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Vitamin C Conjugated Nanoparticle Protects Cells from Oxidative Stress at Low Dose but Induces Oxidative Stress and Cell Death at High Dose Atanu Chakraborty, and Nikhil R. Jana ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b16055 • Publication Date (Web): 14 Nov 2017 Downloaded from http://pubs.acs.org on November 14, 2017

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ACS Applied Materials & Interfaces

Vitamin C Conjugated Nanoparticle Protects Cells from Oxidative Stress at Low Dose but Induces Oxidative Stress and Cell Death at High Dose Atanu Chakraborty and Nikhil R. Jana* Centre for Advanced Materials, Indian Association for the Cultivation of Science, Kolkata700032, India *Corresponding authors E-mail: [email protected]

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ABSTRACT: Although antioxidant property of vitamin C is well known for protecting cells from oxidative stress, recent study shows that it can also generate oxidative stress under high intra-cellular concentration and induce cell death. However, poor chemical stability and low biological concentration (micromolar) of vitamin C restrict their function primarily as antioxidant. Here we report two different nanoparticle forms of vitamin C with its intact chemical stability, glucose responsive release from nanoparticle and efficient cell delivery in micro to millimolar concentration. Nanoparticles are composed of silica coated Au nanoparticle or lipophlic polyaspartic acid-based polymer micelle which is conjugated with vitamin C via phenylboronic acid. Surface chemistry of nanoparticle is optimized for efficient cellular interaction/uptake and for cell delivery of vitamin C. We found that vitamin C protects cell from oxidative stress at micromolar concentration but at millimolar concentration it induces cell death by generating oxidative stress. In particular high dose vitamin C produces H2O2, disrupts the cellular redox balance and induces cell death. This study highlights the concentration dependent biological performance of vitamin C and requirement of high dose cell delivery approach for enhanced therapeutic benefit. KEYWORDS: vitamin C, gold nanoparticle, polymer micelle, antioxidant, reactive oxygen species, oxidative stress, glucose responsive drug delivery


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INTRODUCTION Eukaryotic cells produce energy through respiration where oxygen is used for degradation of food.1 During this respiratory process different oxygenic free radicals (e.g. superoxide, peroxide) are formed within the mitochondria which are commonly known as reactive oxygen species (ROS).2 The concentration of ROS in the normal cells is always maintained to a certain level in order to have uninterrupted cellular activities.2 If the ROS concentration is increased, cells are exposed under oxidative stress which leads to mitochondrial dysfunction and other related diseases such as Alzheimer’s, Perkinson’s, aging and cancer.3 In live cell ROS concentration is maintained through different intracellular enzymes (e.g. superoxide dismutase, peroxidise, catalase) and extracellular antioxidant chemicals such as vitamin E, vitamin C and coenzyme Q.3 These enzymes/chemicals scavenge the oxygenic radicals and minimize the toxic effect caused by ROS. However, these enzymes/chemicals often have poor bioavailability when on demand; either due to poor production, low chemical stability, poor solubility and poor cell uptake.4 For example, in diabetes this antioxidant defence network breaks down and causes oxidative stress within the mitochondria that leads to mitochondrial dysfunction.5 Vitamin C is a well known antioxidant that reacts with ROS and prevents cells from their toxic effect.6,7 Linus Pauling first showed that high dose vitamin C increases the survival rate of cancer patients and explained due to their antioxidant property.8 However, oral administration of high dose of vitamin C showed no therapeutic benefit for cancer patients.9 Subsequent works show that biological performance of vitamin C depends on the mode of delivery, dose, presence of metal ions and other extracellular conditions.6 At the physiological pH, vitamin C remains as mono-anion which is chemically unstable and acts as reducing agent (antioxidant).6 However, vitamin C spontaneously oxidizes at high 3 ACS Paragon Plus Environment


