavior of HPC-PeEt was greatly dependent on the CLC orientation state, similar to the case of esterified HPC derivatives. This report will contribute to the detailed comprehension of the correlation between CLC and rheological properties for various cellulose materials.

Experimental Section
HPC (viscosity of 2.0 wt% aqueous solution: 2.0-2.9 mPa·s) as the starting material was purchased from FUJIFILM Wako Pure Chemical Co (Osaka, Japan). The number average molecular weight (M n ) and weight average molecular weight (M w ) were 2.68 × 10 4 and 6.37 × 10 4 , respectively, as determined by measurement of size exclusion chromatography (SEC) equipped with a refractive index detector (RI-4030 and PU-4180, JASCO, Tokyo, Japan) calibrated using the polystyrene standards. In the SEC measurements, tetrahydrofuran (THF) was used as the eluent. HPC was dried in vacuo overnight before use. When the 1 H-NMR spectrum of this pristine HPC was measured in CDCl 3 , the molar amount of chemically combined propylene oxide per anhydroglucose unit, that is, the molar substitution (MS), was found to be 3.98. Therefore, the average molecular weight per anhydroglucose monomer unit could be calculated to be 394. Subsequently, the changes in 1 H-NMR spectrum of pristine HPC were evaluated during titration with trichloroacetyl isocyanate. From the 1 H-NMR spectral changes, the number of hydroxy groups substituted per anhydroglucose unit, that is, the degree of substitution (DS) was estimated to be 2.40, according to the previous report [25]. Dehydrated N-methyl-2-pyrrolidone (NMP) was obtained from Kanto Chemical Co., Inc. (Tokyo, Japan). 1-Bromopentane (PeBr) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Sodium hydroxide and potassium iodide were acquired from FUJIFILM Wako Pure Chemical Co. These reagents were used as received.
HPC-PeEt was synthesized by Williamson ether synthesis as reported in our previous study [11]. First, 5.00 g of HPC was completely dissolved in 80.0 mL of dehydrated NMP, and was subsequently added by 23.6 mL of PeBr (5.00 eq. to hydroxy groups of HPC). After stirring for 30 min at 65 • C, 7.60 g of powdered sodium hydroxide (5.00 eq. to hydroxy groups of HPC) and 1.58 g of potassium iodide (5.00 mol% to PeBr) were added into the reaction solution. After that, this reaction mixture was stirred at 65 • C for 48 h. Then, the reaction mixture was purified by two rounds of centrifugation at 1.0 × 10 4 rpm for 5 min to remove any sediment such as sodium bromide. PeBr was evaporated from the supernatant by heating at 60 • C in vacuo for 30 min. The residue was dialyzed against an equivolume mixture of methanol and water for 3 h, and the dialysis was continued for additional 48 h in an equivolume mixture of THF and methanol. The product was obtained by reprecipitation to a mixture of water and methanol at the volume ratio of 9:1, respectively, and was finally freeze-dried to afford the purified HPC-PeEt. The characterization of HPC-PeEt was carried out by FT-IR spectroscopy using an attenuated total-reflection module (FT-IR4700 and ATR PRO ONE, JASCO) and 1 H-NMR spectroscopy (JNM-ECZ400S, JEOL, Tokyo, Japan) for the molecular structure as well as SEC analysis for the M n and M w values.
In order to measure the optical properties, a CLC cell was fabricated by sandwiching HPC-PeEt between a pair of glass substrates. The cell gap was adjusted by polytetrafluoroethylene film spacers to a thickness of~200 µm. Transmission spectra of the CLC cell were taken on a compact charge-coupled device (CCD) spectrometer (USB2000+, Ocean Optics, FL, USA) equipped with an optical fiber. The CLC cell was illuminated with white light from a tungsten halogen light source (Ocean Optics, HL2000). The collinearly transmitted light from the CLC cell was focused through two pieces of achromatic doublet lenses and collected into the entrance of optical fiber connected with the CCD spectrometer. The temperature of the CLC cell was precisely controlled using a hot-stage system (HS82 and HS1, Mettler Toledo, OH, USA).
The viscosity measurements were conducted using a stress-controlled rheometer (MCR102, Anton Paar, Graz, Austria) equipped with a stainless-steel parallel plate with a diameter of 25 mm and a forced convection oven (Anton Paar, CTD450) for controlling the temperature. In the measurements, HPC-PeEt was sandwiched at a gap of~500 µm. Angular frequency (ω) dependence of HPC-PeEt was analyzed using the above-mentioned rheometer. Both the storage modulus (G ) and loss modulus (G") were measured at the range of ω between 0.1 rad/s and 100 rad/s at 25 • C. The strain amplitude was adjusted in the range between 0.2% and 1.2%, which is small enough to measure the linear viscoelasticity. Prior to this measurement, HPC-PeEt was either pre-heated or pre-sheared in order to erase any orientational history of CLC structures. As the pre-heat treatment, HPC-PeEt was heated at 155 • C for 10 min in the rheometer. This temperature was higher than the isotropic phase transition temperature of HPC-PeEt, corresponding to 150 • C, to retain the random orientation of cholesteric texture as confirmed by the polarized optical microscopic observation. On the other hand, the pre-shear treatment was conducted by shearing HPC-PeEt at a constant shear rate of 1.0 s −1 for 5 min at 25 • C, followed by leaving it to stand for 7 min at the same temperature after stopping the shearing force.

