Gold-Nanocluster-Embedded Mucin Nanoparticles ... - ACS Publications

Subscriber access provided by BUFFALO STATE

Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Gold Nanoclusters Embedded Mucin Nanoparticles for Photodynamic Therapy and Bioimaging Deepanjalee Dutta, Sunil Kumar Sailapu, Anitha T Simon, Siddhartha Sankar Ghosh, and Arun Chattopadhyay Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b00998 • Publication Date (Web): 10 Jul 2019 Downloaded from pubs.acs.org on July 17, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Gold Nanoclusters Embedded Mucin Nanoparticles for Photodynamic Therapy and Bioimaging Deepanjalee Dutta, ∥, 1 Sunil Kumar Sailapu, ǂ, 1 Anitha T Simon, 1 Siddhartha Sankar Ghosh*, 1, 2 and Arun Chattopadhyay*, 1, 3 Centre for Nanotechnology,1 Department of Biosciences and Bioengineering2 and Department of Chemistry3 Indian Institute of Technology Guwahati, Guwahati – 781 039; India *Email ID: [email protected], [email protected]

KEYWORDS: Mucin, Nanoparticles, Gold nanoclusters, Methylene blue, Photodynamic therapy.

ABSTRACT: Effective delivery of a photosensitizer with the ability to trace its eventual progress forms an important aspect in photodynamic therapy (PDT). Further, the delivery mechanism might require to possess the ability to traverse through complex mucus barrier that offers retention of therapeutic molecules. In this work, gold nanocluster (Au NC) embedded mucin nanoparticles were synthesized by a rapid green synthetic procedure for application as a nanocarrier and to achieve image guided PDT. The mucin based nanocarrier exhibited excellent biocompatibility towards normal cells (HEK 293T). The photosensitizer methylene blue (MB) was loaded onto these Au NC-mucin nanoparticles (NPs). HeLa cancer cells were treated with MB loaded Au NCmucin nanoparticles under irradiation of 640 nm light. The cell viability assay revealed that the viability of HeLa cells was reduced to 50 % after treatment with MB loaded Au NC-mucin NPs

ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

kept under 640 nm irradiation. The luminescence exhibited by Au NCs in the nanocarrier was applied for tracking the delivery of MB inside HeLa cells using confocal microscopy. The flow cytometry assays elucidated the mechanism of cell death.

Introduction

Photodynamic therapy (PDT) has become an important non-invasive therapeutic modality where photosensitizers (PS) are activated upon light irradiation, thereby resulting in effective treatment of various diseases including cancer via cytotoxic singlet oxygen generation. (1) PSs have been synthesized in the last few decades, among which a few have been given the regulatory approval by Food and Drug Administration (FDA) for clinical application. However, some of the notable issues that limit the usage of the current PSs include poor accumulation in tumor tissues, low solubility in physiological media, prolonged cutaneous photosensitivity and inherent hydrophobic nature leading to its pronounced self-aggregation in aqueous environment. These factors ultimately contributed to lowering their photodynamic efficacy and compromised photophysical characteristics. (2-4) Hence, an interest to develop improved delivery systems for PSs that can entrap the PS without its activity being lost or altered have been accelerated, in addition to efforts towards addressing solubility issues and ensure delivery through accumulation within the target tissues. Nanocarriers, for example, polymeric nanoparticles, ceramic nanoparticles, liposomes, proteins, carbon nanomaterials, gold nanoparticles, quantum dots (QDs), magnetic nanoparticles (MNPs) and upconversion nanoparticles (UCNPs) have been formulated to attain stable dispersions of PSs in aqueous media for their effective delivery. Additionally, nanoparticles possesses several advantages like preventing the interference of intracellular environment with loaded cargo, improving the incorporated drug’s solubility and rendering extended half-life during circulation in


Page 2 of 31

Page 3 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

the blood with reduced side effects.(5,6) However, while developing nanocarriers, ensuring penetration into tumor tissues is a challenge to be addressed, in addition to having biocompatible and biodegradable characteristics of the same.(7) Importantly, designing a putative drug delivery nanoparticle requires that it possess the ability to traverse through complex mucus barrier and the rate of penetration of nanoparticles through mucus should exceed its characteristic clearance rate.(8) For instance, mucin, a family of high molecular weight glycosylated proteins, is the major component of mucus (found in all wet epithelial cells/tissues, including nasal cavity, oral cavity, gastrointestinal tract, lungs and female genital tract) (9) and has been predominantly equipped with an optimized and manifold chemistry for providing protection against pathogenic viruses, bacteria and minute particles.(10,11) Thus, the unique chemistry and the intricate composition of mucus leads to binding and retention of the therapeutic molecules within the complex matrix and this makes mucus play an important role as biophysical barrier to most of the uptaken drugs. Until recent times, it was established that mucus acts as protective layer against harmful foreign entities. However, the research advances have revealed the possible multiple interactions of mucins or mucus glycoproteins with many biologically significant molecules like enzymes, drugs, cell surfaces molecules as well as viruses and bacteria in numerous ways.(12) Though mucosal barrier creates a critical problem for drug delivery, an interesting approach can be introduced using this tenacious nature of mucin by exploiting it as a carrier to encapsulate drug molecules – via the possible mucin–drug interactions in order to achieve effective drug delivery by surpassing the mucus barrier. The ability of bovine submaxillary mucin to stabilize carbon nanomaterials and the reduced opsonization effect with promising haemo- and cyto-compatibility of mucylated nanocarriers (e.g., polylactic-co-glycolic acid) have been investigated in earlier studies.(13,14) The coating of mucin on biomaterials can considerably minimize host immune