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concentration (> millimolar) or at high pH or in presence of catalytic metal and acts as a source of H2O2.6 Thus predominate antioxidant property of vitamin C is ensured by lower plasma concentration ( ~40-80 micromolar) and mono-anionic forms.10-13 In contrast high dose (> millimolar concentration) of vitamin C is shown to have pro-oxidant property via generating H2O2 and has been used to kill cancer cells.14-21 In the cellular label excess of H2O2 increases the oxidized glutathione that changes the cellular redox balance (decreases the ratio of glutathione to reduced glutathione), influences metabolic alteration and inhibits the progression of cancer cells.15,21 Other report shows that vitamin C enters into cell in its oxidized form (dehydroascorbic acid) through glucose transporters and reduces back to vitamin C by consuming glutathione.20 As a result the cellular glutathione level gets lowered, ROS gets accumulated and results cell death.20 This approach has been exploited for selective killing of cancer cells that have glucose transporters.20 All these studies clearly highlight the importance of cellular delivery of vitamin C with varying dose. However, high dose and controlled cellular delivery of vitamin C is very challenging due to their poor chemical stability and non-availability of appropriate delivery carrier. Most of the earlier study directly uses molecular form of vitamin C that decreases their bioavailability due to spontaneous oxidation to dehydroascorbic acid.6,14 Experimental work shows that high plasma concentration (> millimolar) of vitamin C can only be achieved via intravenous injection and not via oral or dietary approach, mainly due to poor chemical stability of vitamin C.6,15,20 Several vitamin C delivery nanocarriers have been proposed which include vitamin C loaded mesoporous silica22/poly lactic acid glycolic acid (PLGA)23/lipid,19 lipophilic vitamin C based micelle/liposome11,16,24 and electrostatically bound vitamin C with chitosan.25 Although these nanoplatforms enhance the delivery performance of vitamin C as compared to molecular form of vitamin C, further improvements


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are required for controlled delivery, responsive delivery and enhanced delivery with intact chemical stability. Here we report vitamin C conjugated nanoparticle as vitamin C delivery platform. The nanoparticle is either composed of silica coated Au nanoparticle (control 1) or lipophilic polyaspartic based polymer micelle (control 2) and they are conjugated with ~2-13 wt % of vitamin C via phenylboronic acid. Surface chemistry of nanoparticle is optimized for efficient cellular interaction/uptake and cell delivery of vitamin C. Au nanoparticle-based system has been selected as it has optical property suitable for imaging/detection and is widely exploited in various biomedical applications.26,27 It is reported that Au nanoparticle can produce ROS and induce cytotoxicity in presence of light, X rays or pulse lasers.28 In the addition bactericides conjugated Ag nanoparticle is shown to provide enhanced antimicrobial property.29 Polyaspartic acid based micelle has been selected as it is biocompatible, non-toxic and functionalization chemistry is well developed.30 The presented nanoparticle-based vitamin C delivery platforms have three distinct advantages which are not reported in earlier approaches. First, nanoparticle form provides chemical stability of vitamin C under physiological condition and offers its delivery with intact molecular form. Second, nanoparticle form offers efficient cellular delivery of vitamin C in a wider concentration range --- from micro to millimolar concentration. Third, nanoparticle form offers slow and continuous vitamin C delivery option depending of surrounding glucose concentration. Using our nanoparticle system we have observed that low dose vitamin C protects cells from oxidative stress but induces oxidative stress and cell death at high dose.



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EXPERIMENTAL SECTION Chemicals. Didodecyldimethylammonium bromide (DDAB, Sigma), mercaptopropyl silane (MPS, Sigma), tetrabutylammonium borohydride (TBAB, Sigma), anhydrous AuCl3 (Sigma), 3-(2-aminoethylamino) propyl dimethoxy methyl silane (AEAPS, Sigma), vitamin C (ascorbic

acid,

Sigma),

3-carboxy

phenylboronic

acid

(PBA,

TCI),

1-(3-

dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, TCI), L-aspartic acid (Sigma), octadecylamine (ODA, Sigma), 4-formylphenylboronic acid (PBA, Sigma), ethylenediamine (EDA, Sigma), 2′,7′-dichlorodihydrofluorescein diacetate (DCF, Sigma), propidium iodide (PI, Sigma), mitotracker orange (Thermo Fischer Scientific), 2-(Nmorpholino)ethanesulfonic acid (MES, Sigma) and dehydroascorbic acid (DHAA, Sigma) were used as received. Synthesis of silica coated Au nanoparticle (control 1). We have used our reported method as described earlier.31 In brief toluene solution of 5 mL AuCl3 (0.01 M) was prepared in presence of equimolar DDAB. Next, 100 µL toluene solution of MPS (0.1 M) was added to it. In a separate vial 12.5 mg TBAB and 12.5 mg DDAB were dissolved in one mL toluene and added to the AuCl3 solution under stirring condition to reduce the gold salt. After that 2 mL toluene solution of AEAPS (0.1M) was added and heated to 65 °C. Particles started to precipitate slowly and after 15 min completely precipitated from the solution. The precipitate was washed with chloroform and ethanol and then it was dissolved in 2 mL distilled water. Preparation of vitamin C conjugated Au nanoparticle i.e. NP1. 500 µL of aqueous solution of control 1 was taken in a 2 mL vial. Next, 0.05 mmole (6 mg) of PBA was dissolved in 100 µL bicarbonate buffer (pH 10) and mixed with control 1 solution. Next, 15 mg EDC was added to the mixture and the whole solution was stirred overnight at 4 ºC. Then


