REDOX Responsive Fluorescence Active Glycopolymer Based

Article Cite This: ACS Appl. Bio Mater. 2019, 2, 2587−2599

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REDOX Responsive Fluorescence Active Glycopolymer Based Nanogel: A Potential Material for Targeted Anticancer Drug Delivery Koushik Bhattacharya,† Sovan Lal Banerjee,† Subhayan Das,‡ Sarthik Samanta,† Mahitosh Mandal,‡ and Nikhil K. Singha*,†,§ †

Rubber Technology Centre, ‡School of Medical Science and Technology, and §School of Nanoscience and Technology, Indian Institute of Technology, Kharagpur, West Bengal 721302, India

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S Supporting Information *

ABSTRACT: A well-defined glycopolymer based fluorescence active nanogel has been prepared via the combination of reversible addition− fragmentation chain transfer (RAFT) polymerization and Diels−Alder (DA) “click” chemistry. To prepare the nanogel, initially, a functional AB block copolymer (BCP) poly(pentafluorophenyl acrylate)-b-poly(furfuryl methacrylate) (PPFPA-b-PFMA), having activated pentafluorophenyl ester group, was synthesized via RAFT polymerization. The activated pentafluorophenyl functionality was replaced by the amine functionality of glucosamine to introduce the amphiphilic BCP poly[2-(acrylamido) glucopyranose]-b-poly(furfuryl methacrylate) (PAG-b-PFMA). Furthermore, the terminal acid (−COOH) functionality of the RAFT agent was modified by gelatin QDs (GQDs) to generate fluorescence active glycopolymer. An anticancer drug, Doxorubicin, was loaded in the micelle via the successive addition of the drug molecule and cross-linking using dithio-bismaleimidoethane (DTME), a REDOX responsive cross-linker. The anticancer activity of the drug loaded nanogel was observed over MBA-MD-231, human breast cancer cell line, and monitored via fluorescence spectroscopy and flow cytometric analyses (FACS). The cytotoxicity of the prepared glycopolymer based nanogel over the MBA-MD-231 cell line was assessed via MTT assay test, and it was observed that the synthesized nanogel was noncytotoxic in nature. KEYWORDS: glycopolymer, REDOX responsive, fluorescence active, nanogel, anticancer activity

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INTRODUCTION Efficient treatment of cancer by chemotherapy with minimum side effects has been very challenging. In the conventional treatment process via chemotherapy, hydrophobic drugs, like doxorubicin, paclitaxel, cisplatin, and camptothecin, have been used to kill cancer cells.1 However, these hydrophobic drugs possess poor water solubility, multidrug resistance, and nontarget specific nature, which limit their therapeutic efficacy.1−3 Tremendous efforts have been made to develop various types of drug-loaded nanocarriers that can overcome these shortcomings. Over the past decade, polymeric nanocarriers have got great attention because of their flexibility, ease of functionalization, drug loading capacity, water solubility, and prolonged blood circulation time.4,5 It is noteworthy that the uncontrolled proliferation of cancer cells leads to abnormal tumor microenvironments such as acidity and hypoxia.6 Owing to the distinct tumor microenvironment, several stimuliresponsive polymeric micelles have been developed, which can release drug under certain stimuli like pH,7,8 temperature,9,10 glutathione,11 etc. Recent studies reveal that presence of the carbohydrates increases the uptake toward cancer cells as they can act as the principal energy source for cell proliferation leading to enhanced uptake of sugar by cancer cell as compared with normal cells.12,13 Glycopolymers are the © 2019 American Chemical Society

synthetic analogue of polysaccharides having pendant carbohydrate moieties. Now-a-days, glycopolymers have received huge attention due to their biocompatibility, biological recognition such as cell growth regulation, cancer cell metastasis, and through specific interactions with lectins.12,14,15 Chen et al.16 have reported polycaprolactone (PCL)glycopolymer based block copolymer (BCP) for the delivery of anticancer drug DOX. The foremost limitation of the micellar system is their stability in the bloodstream. Because of the presence of the enzymatic reactions, polymeric micelles lose their stability in the bloodstream leading to the burst release of the drug. Core-cross-linking of the micelles is one of the best adopted solutions to restrict the undesired degradation of the micelle, and it also facilitates triggered release of the drug depending on the chemical nature of the cross-linker. It has been reported that core-cross-linking of the BCP leading to the formation of the nanogels. Nanogel is highly cross-linked material, which has size of about 20−200 nm and is usually prepared via in situ polymerization using a suitable cross-linker.17,18 It may be cross-linked micelle or Received: March 29, 2019 Accepted: May 21, 2019 Published: May 22, 2019 2587