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 31

response resulting from biomaterials.(15) Mucin based composites complexed with biomaterials like lysozymes, chitosan, or lectins can be fabricated by layer-by-layer assembly.(16,17) Another strategical

development

has

been

evolved

where

mucin–alginate(18)

or

mucin–

gelatin(19) complexes have been synthesized to design microparticles. Macroscopic covalently cross-linked mucin hydrogel was developed to investigate the drug binding and release efficacy, and further a mucin particle for enzyme encapsulation and release has also been reported. (20,21) These mucin-based scaffolds, were suggested to be capable of loading both hydrophilic and hydrophobic drugs, offering sustained drug release. However, majority of the previous reports involved time consuming protocols, multiple precursors for synthesis of mucin based nanocarriers. Moreover, no previous investigation has been carried out to exploit the potency of solely mucinbased biomaterials assembled into nanocarriers produced by a facile one pot synthetic route for its application in retention and release of drug molecules. A major weakness in the current procedures of photodynamic therapy also arises from the inability to trace the photosensitizers under conventional tracking techniques.

(22)

The lack of

successive follow-up of therapy after administration of PS leads to limited use of such a therapeutic practice. Nanoparticles can act as a multimodal platform with imaging property equipped within it, thereby qualifying the drug delivery systems based on these as improved theranostic systems. In this regard several multifunctional systems have been realized for image-guided photosensitizer delivery. (22,23) A wide variety of organic dyes as well as luminescent materials including QDs, and UCNPs (upconversion nanoparticles) have been developed as multifunctional platform for both optical imaging and drug/gene delivery. However, many of the organic dyes are known to be cytotoxic, have poorly water solubility and known to induce DNA damage, Also, composition of heavy metal elements (such as Cd2+ and Pb2+) that are harmful to biological systems and pose


Page 5 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

environmental issues limit the application of QDs in the field of theranostics.(24) Further, the poor thermal stability and mechanical stability exhibited by lanthanide complexes in UCNPs reduce their application potential.(25) Noble metal nanoclusters, such as gold nanoclusters (Au NCs) have gained wide attraction in biological applications due to their high luminescence, excellent photostability, negligible toxicity, good biocompatibility and aqueous dispersibility.(26,27) Hence, photosentizing agents, conjugated with these luminescent metal nanoclusters could offer significant advantages for efficient image guided photodynamic cancer treatment.(28,29) Thereby, development of a luminescent Au NCs embedded composite nanocarrier encapsulated with PSs based on mucin template via rapid and green approaches is important to effectively achieve both PS delivery and tracking. In this regard, it is highly advantageous that the synthesis and formulation be achieved without involving multistep reactions/harsh reducing agents and employing minimum requirement of precursors to ensure a simplified approach. Herein, a facile and green synthesis of a mucin based nanocarrier embedded with luminescent Au NCs is reported. The cationic photosensitizer methylene blue (MB) was loaded onto the Au NC-mucin nanoparticles (NPs) to achieve photodynamic therapy in HeLa cancer cells and its simultaneous tracking was achieved by bioimaging the luminescence of Au NCs (Scheme 1). The role of singlet oxygen generation and subsequent cell death pathway were elucidated by flow cytometry based assays.


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Scheme 1. Schematic representation of synthesis of MB loaded Au NC-mucin NPs and photodynamic therapy in cancer cells.

Materials and Methods

Chemicals: Methylene blue (Merck), Type III mucin (source: porcine stomach; SigmaAldrich), HAuCl4 (Au, 99.99%, 17 wt % in dilute HCl; Sigma-Aldrich), mercaptopropionic acid (MPA; Sigma-Aldrich) and water (Milli-Q-grade, >18 MΩ/cm) were utilised devoid of any alteration.

Synthesis of Au NCs-mucin nanoparticles: For the generation of mucin templated luminescent Au NCs, a slightly modified synthesis protocol based on our previously reported method in a different system was employed.(26) For synthesis, an amount of 8 μL of MPA (0.11 M), 20 μL of HAuCl4 (10 mM) and 1 mL of 1 mg/mL mucin were mixed together. The solution was subjected to heating at 95 °C for 2 min, and then was quickly transferred to 4 °C. The sample was then subjected to centrifugation for 5 min at 6000 rpm. The resuspension of the pellet was done in water for further use.


Page 6 of 31

Page 7 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Loading efficiency: MB (3.12 mM) was added to the Au NC-mucin NPs with concentrations varying from 0 mg/mL to 0.25 mg/mL and incubated for 1 h at 37 °C. The samples were the centrifuged at 6000 rpm for 5 min and the intensity of free MB was probed using PerkinElmer LS55, fluorescence spectrophotometer. The loading efficiency of the composite NPs was calculated with following formula.

Loading Efficiency (%) =

Total MB − MB in supernatent 𝑋100 Total MB

The concentration of MB used in the all characterisation experiments is 2.2mM and Au NC-mucin NPs is 1mg/mL unless otherwise mentioned.

Luminescence measurements: The luminescence spectra were obtained by PerkinElmer LS55, fluorescence spectrophotometer.

UV–visible spectroscopy: Jasco V-630 was applied to obtain the absorbance of all samples.

X-ray photoelectron spectroscopy (XPS): XPS analysis was carried out using a ESCALAB Xi+ (Thermo Fisher Scientific Pvt. Ltd, UK) X-ray photoelectron spectrometer. The X-ray source used was monochromatic Al Kα (1486.6 eV).

CD spectroscopy: For CD spectroscopy, the samples were evaluated using JASCO-815 spectrometer (Jasco, Japan). To carry out the measurements under constant nitrogen gas purging at a flow rate of 5 L/min at 25 ºC, 0.2 cm path length cuvette was utilized. The


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

spectra were recorded from wavelength 240 nm to 190 nm with four accumulations and background spectrum subtraction was performed with solvent.

