Novel bodipy—cellulose nanohybrids for the production of singlet oxygen

Abstract

A novel hydrophilic carboxylated bodipy dye was synthesized and characterized by different spectroscopic techniques. The new nanomaterial was prepared by covalently conjugating it to amino nanocellulose, which was capable of producing singlet oxygen using a white light source.

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Singlet oxygen or the reactive oxygen species is produced by a sensitizing drug which is the key component of Photo Dynamic Therapy (PDT); wherein it comes into action together with use of oxygen and light to kill tumour producing cancer (oral and skin) cells.¹ The selection of these photosensitizing drugs which are suitable for PDT are quite challenging: which often requires the limited or negligible use of non-aqueous solvent for its mode of action and ease of removal of the drug after the completion of treatment, without affecting other cells. Thus, the search for a novel and an effective photosensitizer has drawn significant interests among the researchers in the past two decades.² The fluorescent bodipy's which are also known as the “small- or half-sisters” of the porphyrin macromolecule have been used as an active reactive chromophore capable of producing singlet oxygen for PDT treatments.³ Because of the rich chemistry of these bodipy dyes and its easily amenable photo physical properties, they are now actively employed in different applications such as sensors, imaging (for labelling other reagents), fluorescent laser dyes and sensitizer.⁴

In the past few decades, nanocellulose (or nano crystalline cellulose) has been a flourishing nanomaterial that has drawn a significant attention because of its outstanding mechanical, physical and chemical properties.⁵ These properties originate from the strong hydrogen bonding (inter- and intra-molecular) networks present in the cellulose chains. The cellulose nanocrystals (NC) are usually formed by the mild acid treatment of the cellulose material, wherein the preferential hydrolysis takes place on the microfibrillar amorphous zones, thus keeping crystalline part intact giving rise to a typical rod-like morphology (having a varying length from few hundreds to 1000 nm and a cross sectional width ranging between 10 to 50 nm).⁶ Availability, biodegradable, and eco-sustainability features make NC an attractive material to be explored. Indeed, the NC core is filled with hydroxyl groups, yet only a few of them are reactive due to its limited accessibility. NC can be utilized for different applications, by introducing other reactive chemical groups.⁷

Surprisingly, till date there is only one report dealing with a fluorescent bodipy derivative covalently linked to a cellulose material (paper) and discusses its use in antimicrobial efficacy.⁸ Thus, herein we first describe the successful synthesis of a novel hydrophilic sulfonated bodipy (SB) dye and then its successful covalent attachment onto modified (amino) cellulose nanocrystals (NC). We proved that the nanocellulose-bodipy hybrid nanomaterial retains the molecular properties of the bodipy: able to serve as an efficient photo-catalyst to carry out photo-oxygenation reaction in the aqueous medium and its capacity to act as photosensitizers for the production of singlet oxygen.

The construction of a novel hydrophilic sulfonated bodipy dye was achieved in two steps, as described in Scheme 1. At first, the bodipy core was prepared using previously described procedure (ESI†).⁹ In brief, the carboxylated bodipy was prepared by condensation of 2,4-dimethyl pyrrole and p-formyl benzoic acid, followed by oxidation with dichloro dicyanobenzoquinone (DDQ). Then, it was neutralized with triethyl amine and finally coordination of nitrogen atom with boron by treatment with boron trifluoride diethyl etherate. In the second step, the carboxylated bodipy (1) formed as result of first step was treated with chlorosulfonic acid¹⁰ to introduce the hydrophilic sulfonate groups on pyrrole ring; thus forming a novel water soluble bodipy dye (SB) bearing reactive carboxylic group. The newly constructed bodipy was well characterized by ¹H NMR spectroscopy shows the elimination of protons present on the pyrrole rings in comparison to the starting and which was further confirmed by (MALDI) mass analysis (see ESI†).

Scheme 1 Preparation of sulfonated bodipy (SB): (a) trifluoro acetic acid (TFA), anhydrous CH₂Cl₂, 20 hour, room temp.; DDQ, Et₃N, BF₃·Et₂O, 12 hour, yield: 12%; (b) chlorosulfonic acid, −40 °C; NaHCO₃, yield: 40%.

The cellulose nanocrystals (NC) used in this study was prepared from the commercially available microcrystalline cellulose (MCC, 80% crystallinity) from cotton linters; by performing a mild hydrolysis with 63% sulphuric acid at 45 °C for 2 hours.¹¹ The foundation of primary amino group on the nanocellulose surface was achieved via a two-step reaction (see ESI†).¹² This requires activation of OH group on the nanocrystalline surface of cellulose by treatment with epichlorohydrin; which introduces epoxy group and finally ring opening of the epoxy by treatment with ammonium hydroxide (Scheme 2), wherein some reactive primary amino group was introduced on the cellulose nanocrystals (NC-NH₂). The success of the amino functionalization was confirmed by Kaiser test (detection of primary amino group), which shows the appearance of Ruhemann's blue/purple colour. This test can be further used to estimate the degree of amino groups present on the NC by using the UV-visible spectroscopy.¹³ Using the Kaiser test, the degree of substitution of primary amino groups was found to be 0.60 mmol NH₂ per g of NC (see ESI†). Finally, the conjugation of sulfonated bodipy (SB) and amino nanocellulose (NC-NH₂) was achieved by using the carbodiimide chemistry (Scheme 2, amide linkage) in an aqueous solution of 2-(N-morpholino)-ethanesulfonic acid (MES) in the presence of coupling reagents (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl) and N-hydroxy succinimide (NHS)).¹⁴ Stirring the suspension for 4 days at room temperature, NCC-SB was recovered by centrifugation at 10 000 rpm, which was further washed with water and dialysed against milliQ water for 3 days. The dark red colour solid NC-SB was obtained by lyophilization (inset, Scheme 2).

