Bovine Serum Albumin Amplified Reactive Oxygen Species

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Functional Nanostructured Materials (including low-D carbon)

BSA amplified ROS generation from Anthrarufin derived Carbon dot and concomitant Nanoassembly for combination antibiotic-photodynamic Therapy application Saptarshi Mandal, Surendra Rajit Prasad, debabrata mandal, and Prolay Das ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b12455 • Publication Date (Web): 21 Aug 2019 Downloaded from pubs.acs.org on August 22, 2019

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ACS Applied Materials & Interfaces

BSA amplified ROS generation from Anthrarufin derived Carbon dot and concomitant Nanoassembly for combination antibiotic-photodynamic Therapy application Saptarshi Mandal1, Surendra Rajit Prasad2, Debabrata Mandal2 and Prolay Das1* 1

Department of Chemistry, Indian Institute of Technology Patna, Patna – 801103, Bihar, India

2

Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER)- Hajipur, Bihar-844102, India

Abstract Amplification of Reactive Oxygen Species (ROS) generation through covalent conjugation of Bovine Serum Albumin (BSA) with newly synthesized, ROS producing Carbon Dot (CD) upon visible light irradiation is reported for the first time. Derivatization of surface carboxyl functional groups of Anthrarufin derived green-emitting CD with amine functionality of BSA usher distinct changes in the photophysics of CD including an unprecedented ~50 nm shift in its excitation maxima, decrease in fluorescence lifetime and concomitant increase in ROS generation. Substantial conformational changes of BSA was witnessed upon conjugation with CD, rendering the BSA-CD conjugate resistant to pepsinolysis. A protease-proof nano-assembly was derived from the BSA-CD conjugate through desolvation that simultaneously hosts a prototype antibiotic and generates ROS with excellent efficiency, making it an attractive platform for antibacterial Photodynamic Therapy (A-PDT) applications. Systemic annihilation of both gram-positive and gram-negative bacteria was achieved with the BSA-CD nano-assembly and envisioned as alternatives to traditional photosensitizers.

Keywords: Carbon Dot; Bovine Serum Albumin; Nanoparticle, antibacterial Photodynamic Therapy, Reactive Oxygen Species

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1. Introduction Carbon Quantum Dot (CD), an incipient member in the arsenal of carbon nanomaterials, have attracted tremendous attention for diverse biomedical applications with intriguing prospects in bioimaging,1-2 biosensing,3-4 drug and gene delivery.5-6 To add to the repertoire of the myriad applications of CD, some photo-excited CDs have been shown to generate ROS that fundamentally point towards their potential use in PDT, a treatment modality where PS generates ROS to kill tumor or microbial cells (Antimicrobial-PDT, A-PDT) upon photoexcitation.7-10 In an obvious consequence, CDs are being envisioned as a better substitute to traditional PS, to address some of their perennial issues like low aqueous solubility, photobleaching effect, aggregation-induced selfquenching, and poor cellular penetration.11-12 However, the challenges of striking a fine balance between ROS generation efficiency and photo-stability of the CD, coupled with the design of an effective formulation involving the CD for its practical use in PDT are quite wide open and far from being convincingly accomplished. Herein, we report for the first time the creation of a CDBSA covalent conjugate that endorses the CD as an efficient visible-light-induced ROS generator and BSA as a simultaneous amplifier of ROS generation efficiency and photostability. A further interesting proposition is to convert the CD-BSA conjugate into a well-defined and highly stable functional nano-assembly to host a drug molecule for its sustained release for potential antibiotic + A-PDT combination therapy application. The efficiency of ROS generation from CD has been shown to be directly linked with the available surface functionality on CD, where mostly ketonic carbonyl and carboxyl groups play a major role.13-14 In addition to its destined activity, the ROS generated could reduce the photostability of the CD itself and result in decreased photoluminescence and loss of other photophysical characteristics.15 This call for systemic tuning of CD surface functionality that is