DOI: 10.1021/acsabm.9b00267 ACS Appl. Bio Mater. 2019, 2, 2587−2599

ACS Applied Bio Materials

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conventional cross-linked polymer like polystyrene, etc. Crosslinked micelles are self-assembled materials that undergo welldefined micelle formation, and they may be cross-linked in the core19 or in the shell.20 In this case, we have nanogel based on core-cross-linked micelle. Lou et al.21 have reported glycopolymer nanogel having disulfide linkage for stimuli responsive delivery of anticancer drug synthesized via atom-transfer radical polymerization (ATRP) process. It has been reported that the cancer cell has higher glutathione (GSH) content as compared to the normal cells.22 Therefore, utilization of the disulfide bonds in the anticancer drug career system provides a liberty of specific delivery of the drug to the cancer cells as the presence of GSH can accelerate the reduction of disulfide bonds leading to release of the drug. Herein, we have prepared a glycopolymer based tailor-made “core-shell” BCP poly[2(acrylamido) glucopyranose]-b-poly(furfuryl methacrylate) [PAG-b-PFMA] via reversible addition−fragmentation chain transfer (RAFT) polymerization technique followed by crosslinking of the core with a disulfide containing cross-linker via Diels−Alder (DA) “click” chemistry. Along with the target specific drug delivery, polymeric micelles with fluorescence activity can enhance its acceptability in biological applications due to a detailed understanding of the cellular uptake process of the drug. Several fluorescent probes, such as quantum dots (QDs),23 organic dyes,24 fluorescent protein,25 and rare-earth-doped nanoparticles,26 have been reported for cellular imaging. Recently, QDs have received enormous attention as biomarker owing to their narrow emission and high brightness.27 However, the toxicity of the inorganic QDs, such as CdS, CdSe, PbS, and Ag2S, is the major problem that limits their use for biomedical applications. Therefore, we are interested in the synthesis of QDs derived from the biological resource. Herein, we have prepared the QDs using gelatin, a green and natural material via hydrothermal process using the previously reported method by Liang et al.28 Gelatin is a combination of proteins and peptides and is obtained from partial hydrolysis of collagen extracted from the bones, skin, and connective tissues of animals. In this case, to prepare the targeted block copolymer PAG-bPFMA, a well-defined functional BCP poly(pentafluorophenyl acrylate)-b-poly(furfuryl methacrylate) (PPFPA-b-PFMA) having activated pentafluorophenyl ester group has been prepared via RAFT polymerization followed by the replacement of the activated pentafluorophenyl functionality by the glucosamine. The BCP was functionalized with gelatin QDs (GQDs) via EDC/NHS reaction between the terminal acid (−COOH) functionality of the RAFT reagent and the amine (−NH2) containing GQDs. The amphiphilic block copolymer was corecross-linked via DA reaction using dithio-bismaleimidoethane (DTME), a REDOX-responsive cross-linker. Doxorubicin (DOX), an anticancer drug, has been introduced in the core of the nanogel during the cross-linking of the core. The hydrophobicity of the drug molecules facilitates the encapsulation of the drug in the hydrophobic core of the nanogel, and presence of the hydrophilic glycopolymer shell imparts water solubility and biocompatibility. The anticancer activity of the drug loaded nanogel was assessed over MBA-MD-231, human breast cancer cell line, and monitored using confocal fluorescent microscopy and flow cytometric (FACS) analyses. Moreover, the noncytotoxicity of glycopolymer based nanogels has been confirmed via MTT assay test over MBA-MD-231 cell line.

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EXPERIMENTAL SECTION

Materials Used. 2,3,4,5,6-Pentafluorophenol (PFP), furfuryl methacrylate (FMA), 4-cyano-(phenylcarbonothioylthio) pentanoic acid (CPPA) (RAFT reagent), and 4,4′-azobis(4-cyanovaleric acid) (ABCVA) (thermal initiator) were purchased from Sigma-Aldrich. Acryloyl chloride, D-glucosamine hydrochloride, and cystamine dihydrochloride were supplied by Alfa Aesar. Maleic anhydride, 1,4dioxane, triethylamine (NEt3), dimethyl sulfoxide (DMSO), and dimethylformamide (DMF) were purchased from Merck. TrypsinEDTA and 10% FBS were procured from Himedia. Propidium iodide (PI), doxorubicin hydrochloride, and Dulbecco’s minimum essential medium (DMEM) were purchased from Sigma-Aldrich and Gibco, respectively. Rhodamin-phalloidin solution, 4′,6-diamidino-2-phenylindole (DAPI), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were supplied by Thermo Fischer Scientific. All the reagents were used as received without further purification. Synthesis of Pentafluorophenyl acrylate (PFPA). Pentafluorophenyl acrylate monomer has been synthesized following the procedure described by Woodfield et al.29 Typically, 2,3,4,5,6pentafluorophenol (3.1 g, 16.8 mmol), triethylamine (2.4 mL, 17.3 mmol), and dry dichloromethane (15 mL) were mixed in a 100 mL round-bottomed flask and placed in an ice bath. Acryloyl chloride (1.5 mL, 18.4 mmol) was then added dropwise at 0 °C, and the solution was vigorously stirred for 24 h at room temperature. After the completion of the reaction, the white precipitate of triethylammonium chloride salt, formed due to the esterification reaction, was taken out by the filtration, and the solid mass was washed with DCM (30 mL) to extract out the desired material. The DCM solution was treated with acidic water (10 mL, pH = 2.0) (twice) followed by a basic solution (20 mL, NaHCO3) (twice) and finally, twice with distilled water (30 mL). The organic phase was dried afterward with anhydrous sodium sulfate (Na2SO4), and then DCM was removed by the rotary evaporator to yield a pale yellow liquid with 80% yield. 19 F NMR (CDCl3, 400 MHz) δppm: −152.7 (m, 2 F, ortho), −158.2 (t, 1 F, para), −162.5 (m, 2 F, meta) (Figure S1). 1H NMR (CDCl3, 600 MHz) δppm: 6.70 (CH2, 1H, d), 6.36 (CH2, 1H, dd), and 6.16 (CH, 1H, dd) (Figure S2). Synthesis of Poly(pentafluorophenyl acrylate) (PPFPA). In a typical synthesis process, PFPA monomer (1 g, 4.2 × 10−3 mol), CPPA (39 mg, 1.4 × 10−4 mol), ABCVA (9.8 mg, 3.5 × 10−5 mol), and 1.5 mL of dry 1, 4-dioxane were added to a Schlenk tube equipped with a stir bar. The tube was sealed with a silicone septum, and the solution was purged with nitrogen gas for 30 min to remove the dissolved oxygen. The tube was then immersed into a thermostated oil bath at 70 °C for 6 h. After 6 h, the reaction mixture was placed on an ice bath to stop the reaction, and the product was precipitated in methanol. The final product was obtained via successive stage of centrifugation followed by drying under vacuum at room temperature. The precipitation and the reprecipitation methods were used thrice to purify the synthesized product. The pink powdery product was obtained as a final product (yield = 75%). 19 F NMR (CDCl3, 400 MHz) δppm: −150.3 and −151.3 (2 F, ortho), −156.9 (1 F, para), −162.1 (2 F, meta). 1H NMR (CDCl3, 600 MHz) δppm: 3.10 (broad, CH2CH), 2.51 (broad, CH2CH), 2.13 (broad, CH2CH). FT-IR (υ/ cm−1) = 1782 (>C=O stretching of PPFPA) and 1516 (>C=C< stretching of aromatic ring of PPFPA). Synthesis of Poly(PFPA)-b-Poly(FMA) (PPFPA-b-PFMA). PPFPA, macro-RAFT agent (0.22 g, 4.77 × 10−5 mol), was dissolved in 6 mL of dry 1,4-dioxane in a reaction tube equipped with a magnetic stirring bar. The monomer FMA (0.19 g, 1.14 × 10−3 mol) and initiator ABCVA (3.4 mg, 1.19 × 10−5 mol) were added to the reaction tube. The tube was sealed with a silicone septum, and the solution was purged with nitrogen gas for 30 min before being placed in an oil bath preheated at 70 °C. Polymerization reaction was conducted for 12 h, and the reaction mixture was placed in an ice bath to stop the reaction. The polymer solution was precipitated in nhexane, centrifuged, and finally dried under vacuum at room temperature. The dried copolymer was then dissolved in THF and precipitated again in n-hexane. This procedure was repeated two 2588