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the solution was centrifuged at 12000 rpm for 5 min and resultant boronic acid functionalized Au nanoparticle precipitate was dissolved in fresh distilled water. In order to conjugate vitamin C, one mL boronic acid functionalized Au nanoparticle was taken in a 2 mL vial and then 500 µL vitamin C (0.1 M) solution was added drop wise followed by adding base to maintain the pH 7.4.32-34 The solution was stirred for 3 h and then it was centrifuged at 12000 rpm to collect the vitamin C conjugated nanoparticle i.e. NP1 as precipitate. Particles were washed with water for 2-3 times and finally dissolved in distilled water with the concentration of ~ 2 mg/mL. Amount of vitamin C conjugated to NP1 was estimated by UV-visible spectroscopy. At first a calibration graph was prepared using the absorbance at 260 nm against vitamin C concentration. Next, 0.02 mg/mL glucose solution was added with colloidal solution of NP1 and stirred for 60 min. Next, particles were separated by centrifugation and the supernatant solution containing the released vitamin C was estimated by UV-visible spectroscopy. Typically, 20 mg of NP1 was taken in 10 mL water and incubated with glucose solution to release vitamin C. Next, released vitamin C was estimated and particles were weighted after centrifuge and drying. Alternatively, 20 mg of functionalized Au nanoparticle was taken in 10 mL water, incubated with vitamin C (0.02M) solution for 3 h and then NP1 was separated by centrifuge. Next, remaining vitamin C was estimated and collected NP1 was weighted after drying. We have estimated that 4-8 wt % of vitamin C is present in NP1. Similarly, we have also synthesized dehydroascorbic acid loaded Au nanoparticles i.e. NP1(control). Typically aqueous solution of dehydroascorbic acid was added to one mL of phenylboronic acid conjugated Au nanoparticle followed by addition of base to maintain the pH 7.4 and stirred for 2-3 h then it was centrifuged. The NP1(control) was precipitated and washed 2-3 times with distilled water and dissolved in fresh distilled water. The 7 ACS Paragon Plus Environment


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dehydroascorbic acid has lower affinity to bind with phenylboronic acid due to absence of enediol.35 Synthesis of phenylboronic acid conjugated polyaspartimide (control 2). Polyaspartimide was synthesized following the previously reported method with some modifications.30 In brief, 3 g of L-aspartic acid was suspended in 10 mL of mesitylene under inert conditions, mixed with 165 μL of phosphoric acid (88 %) and heated to 150 °C for 4 h. The reaction mixture was cooled to room temperature and the white colour residue was collected and dissolved in dimethylformamide (DMF) and then excess water was added to precipitate the polysuccinimide. The precipitate was washed with water for a number of times to remove phosphoric acid and DMF and then washed with methanol several times. Finally, solid polysuccinimide was dried in vacuum. Next, 250 mg polysuccinimide was dissolved in 20 mL dry DMF, mixed with 135 mg octadecylamine and heated at 80 °C for 24 h under inert atmosphere. The solution was cooled to room temperature and mixed with 170 µL of ethylene diamine and heated further at 80 °C for 24 h under inert atmosphere. After that 150 mg of 4-formyl phenylboronic acid (PBA) was added and stirred for 12 h at room temperature. Resultant PSI-PBA was dissolved in 510 mL DMF for further use. Preparation of vitamin C conjugated polyaspartimide (NP2). Typically, 0.1-1.0 mL of freshly prepared DMF solution of control 2 was added to 5-50 mL water under vigorous stirring condition. Next, 0.5-5.0 mL vitamin C solution (0.1 M) was added drop wise followed by adding base to maintain the pH 7.4.27 The solution was stirred for 3 h and then resultant NP2 was precipitated by adding acetone and centrifuged at 12000 rpm to collect NP2 as precipitate. Precipitate was washed with methanol and dissolved in water with