Scheme 2 Synthesis of nanocellulose-bodipy conjugate: (a) epichlorohydrin, NaOH, 60 °C, 2 hours; NH₄OH, 60 °C, 2 hours; (b) SB, EDC–NHS, MES buffer pH-4, room temp, 4 days. Inset: digital picture of NC-SB solid.

The NC-SB hybrid nano-material was well characterized by different spectroscopic techniques as follows. The NC-SB forms a stable suspension in aqueous solution upon mild sonication. Fig. 1 shows the UV-visible spectrum of 0.25 mg mL⁻¹ suspension of NC-SB (blue) and NC (red) in water at neutral pH. The typical spectroscopic signals of a bodipy compound are observed. It shows the appearance of a Soret band at 500 nm (blue), which confirms the attachment of bodipy dye SB on the cellulose surface. On the contrary, the NC (red) does not show any variation in the UV-visible spectra. With the use of inductive coupled plasma atomic emission spectroscopy (ICP-AES), specifically, NC-SB confirmed the presence of boron and fluorine in the 1 : 2 ratio which could only arise from the bodipy core. Further, in-depth analysis using X-ray photoelectron spectroscopy (XPS) spectroscopy, which accounts for individual metal atomic composition present in the NCC-SB (see ESI†). Collectively using the ICP-AES and XPS spectroscopic techniques, the degree of loading of bodipy was found to be 2.6 × 10⁻³ mmol per g of NC. As the linkage takes place on amino group present on the NC, the corresponding ratio of bodipy/amino groups was found to be 0.0044, which means 1 out of every 230 amino was functionalized with the bodipy moiety. The relative low loading of bodipy dye was in accordance with a previous report wherein a porphyrin dye was covalently linked to nanocellulose.^14b The Thermo Gravimetric Analysis (TGA), indicated that SB conjugation onto the NC surface did not significantly change thermal behaviour (see ESI†). It was found that both the NC and NC-SB started to decompose above 270 °C under nitrogen environment and it was quite stable under the oxygen environment too. The nano-crystalline structure of NC (Fig. 2a) was confirmed by Transmission Electron Microscopy (TEM), which showed the typical ‘whisker’ like morphology. The measurement of the NC-nanowhisker gave an average length of 162 ± 40 nm, while the ζ-potential value was found to be −26 ± 6 mV which is in accordance with previously reported study.⁷NC-NH₂ obtained by the amino functionalization of nanocellulose confirms the similar whisker like morphology (Fig. 2b) which shows the average length of 138 ± 20 nm, with a change in ζ-potential value to −29 ± 5 mV due to introduction of amino groups. Fig. 2c shows the TEM image of NC-SB, had an average length of 134 ± 30 nm and had a much more negative ζ-potential value of −32 ± 5 mV, which arises from the anionic sulfonate groups from the bodipy dye.

Fig. 1 UV-Visible spectrum of 0.25 mg mL⁻¹ suspension of NC (red) and NC-SB (blue) in neutral water.

Fig. 2 TEM images of cellulose: (a) NC, (b) NC-NH₂, (c) NC-SB.

To verify the potential of NC-SB as a source for production of singlet oxygen; two individual experiments were carried out under the source of light and oxygen. In the first, singlet oxygen production with NC-SB was confirmed using 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA)¹⁵ which is known to react with singlet oxygen to yield an endoperoxide. In brief, the in situ production of endoperoxide was examined by a significant 10% decrease in UV absorption at 380 nm, when ABDA is irradiated in the presence of NC-SB against the white light source (75 ± 5 mW cm⁻² Hg) lamp (filter > 375 nm) for 5 min (see ESI†). While, the another experiment was carried to demonstrate the capacity of NC-SB as an efficient photo-catalyst to carry out photooxygenation reaction for L-methionine methyl ester.¹⁶ For this purpose, a suspension containing 1 mg of NC-SB and 20 mg of L-methionine methyl ester in oxygenated solution of D₂O (0.75 mL) was irradiated against white light source (75 ± 5 mW cm⁻² Hg) lamp (filter > 375 nm). After the 30 minutes irradiation, the slow formation of corresponding sulfoxide can be confirmed by ¹H NMR spectroscopy (see ESI†). Fig. 3 demonstrate the variations in ¹H NMR spectrum of L-methionine methyl ester with time, when irradiated in presence of NC-SB. In fact, in 210 minutes time duration it was completely transformed to the sulfoxide product. Fig. 4, depicts the graphical representation of L-methionine methyl ester conversion in percentage with respect to time. Interestingly, it was found that NC-SB can be recycled and reused up to 7 times, without significant loss in the activity.

Fig. 3 ¹H NMR spectrum of L-methionine methyl ester conversion to corresponding sulfoxide on treatment with NC-SB with the increasing irradiation time.

Fig. 4 Time dependent conversion of L-methionine methyl ester to L-methionine methyl ester sulfoxide in presence of NC-SB.

In conclusion, we have demonstrated that nanocellulose can be used as a scaffold to covalently append the bodipy units which serves as an efficient photo-catalyst to carry out photooxygenation reaction in the aqueous medium and capable to act as efficient photosensitizers for the production of singlet oxygen, which in turn can be termed as an excellent candidate for the therapeutic applications.

Acknowledgements

Authors gratefully acknowledge the financial support from (NSERC) Natural Sciences and Engineering Research Council of Canada: NSERC Discovery and NSERC Discovery Accelerator Supplements Programs.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Experimental details on the synthesis of cellulose nanocrystals and its functionalization with bodipy. See DOI: 10.1039/c6ra04275a

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