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ACS Applied Bio Materials times. The yellow powdery product was obtained as 92% yield. 1H NMR (CDCl3, 600 MHz) δppm: 7.5 (1H, −CH−O− of furan ring), 6.4 (2H, =CH−CH= of furan ring), 4.9 (2H, O−CH2− of PFMA), 3.10 (CH2−CH of PPFPA segment), 2.51, 2.13 (CH2−CH of PPFPA segment), 1.25−1.6 (different aliphatic protons of the methacrylate unit). FT-IR (υ/ cm−1) = 1782 (>C=O stretching of PPFPA segment), 1727 (>C=O stretching of PFMA segment), 1516 (>C=C< stretching of aromatic ring of PPFPA segment). Preparation of PAG-b-PFMA via Postpolymerization Modification of PPFPA-b-PFMA by Glucosamine. Postpolymerization modification of (PPFPA-b-PFMA) by glucosamine was performed following the procedure described by Boyer et al.30 with a slight modification. In a typical synthesis process, (PPFPA-b-PFMA) (0.2 g, 4.79 × 10−4 mol of PFPA) was dissolved in 4 mL of DMF. Gluosamine solution was prepared by dissolving glucosamine hydrochloride salt (0.13 g, 6.0 × 10−4 mol) in water (1.5 mL), and triethylamine (90 μL, 6.5 × 10−4 mol) was then added to the glucosamine hydrochloride solution to deprotonate the amine group. The glucosamine solution was then added slowly to the (PPFPA-bPFMA) solution to avoid sudden precipitation of the polymer. The solution was stirred for 6 h at room temperature. The polymer was dialyzed (MWCO = 3500 Da) against water for 48 h to remove any excess of glucosamine and was freeze-dried to achieve the final product. 1H NMR (DMSO-d6, 600 MHz) δppm: 7.98 (1H, −NH of PAG segments), 7.6 (1H, −CH−O− of furan ring), 6.5 (2H, =CH− CH= of furan ring), 4.9 (2H, O−CH2− of PFMA, anomeric H of glucosamine), 3.6 and 3.1 (6H of glucosamine). FT-IR (υ/ cm−1) = 3300 (br, hydroxyl groups of PAG segments and −NH stretching of amide) 1727 (>C=O stretching of PFMA segment), 1652 (amide >C=O stretching), 1570 (−NH bending of amide). Synthesis of Gelatin Quantum Dots (GQD). Gelatin QD has been synthesized according to the procedure reported in literature.28 Typically, 0.4 g of gelatin was dissolved in 20 mL of water and the clear solution was poured into a stainless steel hydrothermal with a Teflon liner of 50 mL capacity and heated at 160 °C for 4 h. After cooling the reactor at room temperature, the resulting light yellow solution was centrifuged at 8000 rpm for 30 min to remove the impurity (precipitate and agglomerated particles) to get a light brown colored aqueous solution of GQDs. Preparation of GQD Tagged BCP (GQD-BCP) via Attachment of GQD with PAG-b-PFMA. In a typical synthesis process, GQD solution (2 mL, solid content 14 mg/mL), EDC (2.6 mg), and NHS (1.5 mg) were added to PBS solution (pH = 7.4, 1 mL), and the reaction mixture was allowed to stir for 30 min at room temperature. Subsequently, the glycopolymer solution (10.0 mg in 10 mL of PBS) was added dropwise to the above PBS solution, and the reaction mixture was stirred at room temperature for 24 h.31 Then the resulting mixture was dialyzed (MWCO 3500) against water for 48 h to remove the undesired side products. Synthesis of Cross-Linker Dithiobis(maleimido)ethane (DTME). The REDOX cross-linker was synthesized using our previous report.32 1 H NMR (CDCl3, 600 MHz) δppm: 6.70 (−CH=CH−), 3.84 (N− CH2−CH2), and 2.92 (−CH2−CH2−S). Preparation of DOX Loaded Core-Cross-Linked (CCL) Micelle of PAG-b-PFMA. In a typical method, DOX·HCl was dissolved in water (1 mg/mL), and sodium hydroxide (NaOH) was added at a molar ratio of 1:3. The solution was stirred in dark condition overnight at room temperature to obtain neutralized DOX. The resulting salt was removed by ultracentrifugation method, and the rest of the solution was lyophilized. A stock solution of DOX (1 mg/ mL) in DMSO has been prepared for further experiments. In a typical procedure, DOX (0.75 mL solution, 0.75 mg), DTME (0.40 mg), and PAG-b-PFMA (20.0 mg) were dissolved in 1 mL of DMSO in a 25 mL round bottomed flask. PBS (pH 7.4, 5.0 mL, 10 mM) was added dropwise to the solution under vigorous stirring for 1 h at room temperature. Then the solution was heated to 60 °C for 12 h to crosslink the micellar core via Diels−Alder (DA) reaction. After 12 h, the resultant solution was transferred into a dialysis bag (MWCO, 3500 Da) and dialyzed against Milli-Q water for 24 h with continuous

changing of water after every 2 h. The DOX loading amount in the nanoparticles was determined by using UV−Visible spectroscopy with a standard calibration curve at 485 nm.33 The drug loading content (DLC) and drug-loading efficiency (DLE) were calculated by the following equations:34 DLC (%) =

DLC (%) =

weight of loaded drug in nanoparticles × 100 weight of drug − loaded nanoparticles

weight of loaded drug in nanoparticles × 100 amount of drug used to load in the nanoparticles