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concentration of ~ 2 mg/mL. Amount of vitamin C conjugated to NP2 was estimated as described above for NP1. Synthesis of fluorescein conjugated NP2. About 500 µL solution of control 2 was taken in a 2 mL vial and 0.5 mL borate buffer (pH 9) was added to it. Next, 100 µL freshly prepared fluorescein-NHS solution (3 mg/mL in DMF) was added to it and stirred for 3 h. Next, the solution was dialysed for overnight in alkaline medium using dialysis membrane (MWCO 12000-14000 Da) in order to remove unreacted reagents. Next, vitamin C is conjugated by previously described method. Glucose responsive vitamin C release study from NP1 and NP2. It is important that vitamin C is transported into cell as chemically intact form and not as oxidized form (i.e. dehydroascorbic acid). We used HPLC to detect vitamin C and dehydroascorbic acid.35 Typically, C18 column was used as solid phase and mobile phase consisted of 50 mM sodium dihydrogen phosphate and acetonitrile in the volume ratio of 6:4. The flow rate is adjusted to one mL/min and the detection wavelength of the UV detector is set at 265 nm for both vitamin C and dehydroascorbic acid. Under this condition dehydroascorbic acid and vitamin C have different retention time and in their mixture they produce reasonably well resolved signals. For glucose responsive vitamin C release study, NP1 or NP2 was taken in phosphate buffer solution (pH 7.4) and mixed with glucose solution (100-200 mg/dL). Next, a part of solution is taken at different time point, nanoparticle is separated by centrifuge whereas PSI micelles are separated by adding acetone and 20 µL of supernatant was injected to the HPLC column to detect vitamin C. We have also studied release of vitamin C from NP2 and NP1 in presence of glucose via UV-visible spectroscopy. Typically, 5 mL of NP2 or NP1 was taken in a dialysis membrane and dipped into 50 mL glucose solution. The glucose concentration was varied 9 ACS Paragon Plus Environment


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from 100 mg/dL to 200 mg/dL. The release of vitamin C is monitored by analyzing the outside solution at different time point via UV-visible spectroscopy. Throughout the process the volume of the outside glucose solution is kept unchanged. Nanoparticle-based cell delivery of vitamin C, in vitro imaging/detection of ROS, quantification of cell death and mitochondrial activity study. CHO/HeLa cells were incubated in DMEM (Dulbecco’s Modified Eagles medium) media in 24 well plate for 24 h. After that the media was replaced by glucose free RPMI (Roswell Park Memorial Institute) media and then 50-100 µL solution of NP1 or NP2 was added. After 3 h of incubation, media was removed and fresh media was added. Next, dark field image or fluorescence image of cells was captured in order to follow the labelling of nanoparticle and delivery of vitamin C. Protective role of vitamin C on the H2O2 induced oxidative stress was studied by estimating ROS using DCF as fluorescent probe.36 DCF is a cell permeable non-fluorescent dye that emits green fluorescent in presence of hydroxyl radicals. Typically, cells were exposed with H2O2, NP1 or NP2 or vitamin C and then incubated with DCF followed by fluorescence imaging to detect ROS inside cells. Usually, 50 µL of NP1/NP2 (with vitamin C concentration of 200 µM) or free vitamin C (200 µM) was added to the cell culture media and incubated for 15 mins. Next, cells were washed and fresh media was added followed by 10 µL of H2O2 (500 µM). Next, 5 µL of DCF (20 µM) solution was added and cells were imaged under fluorescence microscope. Propidium iodide (PI) based method was used for quantification of dead cells.37 In dead cells pores were created in the membranes through which PI goes inside the cells and intercalates with DNA of the nucleus and show fluorescence. Typically, cells were mixed with 10 µL of PI (1µM) and incubated for 15 mins and imaged under microscope or quantified by flow cytometer. For flow cytometry study the cells were detached using 10 ACS Paragon Plus Environment