Transmission electron microscopy (TEM): For TEM imaging of the samples, 7 μL of the sample was drop-cast onto the carbon-coated copper TEM grid. It was then air-dried and viewed under a transmission electron microscope (JEM 2100; JEOL, Peabody, MA) at an accelerating voltage of 200 keV.

Dynamic Light scattering analysis (DLS): To measure the hydrodynamic diameter and the zeta potential, Malvern Zetasizer Nano ZS was used. The size and zeta potential measurements were carried out at 25°C in disposable size and zeta cuvettes (Malvern) with water as solvent.

Measurement of quantum yield (QY): The examination of QY was performed via implementing quinine sulfate in a 0.10 M H2SO4 solution as reference. The equation applied for quantification of the QY is:

QY = QYr

m n2 m r n 2r

Here, ‘m’ depicts the slope of integrated luminescence intensity versus absorbance plot, subscript ‘r’ depicts the reference solution of quinine sulfate and ‘n’ indicates the refractive index. The absorbance as well as the luminescence intensity readings were collected consecutively one followed by other using the same solution. The refractive index of water (solvent) is 1.33 and the QY of the standard (QYr) is 0.54.


Page 8 of 31

Page 9 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1, 3 - diphenylizobenzofuran (DPBF) singlet oxygen detection experiment: For this, 100 μL of MB loaded Au NC-mucin NPs were mixed with 100 μL of DPBF (0.08 mM) in dimethyl sulfoxide (DMSO) under dark conditions. Another set containing only DPBF was kept as control. The samples were irradiated for 0 to 4 min in steps of 1 min using LED array with peak emission around 640 nm (2.5 mW/cm2). The luminescence of DPBF was recorded using Perkin Elmer LS 55 fluorescence spectrophotometer.

Cell culture: HeLa (human cervical carcinoma), HEK 293T (human embryonic kidney) cells for cell culture experiments were procured from the National Centre for Cell Sciences, Pune, India. For maintaining the cells, Dulbecco’s modified Eagle’s medium, augmented with l-glutamine (4 mM), penicillin (50 units/mL), 10% (v/v) fetal bovine serum (FBS; PAA Laboratories, Austria) and streptomycin (50 mg/mL, Sigma-Aldrich) was utilised, in a 5% CO2 humidified incubator at 37 °C.

MTT based cell viability assay: The influence on the cell viability was assessed by MTT assay by seeding 1 × 104 cells /well of HeLa or HEK in a 96-well plate and grown for 24 h at 37 °C in a 5% CO2 humidified incubator. The cells were treated with MB, Au NC-mucin NPs and MB loaded Au NC-mucin NPs for 3 h and then irradiated with 640 nm light (2.5 mW/cm2) for 30 min using a custom made LED array device and thereafter incubated for 24 h. Control experiments were carried out by treating the cells with MB and MB loaded Au NC-mucin NPs without irradiation (in dark). Thereafter, the assay using MTT [3-(4,5dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide] was performed where the MTT is reduced into purple colored formazan by mitochondria in case of live cells giving an absorbance at 570 nm. Thereby, measurement at 570 nm is directly proportional to the live


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 31

cells present. The absorbance at 690 nm (as background interference) was deducted. The percentage of cell viability was calculated as

% of viable cells =

(A570-A690)of treated cells x 100 (A570-A690)of control cells

The concentration of MB used in the cell culture experiments is 6µM and Au NC-mucin NPs is 22µg/mL unless otherwise mentioned.

Confocal microscopy: To perform confocal microscopy analysis, 1 × 105 HeLa cells were seeded on the coverslips in 35 mm culture plates and allowed to grow in a 5% CO2 humidified incubator at 37 °C for 24 h. Thereafter, the cells were treated with MB, Au NCmucin NPs and MB loaded Au NC-mucin NPs for 3 h. After the treatment, the cells were fixed by use of formaldehyde (0.1%) and chilled ethanol (70%). The coverslips were then placed on glass slides followed by sealing of the ends. Control samples without any treatment were processed in a similar manner and all samples were viewed employing a Zeiss LSM 880 microscope (at excitation 405 nm).

Cellular uptake studies: 1× 106 HeLa cells were seeded in a 6-well plate for 24 h and then were

treated

with

Au

NC-mucin

NPs

for

3

h.

The

samples

were

then harvested by trypsinization and analyzed in a FACSCalibur (BD Biosciences, NJ). The fluorescence data were recorded for 15000 cells with the (BD Biosciences) CellQuest program per sample and then analyzed.

Reactive oxygen species (ROS) determination: For ROS production studies, HeLa cells were seeded at 1 × 105 cells/well in a 6-well plate, grown for 24 h, and then treated for 3 h


Page 11 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

with MB or MB loaded Au NC-mucin NPs. Thereafter, the cells were irradiated in a similar manner as described in MTT assay to induce ROS generation. In order to confirm the role of singlet oxygen towards increased ROS levels, the cells were pretreated with 50 mM sodium azide (NaN3) for 1 h before irradiation. Control experiments were carried out by treating the cells with MB or MB loaded Au NC-mucin NPs without irradiation (in dark). The cells, followed by the treatment, were exposed to 5μL of 2,7-dichlorofluorescein diacetate (DCFH-DA; 1 mM, Sigma-Aldrich) in each well for 10 min. The medium was removed and the cells were harvested by trypsinization followed by redispersing in fresh medium. DCFH-DA is nonfluorescent in nature and is converted into DCFH via hydrolysis inside living cells followed by formation of green fluorescent dichlorofluorescein (DCF) through oxidation by ROS. The samples were then examined in FL1-H channel using a FacsCalibur, BD Biosciences, NJ flow cytometer. The fluorescence data was obtained for 15000 cells in each sample using Cell Quest program (BD Biosciences).