Characterization of HPC-PeEt
First, we measured FT-IR and 1 H-NMR spectra to confirm that all hydroxy groups of pristine HPC are substituted with pentyl ether side-chains after etherification. Figure 2A,B show the FT-IR spectra of pristine HPC and HPC-PeEt and 1 H-NMR spectrum of HPC-PeEt, respectively.
spectrometer. The temperature of the CLC cell was precisely controlled using a hot-stage system (HS82 and HS1, Mettler Toledo, OH, USA).
The viscosity measurements were conducted using a stress-controlled rheometer (MCR102, Anton Paar, Graz, Austria) equipped with a stainless-steel parallel plate with a diameter of 25 mm and a forced convection oven (Anton Paar, CTD450) for controlling the temperature. In the measurements, HPC-PeEt was sandwiched at a gap of ~500 µm. Angular frequency (ω) dependence of HPC-PeEt was analyzed using the above-mentioned rheometer. Both the storage modulus (G′) and loss modulus (G″) were measured at the range of ω between 0.1 rad/s and 100 rad/s at 25 °C. The strain amplitude was adjusted in the range between 0.2% and 1.2%, which is small enough to measure the linear viscoelasticity. Prior to this measurement, HPC-PeEt was either pre-heated or pre-sheared in order to erase any orientational history of CLC structures. As the pre-heat treatment, HPC-PeEt was heated at 155 °C for 10 min in the rheometer. This temperature was higher than the isotropic phase transition temperature of HPC-PeEt, corresponding to 150 °C, to retain the random orientation of cholesteric texture as confirmed by the polarized optical microscopic observation. On the other hand, the pre-shear treatment was conducted by shearing HPC-PeEt at a constant shear rate of 1.0 s −1 for 5 min at 25 °C, followed by leaving it to stand for 7 min at the same temperature after stopping the shearing force.

Characterization of HPC-PeEt
First, we measured FT-IR and 1 H-NMR spectra to confirm that all hydroxy groups of pristine HPC are substituted with pentyl ether side-chains after etherification. Figures  2A,B show the FT-IR spectra of pristine HPC and HPC-PeEt and 1 H-NMR spectrum of HPC-PeEt, respectively. The FT-IR spectral results are shown in Figure 2A. The pristine HPC exhibited a broad peak in the wavenumber range from 3000 cm −1 to 3600 cm −1 , which is assigned to the stretching vibration of hydroxy groups. In contrast, this peak disappeared in the FT-IR spectrum of HPC-PeEt, suggesting that the hydroxy groups of pristine HPC are almost substituted with pentyl ether groups [11]. The etherification of HPC was also confirmed from the 1 H-NMR spectral results of HPC-PeEt [11]. Figure 2B represents the 1 H-NMR spectrum of HPC-PeEt in CDCl3 [25]. A sharp peak at 0.99 ppm can be assigned to the terminal methyl groups in the pentyl ether groups of HPC-PeEt. As the quantitative analysis, the etherification degree (DSPe) in a monomer unit of HPC was calculated by the following equation: The FT-IR spectral results are shown in Figure 2A. The pristine HPC exhibited a broad peak in the wavenumber range from 3000 cm −1 to 3600 cm −1 , which is assigned to the stretching vibration of hydroxy groups. In contrast, this peak disappeared in the FT-IR spectrum of HPC-PeEt, suggesting that the hydroxy groups of pristine HPC are almost substituted with pentyl ether groups [11]. The etherification of HPC was also confirmed from the 1 H-NMR spectral results of HPC-PeEt [11]. Figure 2B represents the 1 H-NMR spectrum of HPC-PeEt in CDCl 3 [25]. A sharp peak at 0.99 ppm can be assigned to the terminal methyl groups in the pentyl ether groups of HPC-PeEt. As the quantitative where A is the integrated value of the peak a in Figure 2B and W is the sum of the integrated values of all protons in HPC-PeEt. The mathematical derivation of Equation (2) is available in the Appendix A, as described below. As mentioned in the Experimental Section, MS was determined to be 3.98. It should be noted that the maximal DS Pe value is 3.00 since the monomer unit of HPC has three hydroxy groups, as depicted in Figure 1. By applying the experimental results to Equation (2), the DS Pe value of HPC-PeEt was estimated to be 3.00. This result indicated that all hydroxy groups of pristine HPC are completely substituted with pentyl side-chains through the ether bonds. Moreover, the values of M n and M w of pristine HPC and HPC-PeEt were evaluated by the SEC analysis. As compiled in Table 1, both M n and M w of HPC-PeEt were in the same order of magnitude as those of pristine HPC, implying that no depolymerization in the main-chain of HPC might occur in the etherification process. Previously, Greiner and colleagues have revealed that the reflection peak wavelength of CLC from the esterified HPC derivatives is greatly affected by the esterification degree, that is, the amount of remaining hydroxy groups of the HPC backbone [26]. In our preliminary experiment, such tendency of reflection property was also observed for the etherified HPC derivatives. In addition, the rheological properties of HPC derivatives might be vulnerable to the remaining hydroxy groups of the HPC backbone due to the formation of hydrogen bonds. Therefore, we anticipated that HPC-PeEt synthesized in this study is suitable for investigating the optical and rheological properties for the CLCs of completely etherified HPC derivatives.