All the experiments were carried out in triplicate, and the average data were presented. MTT Colorimetric Assay. The cytotoxicity assay was done following the protocol described by Kumar et al.35 MDA-MB-231 cells were harvested using Trypsin-EDTA (Himedia, TCL144). Cells were counted using hemocytometer. Then the cells were seeded in a 96-well plate. The cell density was about 5000 cells/well. Cells were grown in high glucose DMEM medium (Gibco, 12800−017) with 10% FBS (Himedia, RM9955). After cells were about 75% confluent, media were changed to incomplete DMEM medium for treatment. Nanogel and DOX loaded nanogel were initially dissolved in DMSO and sonicated to form a uniform emulsion. Then they were further dissolved in the incomplete DMEM medium. The final concentration of nanogel was 500 μg/mL, and drug-containing nanogels were adjusted to Doxorubicin concentration of 50 μM. Plates were treated as per following concentration, in triplicate: (a) Only nanogel: 500 μg/mL to 1.95 μg/mL serially diluted. (b) DOX containing nanogel: DOX equivalent dose of 50 μM to 0.024 μM serially diluted. (c) DOX: dose of 50 μM to 0.024 μM serially diluted. The plate was incubated for 48 h, and the incomplete medium was replaced by 100 μL of MTT reagent (Thermo Fischer Scientific, M6494) per well (1 mg/mL). Reading was taken after four h at 550 nm wavelength in a microplate reader (Biorad) to determine the cytotoxicity. The plot of log inhibitor versus normalized response analysis was made to determine the IC50. All statistical analysis was done in Graph Pad Prism. For cytotoxicity of Human dermal fibroblast cells, HiFi adult dermal fibroblasts (HADF) cells were procured from Himedia. The cells were cultured similar to the MBA-MD-231 cells, and all the procedures were similar to the cytotoxicity assay of MBA-MD-231 cell line. Flow Cytometric Analysis (FACS). For flow cytometry analysis, we followed the previously mentioned procedures by Pal et al.36 About 104 MDA-MB-231 cells were seeded in a 100 mm petri dish and cultured with high glucose DMEM media with 10% FBS and 1% antibiotic solution containing 10 000 units/mL of penicillin, 10 000 μg/mL of streptomycin, and 25 μg/mL of amphotericin B in a CO2 incubator at 37 °C. After the cells reached 75% confluence, they were treated with nanogel at 3.6 μg/mL dose. The equivalent dose of doxorubicin was around 900 nM. The control was treated with 1% DMSO. The cells were incubated with nanogel for 24, 48, and 72 h. Then the cells were harvested with Trypsin-EDTA. The cell number was counted, and around 105 cells were washed twice with sterile PBS. Then the cells were fixed with chilled 70% ethanol and kept at −20 °C overnight (12 h). Next, the cells were washed three times with sterile PBS and stained with 1 mL of staining solution containing 10 μg/mL PI (Sigma, P4170) and 1 μg/mL RNase-A (Sigma, R6148) and incubated for 15 min in the dark. The stained cells were analyzed in FACS Caliber form BD, and the data were analyzed later using flowing software. Cell Uptake Study through Fluorescent Optical Microscopy. About 103 cells were seeded on a sterile coverslip and cultured in a 33 mm uncoated petridish containing high glucose DMEM media with 10% FBS and 1% antibiotic solution having 10 000 units/mL of penicillin, 10 000 μg/mL of streptomycin, and 25 μg/mL of amphotericin B in a CO2 incubator at 37 °C. After the cells reached 75% confluence, they were treated with nanogel at 3.6 μg/mL dose. 2589


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Scheme 1. Schematic Representation of Synthesis of (a) Poly(pentafluorophenyl acrylate) Homopolymer (Macro-RAFT Reagent) and Block Copolymer Poly(pentafluorophenyl acrylate)-b-poly(furfuryl methacrylate) (PPFPA-b-PFMA), (b) Poly[2-(acrylamido) glucopyranose]-b-poly(furfuryl methacrylate) (PAG-b-PFMA) via Postpolymerization Modification of PPFPA-b-PFMA and Gelatin QD Tagged PAG-b-PFMA

The control was treated with 1% DMSO. The cells were incubated with nanogel for 8, 16, and 24 h, and after that, the media were discarded, and the cells were carefully washed with sterile PBS three times. Then the cells were fixed with 4% formaldehyde solution for 15 min at room temperature. Then the cells were permeabilized with 1% Tittron-X solution for 10 min. Next, the cells were stained with the

rhodamin−phalloidin solution (Thermo Fischer Scientific, R415) (RH-P) following manufacturer’s instruction. Next, the cells were counterstained with DAPI (4′,6-diamidino-2-phenylindole, Thermo Fischer Scientific catalogue no. D1306) following manufacturer’s instruction. Microscopy was done with a laser scanning confocal microscope (Model NO- Olympus Fluoroview, FV 100) at 60× 2590


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Figure 1. FTIR analysis of (a) homopolymer and block copolymers, (b) PAG20-b-PFMA32, and the core-cross-linked product.

Table 1. Summary of Block Copolymers, PPFPA-b-PFMA Prepared Using PPFPA20 and PPFPA27 as Macro-RAFT Agenta SI. no.

sample composition

[M]/macro-CTA/[I]

Mn/theo (g/mol)

Mn/GPC (g/mol)

PDI

conv (%)

1 2 3 4

PPFPA20-b-PFMA20 PPFPA20-b-PFMA32 PPFPA27-b-PFMA29 PPFPA27-b-PFMA37

96:4:1 152:4:1 144:4:1 192:4:1

8788 11 114 12 382 14 376

8100 10 100 11 200 12 500

1.29 1.37 1.35 1.43

92 90 90 87

Solvent = 1,4-dioxane, time = 12 h; temp. = 70 °C; (Mn/GPC of PPFPA20 = 4800 g mol−1; Mn/GPC = 6300 g mol−1).

a

Figure 2. 1H NMR of poly(pentafluorophenyl acrylate)-b-poly(furfuryl methacrylate) (PPFPA20-b-PFMA32) BCP in CDCl3.

PPFPA, was characterized by 1H NMR, 19F NMR, FT-IR, and GPC analyses. 19 F NMR spectrum of PPFPA showed broad peaks in the regions −150.3 and −151.3 (2 F, ortho), −156.9 (1 F, para), −162.1 (2 F, meta) for the different fluorine atoms attached to the phenyl ring (Figure S3). 1H NMR (Figure S4) shows characteristic proton resonances of the homopolymer PPFPA at δ = 3.1 ppm (labeled as 4) and the RAFT phenyl peaks at δ = 7.4−7.5 ppm (labeled as 2, 3) and methylene (−CH2− CH2−) at δ = 2.7 (labeled as 6, 7). The resonances designated

magnification. The images were further analyzed for uptake of the particles in a time-dependent manner.