Cell cycle analysis: For this experiment, the propidium iodide (PI) assay was implemented. HeLa cells were seeded in 6-well plates at 1 × 105 cells/well and cultured for 24h. Then treatment with MB or MB loaded Au NC-mucin NPs was given in a similar manner as in case of MTT assay. The medium and PBS were accumulated individually for both the control and treated samples after the treatment time was over. The cells were then gathered via trypsinization and subjected to centrifugation at 650 rcf, 6 min. Thereafter, under constant vortexing, the cells were fixed by slowly adding 1 mL of 70% chilled ethanol and then stored at 4 °C. Following this, the cells were centrifuged and washed using PBS (icecold). Thereafter, RNase treatment was carried out for 1 h at 55 °C. Then, to all of the samples, 10 μL of 1 mg/mL PI was added, and incubated for 30 min at 37 °C in the dark.


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The samples were further subjected to analysis in a flow cytometer (BD) and 15000 cells/sample was assessed for PI fluorescence using the program Cell Quest Pro (BD). Assay of Caspase-3: To ascertain the cell death via apoptosis, caspase-3 assay was done wherein 1 × 105 HeLa cells cells/well were cultured for 24 h in 6-well plates and then were subjected to treatment with MB or MB loaded Au NC-mucin NPs in a similar manner as in case of MTT assay. After the treatment, the control as well as treated samples were trypsinized and fixed using formaldehyde (0.1%) for 15 min in dark. Following this, the samples were sub centrifuged at 650 rcf for 6 min, and then resuspended in PBS. Then, the samples were incubated in the dark for 20 min after addition of 0.5% Tween 20. The cells were subjected to washing three times with PBS, followed by addition of PE-conjugated anticaspase-3 antibody (10µL). Then, incubation for half an hour at 37 °C was carried out and 15000 cells/sample were examined employing a flow cytometer (BD) (FL2-H channel) for PE fluorescence. Using Cell Quest program (BD) assessment was performed for all the samples.


Page 12 of 31

Page 13 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

RESULTS AND DISCUSSION

Figure 1. (A) Emission spectra of Au NC-mucin NPs (λem = 580 nm) at 300 nm excitation (inset shows magnified spectrum exhibiting no additional peak at 700 nm). (B) Emission spectra of MB loaded Au NC-mucin NPs (λem = 580 nm, 700 nm) at 300 nm excitation (inset shows magnified spectrum exhibiting additional peak at 700 nm due to MB). (C) TEM image of Au NC-mucin NPs with a scale bar of 50 nm. (D) Magnified TEM image of Au NC-mucin NP with a 20 nm scale bar. The Au NC embedded mucin nanoparticles (Au NC-mucin NPs) appeared as a colourless dispersion with an emission peak at 620 nm when excited at 300 nm, which indicated the formation of Au NCs (Figure 1A). The MB loaded Au NC-mucin NPs exhibited an additional fluorescence


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

peak at around 700 nm, which confirmed the binding of MB to Au NC-mucin NPs (Figure 1B). The UV-Vis absorbance at 280 nm due to the tryptophan residues of the protein was observed in case of Au NC-mucin NPs as well as MB loaded Au NC-mucin NPs (Supporting Information, Figure S1). MB loaded Au NC-mucin NPs in addition exhibited a peak at 660 nm due to the presence of MB, which corresponds to maximum absorption peak of only MB. The absence of plasmonic signature in case of Au NC-mucin NPs at 520 nm indicates that Au NPs were not present (Supporting Information, Figure S1a-c). TEM investigation showed the formation of MB loaded Au NC-mucin NPs with an average size of 139 ± 47 nm. Magnified TEM investigation revealed the presence of Au NCs with an average size of 1.9 ± 0.34 nm inside the mucin nanoparticles (Figure 1C, D). The TEM images of only mucin (control) processed under the same synthesis conditions is shown in Supporting Information, Figure S1e. The loading efficiency of cationic photosensitizer methylene blue loaded on the Au NC-mucin NPs was found to be 70% (Supporting Information, Figure S1d). The hemolysis study and stability of the Au NC-mucin NPs in human serum were carried out. The results showed negligible percentage of hemolysis, indicating the hemocompatibility of the Au NC-mucin NPs and it was observed that the Au NC-mucin NPs were stable in human serum with no reduction of luminescence (Supporting information, Figure S1f and g). These analyses indicated the potential of using this system for in vivo applications. After loading of MB, luminescence of Au NC-mucin NPs was quenched in comparison to the as synthesised Au NC-mucin NPs due to possible photo-induced charge transfer between MB and Au NCs.(30) In case, quenching of the Au NCs (donor) was occurring due to Föster resonance energy transfer (FRET), that should also have resulted in a simultaneous enhancement of emission of the MB dye that is the fluorescent acceptor here, which was not observed as shown in Supporting information, Figure S2a-c. Hence, FRET is possibly not the reason for the observed result in this