Optical Properties of HPC-PeEt
HPC-PeEt exhibited thermotropic CLC phase with visible reflection characteristic. Figure 3 shows changes in the transmission spectrum of a CLC cell comprising HPC-PeEt upon heating process. When the temperature of the CLC cell was adjusted to 25 • C, a reflection peak appeared at 400 nm, corresponding to the blue reflection color. Subsequently, when the temperature was gradually elevated from 30 • C to 120 • C at the intervals of 10 • C, the reflection peak was continuously red-shifted to the near-infrared region of 780 nm. This can be attributed to the increase in p value of CLC as the temperature rises [27]. Successively, the reflection peak was blue-shifted from 780 nm to 400 nm upon cooling from 120 • C to 25 • C. Such a reversible change in reflection peak by temperature is the typical characteristic of thermotropic CLC phase.
These spectral results were good agreement with our previous report on the thermotropic CLC from an HPC derivative completely substituted with pentyl ether groups [11]. In addition, such a wide wavelength shift in reflection peak of CLC, which fully covers the visible wavelength region as well as near infrared region, is in high demand for the technological applications of CLC photonic devices. Considering that the rheological behavior of CLC is greatly affected by the difference of CLC helical structure, it is necessary to choose an etherified HPC derivative with a similar reflection property to that of esterified HPC derivatives for fair comparison of their rheological properties. As another advantage, HPC-PeEt exhibited thermal stability in its reflection properties because there was no distinct wavelength shift in the reflection peak even after heating at 120 • C for 5 h, indicating no thermally-induced decomposition of HPC-PeEt. For these reasons, we concluded that HPC-PeEt is suitable for investigating the rheological properties of cellulose-based thermotropic CLCs similar to the esterified HPC derivatives reported in our previous studies [23,24].
Polymers 2022, 14, x FOR PEER REVIEW based thermotropic CLCs similar to the esterified HPC derivatives reported in ou ous studies [23,24].

Pre-Treatments of HPC-PeEt before Rheological Measurements
For fair evaluation of the rheological properties of CLCs, it is essential to con molecular orientation states at the CLC phase. This is because the rheological pr are greatly affected by the internal molecular structures [23,24]. To overcome this the pre-heat and pre-shear treatment defined in the Experimental Section might b for the rheological measurements, to ensure the disordered and aligned states, tively, of molecular structure at the CLC phase.
The pre-heat treatment was conducted by heating HPC-PeEt at 155 °C for 1 advance. This condition was determined from both transmission spectral measu and polarized optical microscopic observation of the CLC cell as follows: Figure 4A the transmission spectra of the CLC cell of HPC-PeEt measured at 25 °C before a the pre-heat treatment. As mentioned in Section 3.2, the CLC cell showed a reflecti at 400 nm as blue color before the pre-heat treatment ( Figure 4A, gray line). W CLC cell was observed by a polarized optical microscope under crossed Nicols wh ing process, no transmission light was observed at 150 °C. This result indicated birefringence of HPC-PeEt completely disappears due to the thermally-induce transition from CLC to isotropic. Thus, the CLC structure is assumed to be the dis state at this temperature. After the temperature was gradually lowered to 25 °C flection peak could not be revived even though the CLC cell reflected blue light of at the same temperature before heating ( Figure 4A, black line). At this stage, the gence observed by the polarized optical microscope was weaker than before the treatment at 155 °C. These results inferred that the disordered CLC structure can tially preserved even at CLC phase of 25 °C after the pre-heat treatment. This ph non can also be supported by the experimental results that the baselines in the tr sion spectra decline from ~85% to ~60% after the pre-heat treatment, which can be to the light scattering caused by disordered CLC structure ( Figure 4A).
The pre-shear treatment was conducted by shearing HPC-PeEt at a consta rate of 1.0 s −1 for 5 min at 25 °C. Figure 4B shows the changes in viscosity of HPC a function of shearing time. As evident from this experimental profile, the viscosi

Pre-Treatments of HPC-PeEt before Rheological Measurements
For fair evaluation of the rheological properties of CLCs, it is essential to control the molecular orientation states at the CLC phase. This is because the rheological properties are greatly affected by the internal molecular structures [23,24]. To overcome this problem, the pre-heat and pre-shear treatment defined in the Experimental Section might be useful for the rheological measurements, to ensure the disordered and aligned states, respectively, of molecular structure at the CLC phase.
The pre-heat treatment was conducted by heating HPC-PeEt at 155 • C for 10 min in advance. This condition was determined from both transmission spectral measurements and polarized optical microscopic observation of the CLC cell as follows: Figure 4A shows the transmission spectra of the CLC cell of HPC-PeEt measured at 25 • C before and after the pre-heat treatment. As mentioned in Section 3.2, the CLC cell showed a reflection peak at 400 nm as blue color before the pre-heat treatment ( Figure 4A, gray line). When this CLC cell was observed by a polarized optical microscope under crossed Nicols while heating process, no transmission light was observed at 150 • C. This result indicated that the birefringence of HPC-PeEt completely disappears due to the thermally-induced phase transition from CLC to isotropic. Thus, the CLC structure is assumed to be the disordered state at this temperature. After the temperature was gradually lowered to 25 • C, the reflection peak could not be revived even though the CLC cell reflected blue light of 400 nm at the same temperature before heating ( Figure 4A, black line). At this stage, the birefringence observed by the polarized optical microscope was weaker than before the heating treatment at 155 • C. These results inferred that the disordered CLC structure can be partially preserved even at CLC phase of 25 • C after the pre-heat treatment. This phenomenon can also be supported by the experimental results that the baselines in the transmission spectra decline from~85% to~60% after the pre-heat treatment, which can be ascribed to the light scattering caused by disordered CLC structure ( Figure 4A).