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RESULTS AND DISCUSSIONS Herein, we have described a new method of designing biocompatible fluorescence active nanogel and their utilization in the anticancer drug delivery (Scheme 1). For this, poly(pentafluorophenyl acrylate) (PPFPA) was synthesized via RAFT polymerization using CPPA as a RAFT reagent and ABCVA as a thermal initiator. The resulting homopolymer, 2591


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Figure 3. 1H NMR of PAG20-b-PFMA32 in DMSO-d6.

Figure 4. (a) HRTEM image of the GQD; (b) photoluminescence (PL) study of the synthesized gelatin QD (GQD) and GQD tagged block copolymer (GQD-BCP).

using the peaks designated as ‘11’ and ‘4’ and GPC analyses (Mn/NMR = 10 100 g mol−1, Đ= 1.37). The appearance of new absorption band at 1727 cm−1 (ester group vibration band of PFMA segments) along with the characteristic vibration bands of PPFPA segments further confirms the successful incorporation of the PFMA segment in the BCP (Figure 1a). However, the characteristic bands of furan moieties at 1510 and 1012 cm−1 due to furan ring stretching and breathing, respectively, were merged with the FT-IR bands of the homopolymer PPFPA. The pentafluorophenyl ester (activated ester) moieties are well-known for the postpolymerization modification to introduce several functional groups in the polymer backbone.37 In this case, the BCP, (PPFPA-b-PFMA) containing the activated pentafluorophenyl ester group, was reacted with glucosamine hydrochloride in the presence of triethylamine to obtain the amphiphilic BCP, PAG-b-PFMA. The successful postpolymerization modification of pentafluorophenyl ester group by glucosamine was confirmed via 1H, 19F NMR, and

as ‘2, 3’ and ‘4’ have been used to calculate the number-average molecular weight (Mn/NMR = 5639 g mol−1), which is comparatively higher than the Mn obtained from GPC analysis (Mn/GPC = 4800 g mol−1, Đ = 1.13) (Figure S5). The strong absorption bands in the FT-IR spectrum at 1782 and 1516 cm−1 are attributed to the >C=O stretching of activated pentafluorophenyl ester and >C=C< stretching of the pentafluorophenyl group, respectively (Figure 1a). This PPFPA macro-RAFT agent was utilized to polymerize FMA to synthesize the BCP, (PPFPA-b-PFMA) of different block lengths (Table- 1). Figure 1b designates the FTIR analysis of the block copolymer (PAG20-b-PFMA32) and the respective core-cross-linked product. Figure 2 represents the 1H NMR spectrum of the PPFPA20b-PFMA32 BCP. The appearance of the new peaks at δ = 7.4, 6.3, and 5.0 ppm (characteristic peaks of PFMA) confirms the presence of PFMA in the BCP. The molecular weight of the BCP was calculated by 1H NMR (Mn/NMR = 9619 g mol−1) 2592


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ACS Applied Bio Materials Scheme 2. Schematic Representation of DOX Loading and Successive Cross-Linking of PAG-b-PFMA

FT-IR analyses. The disappearance of the peak at 1782 cm−1 (activated ester absorption band) and appearance of a new absorption band at 1652 cm−1 (amide carbonyl stretching) along with the broad band at 3300 cm−1 (hydroxyl groups and stretching of amide −NH of PAG segments) suggests the successful incorporation of glucosamine moieties by replacing the PPFPA segments (Figure 1a). Furthermore, the primary amine functionality of glucosamine will cleave the RAFT endgroup to thiol.30 The emergence of a new broad band at 2520 cm−1 suggests the formation of the thiol (Figure 1b). Disappearance of the resonance at δ = 3.1 ppm (characteristic peak of PPFPA segment) and the emergence of new resonances at the region δ = 7.98 ppm and δ = 3.5 ppm (characteristic peaks of PAG segments) in the 1H NMR spectrum of PAG20-b-PFMA32 also confirms the successful modification of PPFPA20-b-PFMA32 to PAG20-b-PFMA32 (Figure 3). Furthermore, the 19F NMR spectrum of PAG20b-PFMA 32 did not exhibit any characteristic peak of pentafluorophenyl moieties (Figure S6), suggesting complete replacement of PPFPA segment by glucosamine. The schematic representation of the synthesis of block copolymer is shown in Scheme 1.

To make our system a cell tracer, we have introduced fluorescence active GQDs in our polymeric nanogel system. Recently, QDs have received tremendous attention as biomarkers due to their narrow and bright emission. Pei et al.31 reported a carboxylic capped CdTe QD attached with glycopolymer via an EDC-NHS coupling reaction. However, the metal based inorganic QDs suffer from toxicity problem, which limits their uses in bioapplications. In this study, we have prepared a biocompatible QD based on gelatin (GQD), which was coupled with the RAFT end carboxylic acid group (−COOH) of the amphiphilic BCP via EDC-NHS coupling reaction in PBS buffer. Size of the GQD is calculated to be 5 ± 2 nm from TEM analysis (Figure 4a). Successful conjugation of GQD with amphiphilic BCP (GQD-BCP) was confirmed by photoluminescence study (Figure 4b) and DLS analysis (Table S1). It showed larger particle size for GQD conjugated glycopolymer micelle as compared to the free micelle. Both GQD and GQD conjugated BCP showed bluish green fluorescence under a fluorescent lamp having excitation wavelength (λex) of 350 nm. In the emission (λem) spectra, the red shifting of emission maxima (λmax) from 519 nm (for GQD) to 532 nm (for GQD conjugated BCP) and the decrease in fluorescence intensity (fluorescence quenching) of 2593


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Figure 5. 1H NMR of core-cross-linked PAG20-b-PFMA32 in DMSO-d6.