Page 14 of 31

Page 15 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

case although it but might somewhat be responsible for the quenching of luminescence of Au NCs. The zeta potential of mucin (-17.3 ± 2.3 V), Au NC-mucin NPs (-5.5 ± 0.2 V), MB loaded Au NCmucin NP s (-2.8 ± 0.1 V) indicated the binding of MB to the Au NC-mucin NPs (Supporting Information, Figure S3a-c). MB being a cationic dye is expected to bind via electrostatic interactions hence reduction of zeta potential from (-5.5 ± 0.2 V) to (-2.8 ± 0.1 V) after loading of MB is possibly an indication of the binding. The hydrodynamic diameter of the Au NC-mucin NPs was found to be around 284±23 nm, which is larger than the TEM possibly due to swelling of the mucin in presence of water (Supporting Information, Figure S4a). The zeta potential increased to 3.97±0.42 with the decrease in pH and also the size increased with the reduction of pH (Supporting Information, Figure S4b,c). The stability of the MB loaded Au NC-mucin nanoparticles in comparison to standard rhodamine 6G was depicted by photostability analysis. The luminescence intensity decrease rate (F/F0) was 0.27% per min, whereas the rate in the case of rhodamine 6G was 0.80% per min. This indicated that the as prepared Au NC-mucin NPs were more photostable compared to standard rhodamine 6G. A similar behaviour have been reported in earlier works.(26,27) The quantum yield of MB loaded Au NC-mucin NPs using quinine sulphate as standard was obtained to be 3.5% which was deemed applicable for imaging studies (Supporting Information, Figure S5a,b). X-ray photoelectron spectroscopy (XPS) results displayed peaks at 87.9 eV and 84.2 eV, corresponding to Au (4f5/2 and 4f7/2, respectively) (Figure S6a). The peak due to Au+1 was at 85.2 eV. The peak of S (2p3/2) at 162.5 eV corroborated with thiolated Au NCs as per earlier works and also an additional peak corresponding to oxidized sulphur was observed at the binding energy of 168.07 eV as shown in Figure S6b. (31-33) This possibly indicates that the Au nanocluster was bound to the thiol group of MPA. The S (2p3/2) (163.39 eV) corresponded to the S–S bond.(34) The ratio of Au(0) / Au(I), which is found to be 16.91:1 indicated that most of the Au was present


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

in the Au(0) state. The ratio of S-S: Au-S bonds was found to be 9.11:1. The CD spectrum of native mucin represented lack of well-defined secondary structure, characteristic of mucins due to heavy glycosylation.(35) Also, CD spectrum of the Au NC-mucin NPs when compared to native mucin showed nominal changes with a slight increase in the percentage of turns (Supporting Information, Figure S7a,b). This is indicative of the fact that the synthesis method which is devoid of harsh reducing agents do not significantly affect the native mucin’s structure as generally seen in earlier reported works(36) where a significant change in the protein structure was observed after synthesis.

Figure 2. Release profile of MB from Au NC-mucin NPs (1mg/mL) at pH 7.4 and 4.5. The release pattern of the MB from the Au NC-mucin NPs was studied at pH 4.5 and 7.5. It was revealed that about 55% of the drug was released at the end of 24 h at pH 4.5 and release of 26% was observed at pH 7.5 at the end of 24 h. The more amount of drug release in pH 4.5 is beneficial in a way that pH in real tumor microenvironment is also acidic. The


Page 16 of 31

Page 17 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

sustained release pattern followed by initial burst release helps in retention of the photosensitizer for its optimum activity (Figure 2).

Figure 3. Luminescence spectra of (A) DPBF and MB loaded Au NC-mucin NPs irradiated with 640 nm light, (B) only DPBF irradiated with 640 nm light, (C) DPBF and MB loaded Au NC-mucin NPs under no irradiation. The potential of the MB loaded Au NC-mucin NPs in generation of singlet oxygen was determined by DBPF based singlet oxygen detection. The luminescence spectra of DPBF in presence of MB loaded Au NC-mucin NPs (irradiated with 640 nm light), only


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

DPBF (irradiated with 640 nm light) and MB loaded Au NC-mucin NPs (non-irradiated) were analysed. In the presence of MB loaded Au NC-mucin NPs, the luminescence of DPBF was found to quench gradually with time, possibly due to the breakdown of DPBF in presence of the singlet oxygen generated by the MB loaded Au NC-mucin NPs (Figure 3A). On the other hand, in the absence of MB loaded Au NC-mucin NPs and non-irradiated samples, significant decay in luminescence was not observed (Figure 3B, C). The observed results indicated that the MB loaded Au NC-mucin NPs can possibly generate singlet oxygen as shown by the quenching of DPBF dye, hence helping in understanding the potential of the MB loaded Au NC-mucin NPs towards singlet oxygen generation under irradiation at 640 nm wavelength.

Figure 4. Images of HeLa cells obtained by confocal microscopy (A-C) Bright field, fluorescence and overlay images respectively, after treatment with MB loaded Au NCmucin NPs. For the application of the MB loaded Au NC-mucin NPs in bio-imaging and tracking the delivery of MB employing the luminescence of Au NCs, HeLa cancer cells were treated with MB loaded Au NC-mucin NPs for 4 h and thereafter visualization was performed using a confocal microscope. The confocal microscopic images delivered evidence of the


Page 18 of 31

Page 19 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

internalisation of MB loaded Au NC-mucin NPs inside cancer cells (Figure 4A-C) as depicted by the luminescence of Au NCs. The control cell (devoid of treatment) images did not exhibit any luminescence (Supporting Information, Figure S8a-c). The emission spectrum of Au NC-mucin NPs at 405 nm excitation is shown in the supporting information, Figure S8d. The z-stack of the confocal microscopic images demonstrated internalization of MB loaded Au NC-mucin NPs into cancer cells (Supporting Information, Figure S9a-d). The confocal microscopic images of cancer cells treated with Au NC-mucin NPs and only MB are shown in Supporting Information, Figure S10a,b. To support the confocal based uptake analysis, the uptake studies of the Au NC-mucin NPs have been performed probing the intrinsic fluorescence of Au NCs in the Au NC-mucin NPs using flow cytometry. It has been revealed that compared to the control cells there was a higher percentage of uptake as shown in the Supporting Information, Figure S11 in case of Au NC-mucin nanoparticle treated samples. This displayed the potential use of luminescent MB loaded Au NC-mucin NPs in imaging cancer cells as well as tracking the delivery of therapeutic photosensitizer MB.