Rheological Properties of HPC-PeEt
The rheological properties of HPC-PeEt were affected by the difference in the pretreatments of HPC-PeEt. Figures 5A,B show the angular frequency (ω) dependence of storage modulus (G′) and loss modulus (G″) of HPC-PeEt treated by pre-heating and preshearing, respectively. The measurements were performed at 25 °C. Although the rheological measurements of HPC-PeEt were repeated at least three times, we found that the reproducibility is sufficient.
In the case of pre-heated HPC-PeEt, the curves of G′ and G″ can be divided into two regions depending on the G′ and G″ values. In the ω range above 1.0 rad/s, G″ clearly exceeded G′, pronouncing that HPC-PeEt exhibits liquid-like behavior ( Figure 5A). However, G′ and G″ were almost equal in the ω range below 1.0 rad/s, indicative of the solidlike behavior of HPC-PeEt in this ω range. In contrast to the pre-heat treatment, the curves of G′ and G″ for pre-sheared HPC-PeEt were distinct from those of pre-heated HPC-PeEt because G″ was constantly larger than G′ in the entire ω range between 10 −1 rad/s and 10 2 rad/s ( Figure 5B). This difference in the relationship of G′ and G″ in the lower ω region, that is, the solid-like behavior of pre-heated HPC-PeEt, can be ascribed to the disordered state of CLC structure. This is also supported by both transmission spectral measurements and polarized optical microscopic observations, as noted in Section 3.3. Interestingly, HPC-PeEt treated by pre-heating and pre-shearing showed a similar ω dependence at the higher ω region. The slopes of G′ and G″ were estimated to be approximately 1.0 and 0.6, respectively. These values were much smaller than those of the ideal Newtonian fluid, which would be 2.0 and 1.0, respectively. Such smaller ω dependences of G′ and G″ can be attributed to the disturbed flow behavior of pre-heated and pre-sheared HPC-PeEt caused by the CLC structure.
Our previous studies have shown the rheological properties for the thermotropic CLC of an esterified HPC derivative possessing propionyl side-chains (HPC-PrEs) [23,24], as depicted in Figure 1. The rheological properties of both HPC-PeEt and HPC-PrEs can be characterized by the following two aspects. First, the ω dependence of G′ and G″ in the higher ω region was almost same since the G′ and G″ of HPC-PrEs became proportional to the ω 1.0 and ω 0.8 , respectively. Second, the relationship between G′ and G″ in the lower ω region was dominated by the CLC molecular orientation state because it was dependent on the difference of the pre-treatment before the rheological measurements. This tendency was commonly observed for both HPC-PeEt and HPC-PrEs. However, the relationship of The pre-shear treatment was conducted by shearing HPC-PeEt at a constant shear rate of 1.0 s −1 for 5 min at 25 • C. Figure 4B shows the changes in viscosity of HPC-PeEt as a function of shearing time. As evident from this experimental profile, the viscosity gradually decreased upon shearing, and became constant after 80 s reaching its steady state. Such decrease in viscosity can be ascribed to the molecular orientation of CLC by shear-flowing. This suggests that the shearing time needed for the stationary state of CLC orientation of HPC-PeEt is 80 s. Therefore, we determined that the pre-shearing for 5 min is long enough to orient the CLC structure of HPC-PeEt.

Rheological Properties of HPC-PeEt
The rheological properties of HPC-PeEt were affected by the difference in the pretreatments of HPC-PeEt. Figure 5A,B show the angular frequency (ω) dependence of storage modulus (G ) and loss modulus (G") of HPC-PeEt treated by pre-heating and pre-shearing, respectively. The measurements were performed at 25 • C. Although the rheological measurements of HPC-PeEt were repeated at least three times, we found that the reproducibility is sufficient.
OR PEER REVIEW 8 of 10 temperature. Such a quick orientation of CLC of HPC-PeEt is very attractive from the viewpoint of fabricating highly-ordered CLCs of HPC derivatives which are desired for the photonic devices with high performances.