GQD conjugated BCP are due to the reaction between the GQD and the amphiphilic BCP.31 Particle sizes of PAG27-bPFMA29 and PAG27-b-PFMA37 (Table S1) were already much bigger, and henceforth, these two amphiphilic BCPs were not further modified by GQD for its use in cell study. The QD modified amphiphilic BCP (GQD-BCP) had selfassembled in water to form micelle having a hydrophobic core (PFMA segment) and hydrophilic corona (glycopolymer segment). The core cross-linking strategy provides stability to the micelle by preventing their dissociation during blood circulation process. In this case, the core of the GQD-BCP was cross-linked using dithiobismaleimidoethane (DTME) via DA reaction between the furan and the maleimide moieties of the BCP and DTME, respectively (Scheme 2). The CCL micelle (nanogel) was characterized by FT-IR, DLS, TEM, and DSC analyses. 1H NMR analysis of the nanogel was performed using DMSO-d6 as solvent. The characteristics peaks of PFMA segments of the nanogel disappeared, whereas the peaks corresponding to the PAG segments (designated as 4, 5, 14, 15, 16, 17, 18, 18′) were still present in the 1H NMR spectrum (Figure 5). This result can be attributed to the fact that due to the formation of the CCL micelle via DA reaction, the cross-linked PFMA core was insoluble in the solvent (DMSO-d6) and its characteristics resonance peaks were absent in the 1H NMR spectrum.38 The FT-IR analysis also supported the formation of nanogel via DA reaction. A new shoulder was observed at 1704 cm−1 in the FT-IR spectrum (Figure 1b) along with the peak at 1727 cm−1 (>C=O stretching of PFMA segments). Kavitha et al.39 reported, the formation of this shoulder can be attributed to the different carbonyl environment caused by the DA reaction between the PFMA and bismaleimide of DTME. Thermoreversible behavior of the nanogel was studied by DSC analysis (Figure 6). The nanogel was heated from −30 °C to +200 °C at a rate of 10 °C/min. Emergence of a broad endotherm at about 141 °C in the heating curve indicated retro Diels−Alder (rDA) reaction. In the cooling curve, a broad exotherm ranging from 106 to 74 °C suggested the DA

Figure 6. DSC thermogram of core-cross-linked nanogel.

reaction, that is, the reformation of DA adduct between PFMA segment and DTME via [4 + 2] cycloaddition reaction.32,38,40 Particle size and distribution index (PDI) of the nanogel and non-CCL micelle were measured by DLS analysis. It was observed that nanogel and non-CCL micelle from PAG20-bPFMA32 showed a particle size of 235 ± 5 nm (PDI = 0.359) and 197 ± 5 nm (PDI = 0.322), respectively, as shown in Figure 7a and b. The reduction in the particle size of nanogel with respect to the non-CCL micelle is attributed to the better compactness of the nanogel after cross-linking. TEM images of the non-CCL (diameter = 150 ± 10 nm) (Figure 7c) and nanogel (diameter = 80 ± 10 nm) (Figure 7d) also support the particle size data obtained from DLS analyses. The nanogel and non-CCL micelles have spherical morphology as observed from TEM images. However, a relatively lower diameter for the nanogel and non-CCL micelle was observed as compared to the diameter obtained from DLS. This discrepancy in particle size is attributed to the fact that DLS analysis has been performed for the solvated micelle, whereas TEM images were taken for the dry samples.41 2594


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Figure 7. Particle size distribution of PAG20-b-PFMA32 (a) noncore-cross-linked micelle, and (b) nanogel; HRTEM analysis of PAG20-b-PFMA32 (c) noncore-cross-linked micelle and (d) nanogel.

Figure 8. Drug release kinetics study of R116 (PAG20-b-PFMA32) and the DLC/DLE values for the core-cross-linked micelle (nanogel) R115 (PAG20-b-PFMA20) and R116 (PAG20-b-PFMA32).

The presence of −S−S− linkage in the cross-linker imparts REDOX-responsive nature to the nanogel. It would cleave under reductive environment to release the drug in cancer cell, which has high glutathione (GSH) content.42,43 Dithiothreitol (DTT) is a well-known reducing agent that specifically cleaves −S−S− linkage.44 REDOX-responsive nature of the nanogel was analyzed by treating them with 10 mM DTT solution. An increase in the particle size was observed due to the cleavage of disulfide (−S−S−) linkage in the core, which was confirmed by DLS analysis (Figure S7).

A hydrophobic anticancer drug, Doxorubicin (DOX), was chosen as a model anticancer drug to be loaded into the hydrophobic core of the nanogel. The drug loading capacity (DLC) of the nanogel was studied by varying the polymer chain length (Table shown in Figure 8). It was observed that DLC increased with the increase in hydrophobic segment (PFMA segment) of the nanogel. This is due to the greater hydrophobic interaction between the drug and the core, as DOX is a hydrophobic cancer drug.33,45,46 Therefore, we have experienced a higher amount of DLE in case of PAG20-bPFMA32 (R116, 96 wt %) compared to the PAG20-b-PFMA20 2595


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Figure 9. Cytotoxicity study of the (a) DOX loaded nanogels (R115, R116) and (b) pristine nanogels (R117, R118) against MBA-MD-231, breast cancer cell line; flow cytometric analysis of (c) control and DOX loaded nanogel [R116 (PAG20-b-PFMA32)] at (d) 24 h, (e) 48 h and (f) 72 h.

DTT, a burst release of DOX has been observed, and the cumulative DOX release value reached to 46.9% within 10 h. This result can be explained by the rapid release of DOX due to the cleavage of −S−S− linkage of the core in the presence of DTT.49 We had carried out a comparative cytotoxicity study (in vitro) of the DOX-loaded and DOX-free nanogels that would indirectly give us an information about the release of DOX from the nanogel inside cancer cell. From the MTT assay result of the DOX loaded nanogels having a composition of R116 (PAG20-b-PFMA32), it was found that IC50 (concentration of drug at which 50% of the target is inhibited) value has been achieved at a concentration of 0.908 μM drug concentration, present in 14.08 μg/mL of nanogel, whereas free Doxorubicin drug showed IC50 at a concentration of 0.631 μM drug concentration (Figure 9a and Table S2). To get an idea about the cytocompatibility of the pristine polymeric nanogel (R118), MTT assay of the nanogel has also been carried out against the MBA-MD-231 cell line, and it has been found that even up to a concentration of 16 μg/mL, it is nontoxic to the cancer cell (approximately 75% cell viability) (Figure 9b). However, at higher concentrations, both the carriers showed some amount of cytotoxicity, which can be neglected as the actual dose of the carriers required for the treatment is below this range (Table S2). To check whether the cytotoxicity effect is present in normal cells, we also treated human dermal fibroblast cells with R116 (DOX loaded nanogel PAG20-b-PFMA32) and R118 (pristine nanogel