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trypsin-EDTA and dispersed in PBS buffer. Similarly in another set of experiment, mitochondrial activity was measured using mitotracker red. For that 25 µL of mitotracker red (200 nM) was added to the culture media of treated cells. Then cells were imaged under fluorescence microscope. Cytotoxicity study. For cytotoxicity study cells were cultured in 24 well plate for 24 h and then cells were incubated with NP1 or NP2 of varying dose for 3 h. After that the cells were washed with PBS buffer of pH 7.4 and incubated with fresh DMEM media for 24 h. Next, 50 µL of freshly prepared MTT (methylthiazolyldiphenyl-tetrazolium bromide) solution (5 mg/mL) was added and incubated for another 3 h. After that the supernatant solution was discarded leaving the formazan in the plate which is dissolved in SDS solution (8 gm SDS dissolved in 40 mL of DMF-water mixture) and absorbance was measured at 570 nm. Instrumentation. UV visible spectra were measured in Shimadzu UV-2550 UV−visible spectrophotometer. Samples for transmission electron microscopic (TEM) study were prepared by putting a drop of particle dispersion on carbon coated copper grid, dried in air and observed with FEI Tecnai G2 F20 microscope using 200kV electron source. Fourier transform infrared spectroscopy on KBr pellet was performed using Shimadzu FT-IR 8400S instrument. Bright field and dark field images of live cells were performed using Zeiss Axio Observer A1 microscope. Fluorescence images were performed in Olympus IX81 microscope using image-pro plus version 7.0 software. Flow cytometry was studied using a BD Accuri C6 flow cytometer. HPLC equipped with SunFire C18 column and UV detector (Waters 2489) was used for determination of vitamin C and dehydroascorbic acid. RESULTS AND DISCUSSION Design and synthesis of nanoparticle with intact chemical stability of vitamin C and glucose responsive release. We have followed two approaches in preparing vitamin C-based 11 ACS Paragon Plus Environment


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nanoparticles. (Scheme 1) In one approach silica coated and primary amine terminated Au nanoparticles is prepared by reported method.31 Next, it is transformed into phenylboronic acid (PBA) functionalized nanoparticle via EDC coupling with PBA where carboxyl group of it is reacted with primary amines of nanoparticles. Finally, this nanoparticle is conjugated with vitamin C via chelate complex formation between enediol groups of vitamin C and boronic acid group of PBA.32 In other approach polyaspartic acid-based amphiphilic polymer is synthesized by our previously reported method30 and functionalized with phenylboronic acid. (Supporting Information, Scheme S1 and Figure S1) Next, this polymer is reacted with vitamin C to produce chelate complex between enediol group of vitamin C and boronic acid group of PBA. The resultant NP2 self-assembles into 40-80 nm micelle in aqueous phase. Functionalization of PBA and chelation with vitamin C has been determined by NMR and FTIR spectroscopy. (Figure 1) The 1H NMR signal of phenyl hydrogen of PBA is detected in purified nanoparticle. The 1H NMR signal of the -CH protons of vitamin C is detected at 4.5 ppm. The 1H NMR signal of enediol at 11 ppm becomes absent after reaction with vitamin C, confirming the chelate formation. FTIR study shows that carbonyl stretching frequency of vitamin C at 1736 cm-1 is present in NP1 and NP2. On the other hand the hydroxyl stretching of enediol of vitamin C at 1311 cm-1 becomes absent in NP1 and NP2 which suggests that vitamin C is conjugated with nanoparticle through the enediol. Chemical stability of vitamin C in nanoparticle form under the physiological condition and glucose responsive release of vitamin C have been investigated using HPLC. This has been performed by long time preservation of the colloidal solution of nanoparticle followed by glucose induced release of vitamin C under physiological condition and identification of vitamin C and dehydroascorbic acid by HPLC. Typically, colloidal solution of NP1 or NP2 is prepared in aqueous phosphate buffer solution, preserved for 12 h and then it is mixed with 12 ACS Paragon Plus Environment