Conclusions
In this report, we investigated the optical and rheological behavior of a thermotropic CLC of HPC-PeEt, that is, an HPC derivative tethering pentyl ether side-chains. HPC-PeEt exhibited thermotropic CLC phase with visible reflection property in the temperature range between 25 °C and 120 °C. The reflection peak wavelength could be reversibly In the case of pre-heated HPC-PeEt, the curves of G and G" can be divided into two regions depending on the G and G" values. In the ω range above 1.0 rad/s, G" clearly ex- ceeded G , pronouncing that HPC-PeEt exhibits liquid-like behavior ( Figure 5A). However, G and G" were almost equal in the ω range below 1.0 rad/s, indicative of the solid-like behavior of HPC-PeEt in this ω range. In contrast to the pre-heat treatment, the curves of G and G" for pre-sheared HPC-PeEt were distinct from those of pre-heated HPC-PeEt because G" was constantly larger than G in the entire ω range between 10 −1 rad/s and 10 2 rad/s ( Figure 5B). This difference in the relationship of G and G" in the lower ω region, that is, the solid-like behavior of pre-heated HPC-PeEt, can be ascribed to the disordered state of CLC structure. This is also supported by both transmission spectral measurements and polarized optical microscopic observations, as noted in Section 3.3. Interestingly, HPC-PeEt treated by pre-heating and pre-shearing showed a similar ω dependence at the higher ω region. The slopes of G and G" were estimated to be approximately 1.0 and 0.6, respectively. These values were much smaller than those of the ideal Newtonian fluid, which would be 2.0 and 1.0, respectively. Such smaller ω dependences of G and G" can be attributed to the disturbed flow behavior of pre-heated and pre-sheared HPC-PeEt caused by the CLC structure.
Our previous studies have shown the rheological properties for the thermotropic CLC of an esterified HPC derivative possessing propionyl side-chains (HPC-PrEs) [23,24], as depicted in Figure 1. The rheological properties of both HPC-PeEt and HPC-PrEs can be characterized by the following two aspects. First, the ω dependence of G and G" in the higher ω region was almost same since the G and G" of HPC-PrEs became proportional to the ω 1.0 and ω 0.8 , respectively. Second, the relationship between G and G" in the lower ω region was dominated by the CLC molecular orientation state because it was dependent on the difference of the pre-treatment before the rheological measurements. This tendency was commonly observed for both HPC-PeEt and HPC-PrEs. However, the relationship of G and G" in the lower ω region was slightly different, especially when measured after the pre-heat treatment. In the case of HPC-PrEs, G became larger than G" in the lower ω region, while G and G" were almost equal in the case of HPC-PeEt. Considering the fact that the birefringence of pre-heated HPC-PeEt is preserved after the pre-heat treatment, unlike HPC-PrEs, it can be assumed that the CLC of HPC-PeEt is more likely to be oriented than that of HPC-PrEs even after it was heated above its isotropic phase transition temperature. Such a quick orientation of CLC of HPC-PeEt is very attractive from the viewpoint of fabricating highly-ordered CLCs of HPC derivatives which are desired for the photonic devices with high performances.

Conclusions
In this report, we investigated the optical and rheological behavior of a thermotropic CLC of HPC-PeEt, that is, an HPC derivative tethering pentyl ether side-chains. HPC-PeEt exhibited thermotropic CLC phase with visible reflection property in the temperature range between 25 • C and 120 • C. The reflection peak wavelength could be reversibly shifted in the full visible-wavelength range from 400 nm to 780 nm upon heating and cooling processes. Moreover, the rheological properties of HPC-PeEt were found to be dependent on the molecular orientation states at the CLC phase. HPC-PeEt showed solid-like responses when heated above its isotropic phase-transition temperature, followed by cooling to 25 • C, even though it showed liquid-like behavior after shearing at a constant shear rate of 1.0 s −1 . This is because the disordered CLC structures of HPC-PeEt prevent the flow behavior. These experimental results would be useful not only for understanding he fundamental physical properties of cellulose-based CLCs, but also for fabricating the environment-friendly CLC devices by utilizing cellulose derivatives for a sustainable society.

Conflicts of Interest:
The authors declare no conflict of interest.

Appendix A Mathematical Derivation of Equation (2)
As addressed in Section 3.1, Equation (2) enables estimation of the DS Pe value-the average number of the pentyl ether groups in a monomer unit of HPC-PeEt. We derived Equation (2) according to our previously reported procedure [28]. A peak of terminal methyl protons in the pentyl ether groups appeared around 0.99 ppm in the 1 H-NMR spectrum of HPC-PeEt. Then, we defined the integrated value of this peak as A and the sum of intergrated values of the peaks derived from all protons of HPC-PeEt as W. Therefore, the W value obeyed Equation (A1) as follows: Because the DS Pe value was calculated from the ratio of A/3 and W, the relation was expressed as Equation (A2).

DS
Lastly, Equation (A2) was rearranged so as to be Equation (2) relating to the DS Pe value, as stated in Section 3.1.


Feasibility of Hydrogen Production from Cellulose and Prediction of the Product Distribution: Thermodynamics Analysis