based composition (R115, 80 wt %) (Figure 8). This has motivated us to choose R116 as a reference for further application. Comparative study of the IC50 value of the PAG20b-PFMA20 (1.08 μM) and PAG20-b-PFMA32 (0.908 μM) (Figure 9a) based nanogel also shows the superiority of the PAG20-b-PFMA32 (R116) based nanogel composition over the PAG20-b-PFMA20 (R115) based nanogel composition. As per the literature, it is understood that the tumor cells are more acidic (pH ≈ 5.0) and have higher intracellular GSH content (2−10 mM) as compared to the healthy cells (pH= 7.4).47 Here, we have investigated the in vitro drug release study for the nanogel and non-CCL micelle at different pH values in the presence and absence of DTT (Figure 8) to mimic the physiological conditions encountered in the healthy as well as cancer cells. At physiological pH, cumulative DOX release from nanogel and non-CCL micelle is 36% and 39.6%, respectively. At pH = 5.0, the amount of DOX release increased for both the nanogel and non-CCL micelle and reached up to 45.4% and 49.6%, respectively. The enhanced release of DOX at lower pH can be attributed to the higher solubility of DOX due to the protonation of the −NH2 of DOX.48 Again, because of better compactness, cumulative DOX release percentage is lower for nanogel for both the pH conditions. The drug release percentage of the non-CCL micelle at pH = 5.0 in the presence of DTT was almost same as that of non-CCL micelle at pH = 5.0, which suggested no effect of DTT on non-CCL micelle, which is due to the absence of thiol linkage. At pH = 5.0 in the presence of the 2596


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ACS Applied Bio Materials PAG20-b-PFMA32). The MTT study showed that the cytotoxic effect was only present at a higher concentration (125 μg/mL for R116 and 500 μg/mL for R118) (Figure S8). However, this was also negligible since the actual dose of the carriers for cytotoxicity on cancerous cells were also below this range (Table S2). As mentioned earlier, the IC50 value of R116 on MBA-MD-231 cell line was found to be 0.908 μM drug concentration (present in 14.08 μg/mL of nanogel), whereas the IC50 value of R116 on human dermal fibroblast cells was found to be 125 μg/mL of nanogel, which indirectly indicated the target specificity of the glycopolymer nanogel. From the MTT assay, it was found that IC50 value of R116 (0.908 μM) is lower compared to the IC50 value of R115 (1.08 μM). Therefore, we have carried out the rest of the biological analysis using R116 composition. To identify the change in the apoptotic populations, we conducted cell cycle analysis according to the procedure described in the materials and methods (Figure 9c−f). The phases of the cell cycle were denoted in the control group, which served as the reference point for the nanogel alone (Figure 9c) or containing DOX (Figure 9d−f). The sub G0 phase indicates the apoptotic cell fraction in cell cycle analysis by the propidium iodide (PI). The increase in the sub G0 fraction also suggests the efficiency of a drug/nanoparticle to induce apoptosis in a cell population.26 Here we have observed a massive increase in sub G0 fraction in R116 (PAG20-bPFMA32) composition after 48 and 72 h of incubation along with a steady decrease in all other fractions (G0/G1, S, and G2M). The quantitative evaluation of the obtained data has been summarized in Figure S9. There was not much change in the pristine nanogel after 72 h of incubation (Figure S10). Hence, we can infer that the DOX-loaded nanogel induces apoptosis in breast cancer cell line (MDA-MB-231). To monitor the in vitro cell uptake of our synthesized fluorescence active nanogel, we have carried out the confocal microscopy analysis after the treatment of the MDA-MB-231 cells with pristine nanogel and DOX loaded nanogel. For the study of cell viability and apoptosis of pristine nanogel and DOX loaded nanogel treated MDA-MB-231 cells, we have double stained the cells with 4′,6-diamidino-2-phenylindole (DAPI) and rhodamin−phalloidin solution. DAPI is able to stain the cell nucleus, whereas rhodamin−phalloidin stain is used to stain actin filaments of the cell, which form bulk of the cellular cytoskeleton. We observed that there were changes in the cellular morphology after 24 h of the treatment with DOX loaded nanogel. The cellular cytoskeleton arrangement was disrupted and the cells seemed to have nuclear fragmentation (Figure 10). This indicated induction of the early stage of apoptosis. However, cell death was not clearly evident as the incubation period was shorter than the flow cytometry study. The study also delineated the cellular uptake of the nanogel by breast cancer cells. Utilizing the green fluorescence of the nanoparticles, we were able to identify the particles inside the cells by confocal microscopy. Wherever the nanoparticles were taken up by the cells, the green fluorescence of the nanogel and red fluorescence of the rhodamin coincided and gave a yellow fluorescence. We observed that the yellow fluorescence was maximum on 24 h time point, which indicated that maximum uptake occurred by this time. Since cytotoxic effect was also observed at this point of time, we can assume that the doxorubicin was released from the nanogel after being taken up by the cells. As a control, we did carry out the cell uptake study

Figure 10. Time dependent cell uptake study of the DOX loaded fluorescence active nanogel via confocal microscopy analysis. The blue panel denotes nuclear staining by DAPI, red panel denotes cytoskeletal staining by rhodamin−phalloidin, and the green panel denotes the nanoparticles. A merged panel is given and the yellow color in the merged segment denotes the areas where the nanogel has entered the cell (where green and red fluorescence is overlapping).

of the pristine nanogel devoid of DOX drug, and the obtained results have been represented in Figure S11.