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glucose solution. Next, released vitamin C is detected under HPLC at different time points. Under our HPLC detection condition, vitamin C and dehydroascorbic acid have separate retention time and so aerial oxidation of vitamin C can be identified. It has been observed that nanoparticle sample shows dominant peak corresponding to vitamin C, even after 12 h exposure under aqueous phosphate buffer solution. (Supporting Information, Figure S2) This result suggests that vitamin C remains chemically stable in the nanoparticle form and would offer cell delivery of vitamin C without their oxidation. In contrast aqueous solution of vitamin C is prone to aerial oxidation and completely oxidized to dehydroascorbic acid within 12 h. (Supporting Information, Figure S3) We have further investigated glucose concentration dependent release of vitamin C where glucose concentration is maintained with the respective blood concentration of normal and diabetic patients.38 (Supporting Information, Figure S4) In general vitamin C release is faster from NP1 as compared to the release from NP2 and in both cases the release kinetic becomes faster with the increasing glucose concentration. Release of vitamin C from NP2 prolongs for more than 5 h under normal blood glucose concentration (100 mg/dL), the release rate can be increased (prolongs for 2-5 h) by taking food (with corresponding blood glucose concentration of 120-140 mg/dL) and release can be completed within 1-2 h under hyperglycaemic condition (200 mg/dL). Low dose NP1 and NP2 protect cells from oxidative stress. Cell delivery of vitamin C involves simple incubation of cell with NP1 or NP2 in culture media. Dark field imaging of cells confirms the labelling of cells by NP2 nanoparticle. Figure 2 shows the typical image of NP1 nanoparticle labelled cells under dark filed. As Au nanoparticle strongly scatters visible light, cells labelled with them are detected with yellow scattering.39,40 High cell labelling has been ensured by the presence of remaining primary amine groups on nanoparticle surface and the non-specific cellular interaction commonly offers by silica coating.31 Similarly cells incubated with fluorescein conjugated NP2 show bright green emission under fluorescence 13 ACS Paragon Plus Environment


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microscope, suggesting the labelling of cells by NP2. (Figure 2) All these labelling are very rapid, typically membrane labelling occurs within 15 min and cellular entry occurs in few hours. During this cellular interaction, vitamin C is released from the surface of nanoparticle via competitive interaction with glucose. Thus high cell uptake of nanoparticle permits the efficient cellular delivery of vitamin C. The ROS scavenging efficiency of NP1 and NP2 are investigated by exposing cells with H2O2 and observing the cell survival, cell membrane damage and mitochondrial function in the presence of NP1 and NP2. Concentration of NP1 is adjusted to 0.4-0.8 mg/mL and concentration of NP2 is adjusted to 0.2-0.4 mg/mL so that they maintain the cell delivery of vitamin C in the 150-200 µM concentration range. It is known that H2O2 produces hydroxyl radical in presence of Fe3+ or other transition metal ions.41 These hydroxyl radicals are toxic to the cells as they induce lipid peroxidation, mitochondrial dysfunction, DNA damage and ultimately lead to cell death.41 We have observed that exposure with H2O2 leads to shrinking of cells followed by death within 6-12 h. However, if cells are pre-incubated with NP1/NP2 and then exposed with H2O2, they are able to maintain their morphology and survival in presence of H2O2. (Figure 3) Mechanism of action of NP1/NP2 has been investigated by detecting ROS inside cell, investigating cell membrane damage and measuring the mitochondrial membrane potential. Detection and imaging of ROS is performed using DCF which is known to produce green fluorescence in presence of ROS. We have observed strong green emission from cells after their exposure with H2O2. (Figure 4 and Supporting Information, Figure S5) In contrast cells which are pre-incubated with NP1/NP2 and then exposed with H2O2, shows insignificant green emission. This result suggests that H2O2 exposure produces ROS inside cell and presence of NP1/NP2 efficiently inhibits their production. Control experiment shows that 14 ACS Paragon Plus Environment