High carbon emissions, depleting fossil energy reserves have become a global problem. It is necessary to develop renewable energy sources that are environmentally friendly. Hydrogen (H2) is one of the energy sources and carriers that can be developed. This gas can be produced from renewable, sustainable, and economical resource such as biomass that contains cellulose as the main ingredient. This thermodynamic analysis of H2 production from cellulose is necessary as a theoretical study to determine the feasibility of the reaction. The computational thermodynamic was analyzed using Microsoft Excel 2019 and Matlab Program R2013a. Prediction of the equilibrium composition of the substances involved in the reaction was attempted by minimization Gibbs free energy change with Lagrange undetermined multipliers methods. As a result, the value of ΔHr 0; ΔSr 0 and ΔGr 0 are +624,7500 kJ/mol; +2,1491 kJ/mol.K and; -26,1540 kJ/mol, respectively. Analysis of equilibrium constant of this conversion has a large ln K value (> 1). A negative ΔGr 0 value and large ln K indicates that the formation of H2 from (C6H10O5)n is plausible and feasible and reaction product formation is strongly favored at equilibrium. The composition of the substances involved at 298 K from the largest to the smallest is CH4 (4.5 mol), H2O (3 mol), CO2 (1.5 mol), H2 (1.28×10 -5 mol), HCOOH (5.85×10-10 mol), C6H12O6 (3.72×10-10 mol) and C6H12O5 (1.35×10 -10 mol). Interestingly, H2 yield will rise significantly with the increase of reaction temperature. This preliminary study provides an overview of reaction conditions so that H2 production from biomass can be produced maximally.

; ΔS r 0 and ΔG r 0 are +624,7500 kJ/mol; +2,1491 kJ/mol.K and; -26,1540 kJ/mol, respectively. Analysis of equilibrium constant of this conversion has a large ln K value (> 1). A negative ΔG r 0 value and large ln K indicates that the formation of H 2 from (C 6 H 10 O 5 )n is plausible and feasible and reaction product formation is strongly favored at equilibrium. The composition of the substances involved at 298 K from the largest to the smallest is CH 4 (4.5 mol), H 2 O (3 mol), CO 2 (1.5 mol), H 2 (1.28×10 -5 mol), HCOOH (5.85×10 -10 mol), C 6 H 12 O 6 (3.72×10 -10 mol) and C 6 H 12 O 5 (1.35×10 -10 mol). Interestingly, H 2 yield will rise significantly with the increase of reaction temperature. This preliminary study provides an overview of reaction conditions so that H 2 production from biomass can be produced maximally.

INTRODUCTION
The decline in environmental quality due to carbon emission has become a global problem. Based on the Statistical Review of Energy released by BP in 2020, the trend of carbon emission is showed where the average annual growth in carbon emissions during 2018 and 2019 is greater than the 10-year average. The industrial and transportation sectors are the largest energy consumers and contribute to the increase in greenhouse gases. This is because the primary energy source currently used is fossil energy (petroleum, natural gas, and coal) (BP 2020). The use of renewable energy sources that are environmentally friendly is absolutely necessary not only because of high carbon emissions but also depleting fossil energy reserves. Hydrogen (H 2 ) is one of the energy sources and carriers that can be developed. H 2 has high chemical energy per mass (122 kJ/g) (Dincer & Zamfirescu 2016), superior to that of many fossil fuels, and its combustion only results in water and zero carbon (Singh & Dutta 2018). This diatomic gas can be produced from renewable, sustainable, and economical resources such as plant crops, agricultural residues, and woody biomass (Lan et al. 2020). Biomass contains lignocellulose that mainly consists of three polymers which are cellulose (>40%), hemicellulose (20%-40%), and lignin (5%) (Kuehnel & Reisner 2018;Megashah et al. 2018).
Hydrogen can be produced from cellulose (C 6 H 10 O 5 )n in several thermochemical processes such as steam gasification (>1000K), fast pyrolysis (450-650K), thermo-catalytic reforming which is associated with the problems of catalyst deactivation and by-product (e.g. CO) production and hydrolysis by acids or enzymes (Nikolaidis & Poullikkas 2017;Takise & Sekine 2019). Due to high energy intensity and difficulties in H 2 separation and purification from mixture yields, nowadays, preferred H 2 production is from photocatalytic reforming process (photoreforming) (Hao et al. 2018;Lan et al. 2020). This process offers more sustainable and environmentally friendly technology since it employs solar as energy source and photocatalyst to accelerate the reaction. Cellulose can be convert to H 2 according to the following reactions (Hao et al. 2018): (C 6 H 10 O 5 ) n (s) + H 2 O (l) nC 6 H 12 O 6 (l) (1) Therefore, the net reaction will be Some researchers doubt this process can be used for effective H 2 production. This is maybe because of the rigid intra and inter-molecular hydrogen bonds in cellulose, it is difficult to hydrolyze cellulose to platform organic molecules and then converting it to H 2 . Hence, thermodynamic analysis of H 2 production from cellulose is necessary to determine the feasibility of the reaction. Some of the existing thermodynamic studies generally use platform organic compounds (Huang et al. 2020) such as methanol, ethanol, glycerol, and monosaccharides such as glucose. Thermodynamic studies of polysaccharides have not been carried out and existing studies use the glucose molecule as a model because it is a major derivative of cellulose.
This paper discusses the thermodynamic analysis of cellulose reforming to H 2 in the absence of a photocatalyst. This analysis will be a preliminary study to assess the feasibility of this reaction. Using the computational method, this theoretical study can be used to predict the conditions of the cellulose reforming reaction and the distribution of the products obtained.