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CONCLUSIONS In conclusion, a fluorescence active nanogel based on glycopolymer based nanogel was prepared via RAFT polymerization technique and Diels−Alder (DA) “click” chemistry. In this case, a functional block copolymer containing activated pentafluorophenyl and furfuryl group (PPFPA-b-PFMA) was prepared followed by the replacement of pentafluorophenyl with the glucosamine to prepare the PAG-b-PFMA. To introduce the fluorescence activity in the nanogel, the terminal acid (−COOH) functionality of the RAFT agent was modified by gelatin QDs (GQDs), a biocompatible material. Anticancer drug, doxorubicin, was introduced into the micellar system via successive drug loading and cross-linking with dithiobismaleimidoethane (DTME), a REDOX responsive crosslinker via DA “click” reaction. A human breast cancer cell MBA-MD-231 was taken as a model cell line and the anticancer activity of the nanogel over the mentioned cell line was monitored using MTT assay, and FACS analysis. The cell uptake study of the DOX loaded fluorescence active nanogel was monitored using fluorescence confocal microscopy analysis. Integration of all the obtained results indicates that this material has the potential for the targeted anticancer activity.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsabm.9b00267. Characterization procedures; 1H and 19F NMR of monomer and polymers; GPC; DLS analysis; MTT assay of drug loaded and pristine nanogel over human dermal fibroblast cell; % cell death due to drug loaded nanogel in flow cytometric analysis; FACS analysis of pristine nanogel; cell uptake study of pristine nanogel; table of particle size analysis of amphiphilic BCPs; 2597


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(14) Liu, L.; Zhang, J.; Lv, W.; Luo, Y.; Wang, X. Well defined pH sensitive block glycopolymers via reversible addition−fragmentation chain transfer radical polymerization: Synthesis, characterization, and recognition with lectin. J. Polym. Sci., Part A: Polym. Chem. 2010, 48 (15), 3350−3361. (15) Ting, S. S.; Gregory, A. M.; Stenzel, M. H. Polygalactose containing nanocages: the RAFT process for the synthesis of hollow sugar balls. Biomacromolecules 2009, 10 (2), 342−352. (16) Chen, W.; Meng, F.; Cheng, R.; Deng, C.; Feijen, J.; Zhong, Z. Biodegradable glycopolymer-b-poly (ε-caprolactone) block copolymer micelles: versatile construction, tailored lactose functionality, and hepatoma-targeted drug delivery. J. Mater. Chem. B 2015, 3 (11), 2308−2317. (17) Talelli, M.; Barz, M.; Rijcken, C. J.; Kiessling, F.; Hennink, W. E.; Lammers, T. Core-crosslinked polymeric micelles: principles, preparation, biomedical applications and clinical translation. Nano Today 2015, 10 (1), 93−117. (18) Yadav, H.; Al Halabi, N.; Alsalloum, G. Nanogels as Novel Drug Delivery Systems-A Review. J. Pharm. Pharm. Res. 2017, 1 (1), 5. (19) Garcia, F. P.; Rippe, M.; Companhoni, M. V.; Stefanello, T. F.; Louage, B.; Van Herck, S.; Sancey, L.; Coll, J.-L.; De Geest, B. G.; Nakamura, C. V.; et al. A versatile method for the selective corecrosslinking of hyaluronic acid nanogels via ketone-hydrazide chemistry: from chemical characterization to in vivo biodistribution. Biomater. Sci. 2018, 6 (7), 1754−1763. (20) Thurmond, K. B., II; Huang, H.; Clark Jr, C. G.; Kowalewski, T.; Wooley, K. L. Shell cross-linked polymer micelles: stabilized assemblies with great versatility and potential. Colloids Surf., B 1999, 16 (1−4), 45−54. (21) Lou, S.; Zhang, X.; Zhang, M.; Ji, S.; Wang, W.; Zhang, J.; Li, C.; Kong, D. Preparation of a dual cored hepatoma-specific star glycopolymer nanogel via arm-first ATRP approach. Int. J. Nanomed. 2017, 12, 3653. (22) Todorova, V. K.; Harms, S. A.; Kaufmann, Y.; Luo, S.; Luo, K. Q.; Babb, K.; Klimberg, V. S. Effect of dietary glutamine on tumor glutathione levels and apoptosis-related proteins in DMBA-induced breast cancer of rats. Breast Cancer Res. Treat. 2004, 88 (3), 247−256. (23) Jaiswal, J. K.; Goldman, E. R.; Mattoussi, H.; Simon, S. M. Use of quantum dots for live cell imaging. Nat. Methods 2004, 1 (1), 73. (24) Koch, A.; Reynolds, F.; Kircher, M.; Merkle, H.; Weissleder, R.; Josephson, L. Uptake and metabolism of a dual fluorochrome Tatnanoparticle in HeLa cells. Bioconjugate Chem. 2003, 14 (6), 1115− 1121. (25) Zhang, J.; Campbell, R. E.; Ting, A. Y.; Tsien, R. Y. Creating new fluorescent probes for cell biology. Nat. Rev. Mol. Cell Biol. 2002, 3 (12), 906. (26) Liu, M.; Wang, K.; Zhang, X.; Zhang, X.; Li, Z.; Zhang, Q.; Huang, Z.; Wei, Y. Fabrication of stable and biocompatible red fluorescent glycopolymer nanoparticles for cellular imaging. Tetrahedron 2015, 71 (34), 5452−5457. (27) Zhang, Y.; Wang, T.-H. Quantum dot enabled molecular sensing and diagnostics. Theranostics 2012, 2 (7), 631. (28) Liang, Q.; Ma, W.; Shi, Y.; Li, Z.; Yang, X. Easy synthesis of highly fluorescent carbon quantum dots from gelatin and their luminescent properties and applications. Carbon 2013, 60, 421−428. (29) Woodfield, P. A.; Zhu, Y.; Pei, Y.; Roth, P. J. Hydrophobically modified sulfobetaine copolymers with tunable aqueous UCST through postpolymerization modification of poly (pentafluorophenyl acrylate). Macromolecules 2014, 47 (2), 750−762. (30) Boyer, C.; Davis, T. P. One-pot synthesis and biofunctionalization of glycopolymers via RAFT polymerization and thiol−ene reactions. Chem. Commun. 2009, No. 40, 6029−6031. (31) Pei, D.; Li, Y.; Huang, Q.; Ren, Q.; Li, F.; Shi, T. Quantum dots encapsulated glycopolymer vesicles: Synthesis, lectin recognition and photoluminescent properties. Colloids Surf., B 2015, 127, 130−136. (32) Banerjee, S. L.; Bhattacharya, K.; Samanta, S.; Singha, N. K. Self-Healable Antifouling Zwitterionic Hydrogel Based on Synergistic

summary of amount of drug loaded nanogel used for cytotoxicity study (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Subhayan Das: 0000-0002-8628-5128 Mahitosh Mandal: 0000-0003-3861-3323 Nikhil K. Singha: 0000-0003-0935-0157 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are thankful to Prof. Sudip K Ghosh, Department of Biotechnology, IIT Kharagpur for the support in confocal microscopy analysis. K.B. is also grateful to UGC, New Delhi for providing the fellowship.

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REDOX Responsive Fluorescence Active Glycopolymer Based

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