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molecular form of vitamin C is unable to inhibit ROS generation in micromolar concentration, either because of poor cell uptake or due to oxidation to dehydroascorbic acid. This study shows clear advantage of the nanoparticle form of vitamin C in maintaining their chemical stability, efficient cell delivery and consuming ROS. Large amount of ROS can introduce pores on the cell membrane via peroxidation of lipid membrane and lead to mitochondrial dysfunction via lowering of mitochondrial membrane potential.42 We have used propidium iodide (PI) labelling experiment showing that H2O2 exposure create pores on the cell membrane, induces entry of PI into cell and label cell nucleus. However, PI fails to label the cell nucleus if NP1/NP2 is present, suggesting that cells are protected from membrane damage. (Figure 5) In contrast molecular vitamin C or phenylboronic acid functionalized nanoparticle cannot stop ROS induced membrane damage. (Supporting Information, Figure S6) Similarly, labelling experiment with mitotracker red to the H2O2 exposed cells indicate that mitochondria cannot be labelled and results only diffused red fluorescence. In contrast, if cells are incubated with NP1/NP2 before exposed to H2O2, mitochondria can be successfully levelled. This result suggests that destruction of mitochondrial membrane by H2O2 is prohibited by NP1/NP2. (Figure 6) On the other hand molecular vitamin C or control nanoparticle treated cells cannot stop the destruction of mitochondrial membrane in presence of H2O2. ((Supporting Information, Figure S7) All these study clearly shows that low dose of NP1/NP2 with micromolar concentration of vitamin C efficiently protects cells from oxidative stress. High dose NP1 and NP2 induce oxidative stress and cell death. Next, we have studied dose dependent cytotoxicity of NP1/NP2 and in particular investigated the cytotoxicity at higher dose. In this study concentration of NP1 is adjusted to 0.4-2.4 mg/mL and concentration of NP2 is adjusted to 0.2-1.2 mg/mL so that it maintain the cell delivery of 15 ACS Paragon Plus Environment


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vitamin C in a wider concentration range from micro- to millimolar concentration range. Two different cell lines such as HeLa and CHO are used for this study. These cells are selected as both of them are cancer cells and widely used for biological studies. We have found that NP1 releases vitamin C quite rapidly compared to NP2 in presence of glucose and therefore NP1 is incubated for 3 h whereas NP2 is incubated with cells for 24 h to make sure of complete release of vitamin C. Next, cells are washed and cytotoxicity study is performed via MTT assay. As NP1/NP2 has strong interaction with cell, it attach on cell surface within 15 min of incubation and enter into cell in next 3-24 h and during this processes vitamin C gets released from nanoparticle surface via competitive reaction with cellular glucose. As we have varied the concentration of NP1/NP2, cellular concentration of vitamin C is also varied, typically from micro to millimolar concentration. Cell viability (%) against the concentration of vitamin C present in NP1/NP2 is summarized in Figure 7. Results show that NP1 is nontoxic with micro to millimolar concentration of vitamin C but starts showing toxicity as the concentration increases in the range of 2-10 mM. In contrary NP2 shows toxicity at much lower concentration (0.4-0.8 mM) than NP1 which may be due to higher uptake of polymeric micelles. Control experiment shows that similar toxic effect is also observed for molecular form of vitamin C. However, nanoparticle without any vitamin C functionalization is relatively non-toxic in the studied concentration range. Here it is also found that polymeric micelles have relatively less toxicity compared to Au nanoparticle and therefore polymeric micelles can be a better option for delivery of vitamin C than the Au nanoparticle. In order to understand the mechanism of cytotoxicity, cellular ROS detection experiment has been performed. Typically, cells are exposed with high dose NP1/NP2/vitamin C for 3 h and then DCF-based ROS imaging is performed immediately and after 24 h. Results show that NP1, NP2 and free vitamin C can generate ROS at their high dose. (Figure 8, 9 and Supporting Information, Figure S8, S9) In addition it is also observed 16 ACS Paragon Plus Environment

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that NP1/NP2 induced ROS generation occurs for longer time (3-24 h) and with time the extent of ROS is increased. This suggest that cells labelled with NP1/NP2 gradually release vitamin C for generating ROS. In contrast molecular vitamin C generates ROS for shorter time period (2-4 h). This result clearly shows that high dose of NP1/NP2 produces ROS for longer time and induce cytotoxicity. We have performed further control experiment using dehydroascorbic acid. We found that dehydroascorbic acid does not produce ROS either at molecular or nanoparticle form and does not induce cytotoxicity similar to vitamin C. (Supporting Information, Figure S10, S11) This result further confirms that ROS generation performance of vitamin C is linked to their cell delivery in the chemically intact form. Advantage of nanoparticle form of vitamin C. There are three distinct advantages of presented nanoparticle form of vitamin C in the cell delivery application. First, nanoparticle form provides chemical stability of vitamin C under physiological condition. This is because vitamin C is covalently conjugated with the Au nanoparticle and oxidative transformation into dehydroascorbic acid is prohibited. Thus vitamin C can be delivered in their intact chemical form and without any oxidation. Considering the poor chemical stability of vitamin C, presented nanoparticle-based option can be more effective for their cell delivery. Second, nanoparticle form offers efficient delivery of vitamin C into cell, particularly at lower concentration. This is because nanoparticles are larger in size and uptake is favoured by endocytosis approach.42 In contrast cellular uptake of molecular vitamin C rely on sodiumdependent vitamin C transporter or glucose receptor dependent uptake of oxidized form.15,20,21 Third, nanoparticle form offers glucose concentration dependent vitamin C delivery into cell. This option is particularly important for prolonged delivery of vitamin C and for more effective action on cell. In particular continuous exposure with molecular 17 ACS Paragon Plus Environment