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5. (A) MTT assay of HEK 293T cells showing biocompatible nature of the Au NCmucin NPs, (B) MTT assay of HeLa cells treated with MB, MB loaded Au NC-mucin NPs in dark, MB under irradiation, and MB loaded Au NC-mucin NPs under irradiation exhibiting less viable cells in case of samples treated with MB loaded Au NC-mucin NPs under irradiation. The biocompatibility of the Au NC-mucin NPs was determined by treating the HEK 293T human embryonic kidney cell lines with Au NC-mucin NPs for 24 h. The MTT assay


Page 20 of 31

Page 21 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

revealed that more that 80% of the cells were viable after 24 h of treatment (Figure 5A) and hence revealing that the Au NC-mucin NPs were biocompatible in nature. In order to establish the potential of MB loaded Au NC-mucin NPs in photodynamic therapy, the HeLa cancer cells were treated with MB, Au NC-mucin NPs, MB loaded Au NC-mucin NPs in dark as well as under irradiation at 640 nm light for 30 min by using a custom LED array device at specific irradiation doses (as mentioned in the Experimental Section). After the exposure, the cells were further incubated for 24 h. The MTT assay revealed that in case of the MB loaded Au NC-mucin NP irradiated samples, 50% of the cells were viable at MB concentration of 6 μM, whereas 69 % cells were viable in case of only MB. Minimum level of dark toxicity was observed in case of both only MB, MB loaded Au NC-mucin NPs (Figure 5B). Hence, an optimum level of photodynamic effect was achieved by MB, MB loaded Au NC-mucin NPs at low concentrations of drug, which resulted in cancer cell death.


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 6. ROS generation profile of (A) HeLa cells treated with Au NC-mucin NPs, (B) MB and (C) MB loaded Au NC-mucin NPs under different conditions. To establish that the mechanism of cell death initiated by the generation of singlet oxygen, intracellular ROS levels were monitored in HeLa cells treated with MB, MB loaded Au NC-mucin NPs under irradiation with 640 nm light and in dark. Increased levels of ROS was found in case of irradiated samples in comparison to control and samples in


Page 22 of 31

Page 23 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

dark. Further, to ascertain that the behavior exhibited was due to the generation of singlet oxygen, the intracellular ROS levels were monitored in HeLa cells with pretreatment of sodium azide (specific singlet oxygen quencher)37 before MB, MB loaded Au NC-mucin NP treatment under irradiation with 640 nm light and in dark. The results indicated that in case of sodium azide pretreated samples, the ROS levels were considerably lowered in the cells treated with MB, MB loaded Au NC-mucin NPs under irradiation (Figure 6A-C). The enhanced lowering of ROS levels in case sodium azide pretreated samples under irradiation indicated that the cell death initiated under irradiation was possibly due to MB, MB loaded Au NC-mucin NP mediated PDT via generation of singlet oxygen.

Figure 7. Analysis of cell cycle incase of control HeLa cells, HeLa cells treated with MB, MB loaded Au NC-mucin NPs in dark, MB under irradiation, and MB loaded Au NC-mucin NPs under irradiation showing higher population of apoptotic cells (sub G1) incase of samples treated with MB loaded Au NC-mucin NPs under irradiation. The propidium iodide (PI) staining mediated cell cycle assay revealed increase in sub G1 cell population after treatment with MB or MB loaded Au NC-mucin NPs irradiated


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

with 640 nm light as compared to other controls, along with slight changes in other phases of cell cycle (G0/G1, S, and G2/M) as shown in Figure 7. The substantial increment in the sub G1 population is suggestive of apoptosis mediated cell death. Further, the results of caspase-3 assay, performed using an anti-caspase antibody which labels caspase-3 produced by the cells during apoptosis also supported the mechanism of apoptosis mediated cell death. It revealed that MB, MB loaded Au NC-mucin NP treated cells irradiated with 640 nm light showed a considerable increment in the active caspase-3 positive cell population in comparison to other controls (Supporting Information, Figure 12a-e). Hence, summarizing the above results, it is shown that the MB loaded Au NC-mucin NPs effectively delivered the photosensitizer and destroyed the cancer cells by virtue of generation of singlet oxygen. Also, the luminescence property of the MB loaded Au NCmucin NPs imparted biolabeling features, thereby helped in tracking the delivery of MB.

CONCLUSIONS In brief, a mucin based luminescent biocompatible nanocarrier was developed through an easy one-step green synthetic route for the delivery of photosensitizer MB into the HeLa cancer cells. The analysis of MB binding to Au NC-mucin NPs was carried out. Further, the uptake of MB loaded Au NC-mucin NPs was analysed by confocal microscopy. The MB loaded Au NC-mucin NPs delivered the MB successfully and the cancer cells were killed through singlet oxygen generation when irritated with light. Also, the luminescent nature of the MB loaded Au NC-mucin NPs provided a prospect of tracking the delivery of photosensitizer inside the cells without the implementation of organic fluorescence moieties. The pathway of cell death has been explained via flow cytometry based assays.


Page 24 of 31

Page 25 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Hence, MB loaded biocompatible Au NC-mucin NPs has been employed to achieve photodynamic cancer therapy along with bioimaging. ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.”

UV-Visible spectra, Loading and release studies, DLS, XPS, Photostability, quantum yield, CD spectra, confocal microscopy,caspase-3 assay.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected], [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Present Addresses ∥ School of Chemistry, The King's Buildings, University of Edinburgh, EH9 3FJ, Edinburgh, United Kingdom. ǂ Centro Nacional de Microelectrónica (CNM) Instituto de Microelectrónica de Barcelona (IMB), Campus

Universitat

Autònoma

de

Barcelona

(UAB),

Carrer

dels

Til·lers,

08193, Cerdanyola del Vallès, Barcelona, Spain. Notes An Indian patent has been filed based on the device used for in vitro PDT in the present manuscript.