COMPUTATIONAL THERMODYNAMIC ANALYSIS
This thermodynamic study was attempted by means of a computational procedure using Microsoft Excel 2019 and Matlab Program R2013a. The analysis consists of several steps, namely: (1) calculating changes in reaction for both standard enthalpy (ΔH o r ) and entropy (ΔS o r ), and then the change in free energy of reaction Gibbs (ΔG o r ); (2) determine the reaction equilibrium constant (K) at various temperatures (3) determine the change in total Gibbs free energy (ΔG t ); (4) minimization of Gibbs energy and prediction of the equilibrium composition of the substances involved in the reaction at the specified temperature and pressure. This minimization introduces Lagrange undetermined multipliers methods and computed using MatLab R2013a program.
For the first step, we calculate ΔH o r ; ΔS o r and ΔG o r according to (5), (6) and (7) (5) The equilibrium constant (K) determined based on (8): Or by Van't Hoff equation: that can be rewrite as (10) with ΔH r 0 is standard enthalpy; T is reference temperature of reaction (298 K); and T' is determined temperature of the reaction (This thermodynamic analysis take place at T=303-353 K). K' is reference of equilibrium constant. The value of ln K can be plotted to T.
Further thermodynamic analysis step aims to calculate the equilibrium composition by minimizing the free energy of Gibbs. The Gibbs energy minimization is limited or constrained by the number of moles of the substance (atoms) involved in the reaction (atomic balance constraints) (Khonde et al. 2021;Tsanas et al. 2017).
The total Gibbs free energy change can be calculated by (11). (11) where (12) and for gases compounds (13) Hence, the total Gibbs free energy change from the mixture can be written as (14) Subsequently, (14) is the objective function that will be minimized. In a closed system, the number of moles of each atom is fixed during a chemical reaction. The total number of moles of a particular atom present in all chemical species in each chemical reaction must equal the initial mole number of that atom.
which k = 1,2,3,… Or which k = 1,2,3,… Minimization was done by substituting boundary conditions in the objective function. The boundary condition is the number of atoms involved in the reaction based on the proposed mechanism, namely three atoms including C, H, and O. The number of moles of the three atoms is entered in the objective function using the Lagrange Multiplier: λ 1 , λ 2 and λ 3 . Therefore (14) will be: (17) where G t is the total Gibbs free energy; G i 0 is standard Gibbs free energy of species (i); R is the molar gas constant; T is the temperature of the system; f i is fugacity of species (i) in the system, f i 0 is standard state fugacity of species (i) and n i is the number of moles of species (i).
where = 0 + and for gases compounds formation, destruction and rearrangement of chemical bonds of compound involve in the reaction, so there will be a change in energy of the system. The change of energy can be observed from ΔH o r ; ΔS o r ; and ΔG o r . Standard enthalpy change can be useful for determining the temperature effect on the equilibrium constant of H 2 production and thus on H 2 yield. In addition, Gibbs free energy changes can be useful for determining the existence of chemical equilibrium in the system and the influence of process variables on the H 2 yield (Hill & Root 2014). One of the important things in thermodynamics analysis is the availability of thermodynamics data sources. In this analysis, the data is tabulated in Table 1   From (5), (6) and (7), the value of ΔH r 0 ; ΔS r 0 and ΔG r 0 are +624,7500 kJ/mol; +2,1491 kJ/mol.K and -26, 1540 kJ/mol, respectively. Even though the value of ΔH r 0 is positive, the value is too small compared to the positive value of ΔS r 0+ . This resulted in the negative value of ΔG r 0 . With a negative ΔG r 0 value, the reaction to form H 2 from (C 6 H 10 O 5 )n can take place spontaneously under standard conditions (298 K, 1 atm). The reaction takes place exothermically (Hua et al. 2020;Smith et al. 2018). This negative value of ΔG r 0 also indicate that the conversion of cellulose can be accelerated using a catalyst.  A large ln K value (> 1) indicates that the reaction, the formation of H 2 from (C 6 H 10 O 5 ) n can be plausible and feasible. With a negative ΔG r 0 value and large ln K, the product formation at equilibrium is strongly favored at equilibrium (Cheng et al. 2019;Muhammad et al. 2017). The higher the temperature, the greater the value of ln K, which indicates an infinite equilibrium.