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reportedly addressed by blunting the Oxo-functional groups through derivatization.16-17 Moreover, modifications of surface carboxyl groups to amides have a profound influence on the band gap and hence photoluminescence (PL) of CD.18 This apparently points towards a stimulating prospect of using BSA, a nifty naturally adaptive carrier protein having side-chain amine functionality to preserve the photostability of the CD without affecting its ROS generation efficiency. Elsewhere, CD has been functionalized with BSA to deliver therapeutic payloads and detect metal ions, albeit non-covalently.19-20 Depending on the method of synthesis and raw materials used, CD exerts several surface functionalities (-COOH, NH2, SH, C=O) for covalent conjugation to BSA. Lack of precise control over the reactivity of those functional groups of CD and retention of conformational integrity of the proteins including BSA after covalent conjugation may call for a meticulous introspection for the use of CD as a protein-labeling agent.21 However, the covalent association of CD with BSA could be advantageously explored to fine-tune the photophysics of CD in a totally different application area such as its use as a PS in A-PDT, which at present is surprisingly underresearched. In fact, to the best of our knowledge, there is no literature report of augmented ROS generation efficiency from CD after its association with any organic or biomolecules. Herein, we report the synthesis of a novel green-emitting CD derived from Anthrarufin and its subsequent conjugation with BSA. The CD was found to generate ROS upon visible light as well as UV excitation. A significant increase in ROS generation efficiency and retention of photostability after covalent conjugation of BSA with CD was established, a rare feat achieved for tuning photophysics of CD with a biomolecule. The CD-BSA conjugate was consequently fabricated into a prototype protease-resistant nanoassembly to host and deliver ciprofloxacin for intended combined antibiotic + A-PDT application (Scheme 1).



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A-PDT has shown great prospects in effectively annihilating microbes irrespective of resistance and thus impart enormous significance in healthcare, food packaging, and other segments.22-23 In an effort to find alternatives to traditional PS, Nanoparticle (NP) photosensitizers like silica and gold NPs, metal oxides like TiO2, CeO2, Al2O3, ZnO, CuO, Fe2O3, metal sulfides, quantum dots etc. have been researched to address the perennial issues of traditional PS.24-25 Augmented ROS generation from nanoparticulated PS to enhance the efficacy of PDT have been explored by surface modification through a wide range of processes including formation of photosensitizing NPs with controlled aggregation and protected deactivation, amination, metalation, and others.26-27 An interesting observation from recent research indicates that in a combination therapy approach, antibiotics can act as an adjuvant in A-PDT by significantly reducing the PS loading and irradiation time, as well as decrease the chance of recurrence and immune response.28-29 Previously, BSA-CuS nanoparticle has been conjugated to the PS, Ce6 for combined A-PDT + Photothermal therapy (PTT) applications.30 As such, numerous examples in literature do exist for BSA-nanoparticle formation by desolvation method followed by crosslinking with a di-aldehyde like glutaraldehyde for enhanced stability, although neither in combination with CD and nor for intended use in A-PDT.31-32 The fact that organic crosslinkers are not required to stabilize the BSACD nanoassembly studied herein and the same is capable of holding a huge payload of antibiotic only to be released in a sustained manner following zero-order kinetics is quite serendipitous. Effective annihilation of gram-positive and gram-negative bacteria was achieved through visiblelight-induced on-demand ROS generation from CD coupled with the simultaneous release of the antibiotic from the BSA-CD nanoassembly.


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Scheme 1: Scheme for synthesis of CD, conjugation of the CD to BSA and subsequent creation of BSA-CD nanoparticle for visible-light-induced ROS generation and simultaneous release of ciprofloxacin for antibacterial activity

2. Experimental section 2.1 Synthesis of CD CD was prepared by solvothermal method. Briefly, 100 mg 1,5-dihydroxyanthraquinone (Anthrarufin) was dissolved in 3 ml dimethylformamide (DMF) followed by the addition of 100 µl 50% hydrogen peroxide to the solution. The reaction mixture was heated in a Teflon lined stainless still autoclave at 210℃ for 6 h and then cooled to room temperature. After evaporating all the solvent, the residue was dispersed in ethanol (2 ml) and filtered with a 0.22 µm syringe filter to remove large carbonized material. The total volume of the collected solution was reduced to 4 ml and stored at 4℃ for further use. 5 ACS Paragon Plus Environment


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2.2 Synthesis of BSA-CD CD was conjugated with BSA following carbodiimide chemistry.33 CD solution (1 µl, 45 µg) was added to 200 µl water followed by the addition of 1-ethyl-3- [3-dimethylaminopropyl] carbodiimide (EDC) (100 µl, 15 µmol) and N-hydroxysuccinimide (NHS) (100 µl, 23.5 µmol) solution. The pH of the solution was adjusted to around 6.5 and then above 7 after 1 h. The reaction mixture was kept for 4 h with occasional stirring for activation of carboxyl groups on the surface of CD. 10 mg BSA was added to the carboxyl activated CD solution and pH was maintained at 8 with dilute NaOH solution. The reaction mixture was kept overnight at 4oC for conjugation. To remove excess EDC, NHS, CD, and byproducts, the solution was taken in a mini-dialysis unit (MWCO 3.5 kDa) and dialyzed for 16 h against water with occasional changing of water. The dialyzed solution was collected and stored at 4oC for further use.