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACKNOWLEDGMENT We thank the Department of Electronics and Information Technology (No. 5(9)/2012-NANO (Vol. II)) and Centre for Excellence DBT (Department of Biotechnology) programme support, Government of India for financial support. We greatly acknowledge the Sophisticated Analytical Instrument Facility, CSIR-NEIST Jorhat for helping us in XPS analysis. We are thankful to Ayan Pal (Department of Chemistry, IIT Guwahati) for his timely help and valuable suggestions. REFERENCES 1. van Straten, D.; Mashayekhi, V.; de Bruijn, H. S.; Oliveira, S.; Robinson, D. J. Oncologic Photodynamic Therapy: Basic Principles, Current Clinical Status and Future Directions. Cancers (Basel) 2017, 9 (2). 2. Staneloudi, C.; Smith, K. A.; Hudson, R.; Malatesti, N.; Savoie, H.; Boyle, R. W.; Greenman, J. Development and Characterization of Novel Photosensitizer: ScFv Conjugates for Use in Photodynamic Therapy of Cancer. Immunology 2007, 120 (4), 512–517. 3. Huang, P.; Xu, C.; Lin, J.; Wang, C.; Wang, X.; Zhang, C.; Zhou, X.; Guo, S.; Cui, D. Folic Acid-Conjugated Graphene Oxide Loaded with Photosensitizers for Targeting Photodynamic Therapy. Theranostics 2011, 1, 240–250. 4. Liang, X.; Li, X.; Yue, X.; Dai, Z. Conjugation of Porphyrin to Nanohybrid Cerasomes for Photodynamic Diagnosis and Therapy of Cancer. Angewandte Chemie International Edition 2011, 50 (49), 11622–11627. 5. Jawahar, N.; Meyyanathan, S. N. Polymeric Nanoparticles for Drug Delivery and Targeting: A Comprehensive Review. International Journal of Health & Allied Sciences 2012, 1 (4), 217223.


Page 26 of 31

Page 27 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

6. Narvekar, M.; Xue, H. Y.; Eoh, J. Y.; Wong, H. L. Nanocarrier for Poorly Water-Soluble Anticancer Drugs—Barriers of Translation and Solutions. AAPS PharmSciTech 2014, 15 (4), 822–833. 7. Barua, S.; Mitragotri, S. Challenges Associated with Penetration of Nanoparticles across Cell and Tissue Barriers: A Review of Current Status and Future Prospects. Nano Today 2014, 9 (2), 223–243. 8. Lai, S. K.; Wang, Y.-Y.; Hanes, J. Mucus-Penetrating Nanoparticles for Drug and Gene Delivery to Mucosal Tissues. Advanced Drug Delivery Reviews 2009, 61 (2), 158–171. 9. Boegh, M.; Nielsen, H. M. Mucus as a Barrier to Drug Delivery – Understanding and Mimicking the Barrier Properties. Basic Clin Pharmacol Toxicol 2015, 116 (3), 179–186. 10. Zanin, M.; Baviskar, P.; Webster, R.; Webby, R. The Interaction between Respiratory Pathogens and Mucus. Cell Host Microbe 2016, 19 (2), 159–168. 11. Wong, P. T.; Wang, S. H.; Ciotti, S.; Makidon, P. E.; Smith, D. M.; Fan, Y.; Schuler, C. F.; Baker, J. R. Formulation and Characterization of Nanoemulsion Intranasal Adjuvants: Effects of Surfactant Composition on Mucoadhesion and Immunogenicity. Mol. Pharmaceutics 2014, 11 (2), 531–544. 12. Adikwu, M. U. Multifarious Potentials of Tropical Animal-Derived Biopolymers in Drug Delivery: Lessons from the African Snail Mucin. In Biopolymers in Drug Delivery: Recent Advances and Challenges, Adikwu M.U.; Esimone E.O., Ed.; Bentham Science Publishers, 2010; pp 27-38. 13. Belgorodsky, B.; Drug, E.; Fadeev, L.; Hendler, N.; Mentovich, E.; Gozin, M. Mucin Complexes of Nanomaterials: First Biochemical Encounter. Small 2010, 6 (2), 262–269.


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

14. Thasneem, Y. M.; Rekha, M. R.; Sajeesh, S.; Sharma, C. P. Biomimetic Mucin Modified PLGA Nanoparticles for Enhanced Blood Compatibility. Journal of Colloid and Interface Science 2013, 409, 237–244. 15. Sandberg, T.; Ott, M. K.; Carlsson, J.; Feiler, A.; Caldwell, K. D. Potential Use of Mucins as Biomaterial Coatings. II. Mucin Coatings Affect the Conformation and Neutrophil-Activating Properties of Adsorbed Host Proteins—Toward a Mucosal Mimic. Journal of Biomedical Materials Research Part A 2009, 91A (3), 773–785. 16. Ahn, J.; Crouzier, T.; Ribbeck, K.; Rubner, M. F.; Cohen, R. E. Tuning the Properties of Mucin via Layer-by-Layer Assembly. Biomacromolecules 2015, 16 (1), 228–235. 17. Borges, J.; Mano, J. F. Molecular Interactions Driving the Layer-by-Layer Assembly of Multilayers. Chem. Rev. 2014, 114 (18), 8883–8942. 18. Builders, P. F.; Kunle, O. O.; Okpaku, L. C.; Builders, M. I.; Attama, A. A.; Adikwu, M. U. Preparation and evaluation of mucinated sodium alginate microparticles for oral delivery of insulin. Eur J Pharm Biopharm 2008, 70 (3), 777-783. 19. Ofokansi, K. C.; Adikwu, M. U.; Okore, V. C. Preparation and Evaluation of Mucin-Gelatin Mucoadhesive Microspheres for Rectal Delivery of Ceftriaxone Sodium. Drug Development and Industrial Pharmacy 2007, 33 (6), 691–700. 20. Duffy, C. V.; David, L.; Crouzier, T. Covalently-Crosslinked Mucin Biopolymer Hydrogels for Sustained Drug Delivery. Acta Biomaterialia 2015, 20, 51–59. 21. Fukui, Y.; Fukuda, M.; Fujimoto, K. Generation of Mucin Gel Particles with Self Degradable and -Releasable Properties. J. Mater. Chem. B 2018, 6 (5), 781–788.