PRODUCT DISTRIBUTION ANALYSIS
Based on the possible reaction mechanisms as stated by Hao et al. (2018) and Zou et al. (2018), the substances involved in the reaction is served in Table 3 that consists of cellulose ((C 6 H 10 O 5 ) n ), water (H 2 O), glucose (C 6 H 12 O 6 ), formic acid (HCOOH), carbon dioxide (CO 2 ), hydrogen (H 2 ) and methane (CH 4 ).  The value of number of atomic mass k in the system (A k ) that is required can be determined from the initial mole numbers, and the number of atom k per molecule i(a ik )values that comes directly from the chemical formulas of each species in the reaction (as shown in Table 4). Based on computational analysis, the composition of the substances involved in (reaction temperature 298 K from the largest to the smallest are CH 4 (4.5 mol), H 2 O (3 mol), CO 2 (1.5 mol), H 2 (1.28×10 -5 mol), HCOOH (5.85×10 -10 mol), C 6 H 12 O 6 (3.72×10 -10 mol) and C 6 H 12 O 5 (1,35×10 -10 mol). The corresponding product distributions at temperatures between 298-398 K are illustrated in Figures 2 and 3. Figure 2 shows the cellulose, glucose, formic acid and hydrogen distribution as a function of temperature whereas Figure 3 shows methane, water and carbon dioxide as a function of temperature.
The most significant finding from Figure 2 is that hydrogen increased exponentially as temperature increase within the stipulated temperature boundary. At temperature 298 K, 1.28×10 -5 moles of H 2 is produced and when the temperature reached 398 K, H 2 of 3.50×10 -4 moles is produced that is an increase of 27 times. In the other hand, the composition of cellulose, glucose and formic acid decreased insignificantly with increasing temperature. At 298 K, the composition of cellulose, glucose and formic acid are 1.35× -10 mol; 3.72× -10 mol and 5.85× -10 mol, respectively. Meanwhile, at T 398 K the amount was reduced to 1.98×10 -22 mol; 6.37×10 -22 mol and 22×10 -22 mol. It is assumed that decrease in cellulose, glucose, and formic acid take place because that substance was converted to H 2 . According to the insight on the reaction mechanism, hydrogen can be produced from cellulose by forming intermediate products including glucose, formic acid or other oxygenate products (Hao et al. 2018;Lan et al. 2020;Zhang et al. 2016). The more cellulose converted into organic compounds, the easier it is to for H 2 to be produced and this is the reason H 2 formation increased rapidly (Syaahidah et al. 2020). The conversion of cellulose into organic products begins with the dissociation of β-(1,4)-the glycosidic bonds in these polysaccharides into saccharides as their monomers. Such process of attaining hydrogen from biomass is complicated (Ahorsu et al. 2018). This dissociation stage is the rate limiting step for the formation of H 2 from cellulose. Furthermore, these saccharides can be transformed into other intermediate products such as  Products (mol)

Temperature(K)
alcohol or carboxylic acid or furfural and finally into hydrogen and other products within a complex reaction network (Lan et al. 2020;Puga 2016;Speltini et al. 2014;Syaahidah et al. 2020;Zou et al. 2018).
Other chemical compounds involved in the conversion of cellulose to hydrogen are methane, water, and carbon dioxide. From Figure 3, it is predicted that there is no change in their compositions. Methane is obtained as one of the main products in cellulose conversion to hydrogen. This gas may be produced from intermediate product that have alkyl group in their structure. Alkyl (CH 3 -) will react with proton (H + ) resulted CH 4 (Bahruji et al. 2011;Bowker et al. 2014;Hao et al. 2018) following reaction route: From the thermodynamic equilibrium point of view, H 2 was dominantly resulted at high temperature, while CH 4 was produced at low temperature. It is a challenge to eliminate this low hydrocarbon from this reaction. Researchers suggested to carry out methane steam reforming and successive water-gas shift reaction to obtain maximum yield of H 2 (Shimura & Yoshida 2011). The reformation of cellulose to hydrogen also produces large amount of CO 2 . CO 2 is produced by the oxidation of the alkyl groups of the intermediate products. However, CO 2 from biomass is considered more environmentally friendly than CO 2 produced from fossil fuels (Danish & Ulucak 2020; Koruba et al. 2017;Sher et al. 2020). Through photosynthesis, this CO 2 will be reversed reacted into cellulose (biomass) (Chen et al. 2019). In addition, CO 2 and CH 4 tend to produce if the intermediate product contains either glycerol or methanol (Tasleem et al. 2020). Furthermore, the produced CO 2 subsequently transformed to CH 4 and this is the plausible explanation of why CH 4 is largely produced. The proposed reaction mechanism of cellulose to hydrogen is illustrated in Figure 4.

RR'CHOH +H2O
RH +R'H-CO2 + H2 RR'CHOH +H2O RH +R'H-CO2 + H2 Prediction of product distribution in this study can be taken into consideration to increase the amount of hydrogen produced from the conversion of cellulose. Ideally, the reaction equilibriums can be adjusted by manipulating temperature for some objectives: (1) maximize H 2 production, (2) reduce greenhouse gases (CH 4 and CO 2 ) production, and (3) minimize carbon release (Cheng et al. 2019). The manipulating reaction also can be attempt by applied an appropriate catalyst to adjust H 2 production. The catalyst is expected to direct the reaction product so that the main product has a relatively high yield.

CONCLUSIONS
Hydrogen is feasible to be produced from biomass that contains cellulose. The value of ΔH r 0 ; ΔS r 0 and ΔG r 0 are +624,7500 kJ/mol; +2,1491 kJ/mol.K and -26,1540 kJ/mol, respectively. With a negative ΔG r 0 value, the forward reaction to produce H 2 from (C 6 H 10 O 5 )n will proceed spontaneously and exothermic under standard conditions. Analysis of equilibrium constant of this conversion has a large ln K value (> 1). It indicates that the formation of H 2 from (C 6 H 10 O 5 ) n can be plausible and feasible. With a negative ΔG r 0 value and large ln K, the product formation at equilibrium is strongly favored at equilibrium. The composition of the substances involved in (at 298K) from the largest to the smallest are CH 4 (4.5 mol), H 2 O (3 mol), CO 2 (1.5 mol), H 2 (1.28×10 -5 mol), HCOOH (5.85×10 -10 mol), C 6 H 12 O 6 (3,72×10 -10 mol), and C 6 H 12 O 5 (1,35×10 -10 mol). Interestingly, H 2 yield will increase significantly at elevated temperatures.