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vitamin C is limited due to rapid oxidation under physiological condition. In that respect nanoparticle form would be very effective. In earlier reports on nanocarrier based vitamin C delivery, the vitamin C is loaded without any chemical bonding19,22,23 or electrostatically attached25 or vitamin C is transformed into lipophilic vitamin C.11,16,24 Thus spontaneous oxidation issue of vitamin C and their high dose delivery at intact chemical form remain unsolved. In that respect our nanoformulation approach is unique as it can be used for high dose delivery of vitamin C with intact chemical form. Most important aspect of present work is the NP1/NP2 induced cytotoxicity at high dose. Vitamin C is known to spontaneously oxidize and produce H2O2 at high concentration (> millimolar).6 In addition presence of transition metal ions can catalyze the H2O2 formation.6 Use of high dose NP1/NP2 ensures high dose delivery of vitamin C and production of high concentration of H2O2. Under such condition cellular enzymes (e.g. catalase, glutathione peroxidase, peroxiredoxins) fails to eliminate excesses ROS inside the cells and result in cell death. The glucose dependent release of vitamin C provides three additional advantages. First, glucose responsive release offer detachment of vitamin C from nanoparticle after delivery and release them inside cell in the molecular from. Second, the release rate of vitamin C from nanoparticle can be enhanced via food intake. This is because food intake leads to the increase of blood glucose concentration from 100 mg/dL to 120-140 mg/dL and such condition can enhance the vitamin C release kinetics from NP2.(see Supporting Information, Figure S4a) Third, in the case of type II diabetes patient there are evidences that ROS generation is highly related with hyperglycemia38 and presented approach may be useful for glucose responsive release of vitamin C to fight against hyperglycemia. As glucose level is high in hyperglycaemia, release of vitamin C would be higher under such condition that 18 ACS Paragon Plus Environment

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would consume ROS more effectively. This condition cannot be achieved for molecular vitamin C or any other reported delivery system. Despite significant advantage there are several limitations of the present approach that need to be solved. First, nanoparticle should be appropriately functionalized for selective targeting of cancer cell. This may be achieved by conjugation with vitamin, aptamer or other targeting molecule. Second, biodegradable polymer nanoparticle should be used for real application. This may be achieved by using well known polymer that self-assemble into micelle/nanoparticle. Third, loading of vitamin C need to be increased and more parameter should be added for controlled and responsive delivery. Fourth, additional peroxidise like catalyst may be incorporated into delivery carrier so that ROS can be generated more efficiently. With all these achievement we may expect that intravenously injected nanoformulation would target the tumour, deliver high dose vitamin C and induce the cell death.

CONCLUSION We have synthesized two different nanoparticle forms of vitamin C and used them for glucose responsive cell delivery at wider concentration range. The nanoparticle form provides chemical stability of vitamin C under physiological condition and offers glucose responsive efficient delivery of vitamin C into cell. We have shown that nanoparticle form of vitamin C protects cell from oxidative stress at micromolar concentration but at millimolar concentration it induces cell death by generating oxidative stress. In particular high dose vitamin C produces H2O2, disrupts the cellular redox balance and induces cell death. Further research should be directed to produce vitamin C based nanoparticle for targeted and high dose cell delivery and to utilize their therapeutic benefit. 19 ACS Paragon Plus Environment


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ASSOCIATED CONTENT Supporting Information Details of nanoparticle characterization, glucose responsive vitamin C release study, cytotoxicity study of NP1(control) and dehydroascorbic acid and control ROS generation experiments. This material is available free of charge via the Internet at http://pubs.acs.org. Notes The authors declare no competing financial interests. Acknowledgement. The authors acknowledge DST Nanomission (Grant No. SB/NM/NB-1009/2016) and CSIR (Grant No. 02(0249)/15/EMR-II), Government of India for financial assistance. AC acknowledges CSIR, India for providing research fellowship.

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