Page 28 of 31

Page 29 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

22. Nair, L. V.; Nazeer, S. S.; Jayasree, R. S.; Ajayaghosh, A. Fluorescence Imaging Assisted Photodynamic Therapy Using Photosensitizer-Linked Gold Quantum Clusters. ACS Nano 2015, 9 (6), 5825–5832. 23. Huang, P.; Lin, J.; Wang, S.; Zhou, Z.; Li, Z.; Wang, Z.; Zhang, C.; Yue, X.; Niu, G.; Yang, M.; et al. Photosensitizer-Conjugated Silica-Coated Gold Nanoclusters for Fluorescence Imaging-Guided Photodynamic Therapy. Biomaterials 2013, 34 (19), 4643–4654. 24. Stan, M. S.; Memet, I.; Sima, C.; Popescu, T.; Teodorescu, V. S.; Hermenean, A.; Dinischiotu, A. Si/SiO2 Quantum Dots Cause Cytotoxicity in Lung Cells through Redox Homeostasis Imbalance. Chemico-Biological Interactions 2014, 220, 102–115. 25. Chu, Y.; Hao, H.; Xie, H.; Chen, C.; Cai, P.; Seo, H. J. Preparation of Lanthanide (Eu3+, Tb3+)-Complex-Grafted Copolymer of Methyl Methacrylate and Maleic Anhydride Films and the Promising Application as LED Luminous Layers. J Mater Sci: Mater Electron 2017, 28 (7), 5615–5622. 26. Sailapu, S. K.; Dutta, D.; Sahoo, A. K.; Ghosh, S. S.; Chattopadhyay, A. Single Platform for Gene and Protein Expression Analyses Using Luminescent Gold Nanoclusters. ACS Omega 2018, 3 (2), 2119–2129. 27. Dutta, D.; Sahoo, A. K.; Chattopadhyay, A.; Ghosh, S. S. Bimetallic Silver Nanoparticle–gold Nanocluster Embedded Composite Nanoparticles for Cancer Theranostics. J. Mater. Chem. B 2016, 4 (4), 793–800. 28. Kim, T.; Jang, H.; Kim, S.; Lee, J.-H.; Kim, S.-Y.; Jeon, N. L.; Song, J. M.; Min, D.-H. Synthesis of Fluorescent Au Nanocrystals–Silica Hybrid Nanocomposite (FLASH) with Enhanced Optical Features for Bioimaging and Photodynamic Activity. Langmuir 2018, 34 (1), 173–178.


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

29. Gao, F.; Zheng, W.; Gao, L.; Cai, P.; Liu, R.; Wang, Y.; Yuan, Q.; Zhao, Y.; Gao, X. Au Nanoclusters and Photosensitizer Dual Loaded Spatiotemporal Controllable Liposomal Nanocomposites Enhance Tumor Photodynamic Therapy Effect by Inhibiting Thioredoxin Reductase. Advanced Healthcare Materials 2017, 6 (7), 1601453. 30. Rakovich, A.; Savateeva, D.; Rakovich, T.; Donegan, J. F.; Rakovich, Y. P.; Kelly, V.; Lesnyak, V.; Eychmüller, A. CdTe Quantum Dot/Dye Hybrid System as Photosensitizer for Photodynamic Therapy. Nanoscale Research Letters 2010, 5 (4), 753-760. 31. Jiang, J.; Conroy, C. V.; Kvetny, M. M.; Lake, G. J.; Padelford, J. W.; Ahuja, T.; Wang, G. Oxidation at the Core–Ligand Interface of Au Lipoic Acid Nanoclusters That Enhances the Near-IR Luminescence. J. Phys. Chem. C 2014, 118 (35), 20680–20687. 32. Vitale, F.; Fratoddi, I.; Battocchio, C.; Piscopiello, E.; Tapfer, L.; Russo, M. V.; Polzonetti, G.; Giannini, C. Mono- and Bi-Functional Arenethiols as Surfactants for Gold Nanoparticles: Synthesis and Characterization. Nanoscale Research Letters 2011, 6 (1), 103. 33. Leus, K.; Concepcion, P.; Vandichel, M.; Meledina, M.; Grirrane, A.; Esquivel, D.; Turner, S.; Poelman, D.; Waroquier, M.; Speybroeck, V. V.; et al. Au@UiO-66: A Base Free Oxidation Catalyst. RSC Adv. 2015, 5 (29), 22334–22342. 34. Cui, L.; Xue, Y.; Noda, S.; Chen, Z. Self-Supporting S@GO–FWCNTs Composite Films as Positive Electrodes for High-Performance Lithium–sulfur Batteries. RSC Advances 2018, 8 (5), 2260–2266. 35. Perez-Vilar, J.; Hill, R. L. The Structure and Assembly of Secreted Mucins. J. Biol. Chem. 1999, 274 (45), 31751–31754.


Page 30 of 31

Page 31 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

36. Brzezicka, K. A.; Serna, S.; Reichardt, N. C. Fluorescent Neoglycoprotein Gold Nanoclusters: Synthesis and Applications in Plant Lectin Sensing and Cell Imaging. Nanoscale Research Letters 2018, 13 (1), 360.

37. Bancirova, M. Sodium Azide as a Specific Quencher of Singlet Oxygen during Chemiluminescent Detection by Luminol and Cypridina Luciferin Analogues. Luminescence 2011, 26 (6), 685–688.

Table of Contents Graphic


Gold-Nanocluster-Embedded Mucin Nanoparticles ... - ACS Publications

Recommend Documents