2.3 Preparation of nanoparticle with BSA-CD BSA-CD nanoparticle (BSA-CD NP) was prepared by desolvation method with few modifications.31 BSA-CD aqueous solution with 1% protein content in 10 mM NaCl was prepared, and pH was adjusted accordingly with dilute NaOH solution. A desolvating agent, 1 ml of acetonechloroform mixture (90:10), was added dropwise to 1 ml BSA-CD conjugate solution under magnetic stirring and kept overnight at room temperature. The nanoparticles, thus formed, were harvested by ultracentrifugation at 20,000 rpm for 35 minutes. The supernatant was also collected for the calculation of percentage yield and entrapment efficiency of BSA-CD NP. For encapsulation of a drug, 1 mg ciprofloxacin. HCl was dispersed in the organic solvent by ultrasonication for 5 min, and then the dispersion was added dropwise to the BSA-CD solution. Although ciprofloxacin is hydrophobic, since ciprofloxacin.HCl monohydrate salt was used; there


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were no solubility issues in water at the nanomolar range of working conditions. Rest of the procedure was the same as that of BSA-CD NP preparation.

2.4 ROS generation study Dihydrorhodamine 123 (DHR 123, 5 µl, 1 nM) solution was added to respective sample solutions and irradiated with tungsten bulb (Philips, 100 W) separately. DHR 123 was oxidized to rhodamine 123 by ROS species thus generated by the samples and fluorescence of rhodamine 123 was measured by steady-state fluorescence spectroscopy at different interval of time. For this, the excitation wavelength was kept fixed at 488 nm, and fluorescence emission was taken between 500 – 600 nm. As a standard PS, PpIX solution (5 µl 1 mM) was used to perform the same experiment under similar conditions with Dihydrorhodamine 123 (DHR 123) solution.

2.5 In-vitro drug release profile Ciprofloxacin loaded BSA-CD NP (0.5 ml) equivalent to 0.38 mg of ciprofloxacin load was taken

in a mini dialysis unit which was then placed in a small beaker containing 3 ml of phosphate buffer, pH 7.4. The whole system was set on a shaker at temperature 37 0C, and the volume of release medium was maintained at 3 ml by adding fresh buffer whenever needed. The amount of ciprofloxacin released at a series of time intervals was measured by an HPLC system following the Indian Pharmacopoeia (IP) protocol with slight modifications. 25 mM Phosphoric acid previously adjusted with trimethylamine to a pH of 3.0 ± 0.1, and acetonitrile at a ratio of 87:13 (v/v) was used as mobile phase. The flow rate was maintained at 1 ml/min, and UV detection was done at wavelength 278 nm. Since ciprofloxacin.HCl salt was used, we did not require any surfactant for in-vitro drug release, which is otherwise customarily added for hydrophobic drugs.



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2.6 Plate count assay for bacterial viability The plate count method was used for determination of the antibacterial activity of BSA-CD NP and BSA-CD NP loaded with ciprofloxacin after measuring Colony Forming Unit (CFU) of bacteria by visual inspection. E. coli DH5α (gram-negative) and S. aureus ATCC 25923 (grampositive) cells were grown in LB and TSB, respectively till the OD600 value reaches~1.0 (~109 CFU/ml). Cells were pelleted, washed and adjusted to ~1x106 CFU/ml for S. aureus and ~3x107 CFU/ml for E. coli in sterile phosphate buffer (0.1M, pH 7.4) and then 1 ml was added per well in a microtiter plate. Cells were mixed with BSA-CD NP (1.47 µg/ml), BSA-CD NP ciprofloxacin (1.47 µg/ml) and then either kept in the dark or exposed to visible light (100W tungsten bulb, 300900nm, for 1 h). The increase in temperature resulting from the irradiation process was countered by using an in-house developed cooling system using dry ice and portable table fan. Ciprofloxacintreated (1 µg/ml) and untreated cells as control were also included. After incubation 0.1 ml cells were taken and serially diluted (101 to 104) in phosphate buffer and 0.1ml of diluted cells were plated in Luria-Bertani (LB) Agar and Tryptic Soya Agar (TSA) plates for E. coli and S. aureus cells, respectively in triplicates. Agar plates were further incubated for 24 h at 37℃. CFU counted from untreated cells were considered 100% and reduction in CFU for nanoparticles treated cells were presented in percentage scale. Plates containing CFUs >20 but

Bovine Serum Albumin Amplified Reactive Oxygen Species

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