New Magneto-Fluorescent Hybrid Polymer Nanogel for Theranostic

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New Magneto-Fluorescent Hybrid Polymer Nanogel for Theranostic Applications Vineeth M Vijayan, Ansar Ereath Beeran, Sachin J Shenoy, Jayabalan Muthu, and Vinoy Thomas ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00616 • Publication Date (Web): 09 Jan 2019 Downloaded from http://pubs.acs.org on January 12, 2019

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New Magneto-Fluorescent Hybrid Polymer Nanogel for Theranostic Applications

* Vineeth M. Vijayan a,b,c, Ansar Ereath Beerand, Sachin J Shenoye, Jayabalan Muthuc , and *

Vinoy Thomasa,b aDepartment

of Materials Science and Engineering, Polymers & Healthcare

Materials/Devices, University of Alabama at Birmingham, 1150 10th Avenue South, Birmingham, AL 35233, United States bCenter

for Nanoscale Materials and Biointegration (CNMB), University of Alabama at Birmingham, 1300 University Blvd. CH 386 Birmingham, AL 35294, United States Sree Chitra Tirunal Institute for Medical Sciences and Technology, Science Division, 4Bioceramics Laboratory,5 Division of In vivo models and testing. Biomedial Technology Wing, Kerala, India,

cPolymer

Tel: (205) 975-4098; E-Mail: [email protected], [email protected],[email protected] ABSTRACT:

Herein we have reported a new magneto-fluorescent nanogel built on

photoluminescent co-macromer [PEG-maleic acid-glycine], N, N Dimethyl amino ethyl methacrylate and citrate capped superparamagnetic iron oxide nanoparticles (SPION). The nanogel was found to have core-shell morphology (SPION core and PEG shell) with particle size around 80 nm. The cytocompatibility of the synthesized nanogel studied using MTT, live dead assays and flow cytometry. The cellular uptake of the nanogel on cervical cancer cell line Hela evaluated through Prussian blue staining and fluorescence microscopy have revealed good cancer cell imaging capability. Magnetic hyperthermia experiments have shown that the synthesized nanogel caused the lysis of cancer cells. The fluorescence bioimaging capability of the nanogel in murine model has shown good near IR imaging capability. Overall, the reported results suggest that the magneto-fluorescent nanogel shows promising future potential for cancer theranostic applications. Key words: Magneto-fluorescent, theranostics, nanogel


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1. Introduction Multifunctional nanoparticle based imaging agents have emerged as promising imaging tool as they can combine different imaging modalities such as optical and magnetic resonance imaging (MRI). Combination of these kinds of multiple imaging modalities in to single domain can significantly increase the sensitivity and detection limit of clinical diagnostics

1,2.

Developments of nanoformulations which can diagnose and treat tumors are

very important in nanomedicine. This concept was initially introduced by Funkhouser (2002), which have initiated a new era in nanomedicine known as theranostic nanomedicine.3 The reports on various research works on theranostics and challenges are presented elsewhere.4Different varieties of nanomaterials including iron oxide nanoparticles, quantum dots, gold nanoparticles and carbon nanotubes are employed for theranostic applications.5 Since these materials are equipped with various imaging techniques such as MRI and fluorescence optical microscopy, they can be efficiently utilized for tumor imaging. The therapeutic function can be mainly achieved through conjugation of anticancer drugs or genes to these materials. However the therapeutic strategy is not limited with this methodology alone, but some of these nanoparticles including gold and iron oxide nanoparticles exhibit unique capability to destroy cancer cells through

photodynamic therapy and magnetic

hyperthermia.6,7 When the size of iron oxide nanoparticles becomes lesser than 20 nm they exhibits super paramagnetic behavior known as superparamagnetic iron oxide nanoparticles (SPION).8 Among the different types of theranostic agents SPION has received special interest as they have inherent theranostic capability to perform both imaging and therapy through magnetic resonance imaging and hyperthermia effect.9 SPION can convert alternating high frequency magnetic field in to thermal energy. This thermal energy can be used for destroying cancer cells. This phenomenon is termed as magnetic hyperthermia.10 However SPION a have some


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major drawbacks such as toxicity, non biodegradability and short circulation half life in vivo 11-14.Hence

these issues have to be addressed efficiently to be used for biomedical

applications. Nanocarriers combining both magnetic and fluorescence characteristics are known as magneto-fluorescent nanocarriers.15 They have great scope for theranostic applications as they can combine the advantage of both optical and magnetic properties. The current strategy to synthesize this type of carriers is to combine different types of fluorophores such as quantum dots and organic dyes with magnetic iron oxide nanoparticles. There are many reports available on these types of nanocarriers such as magneto-fluorescent nanocarrier based on fluorescent CdS quantum dots and Fe3O4 nanoparticles 16, organic dye Rhodamine B isothiocyanate and iron oxide nanoparticles

17,

CdTe and iron oxide nanoparticles18,

photosensitizer pheophorbide-A and iron oxide nanoparticles.19 Even though all of these reported magneto-fluorescent nanocarriers have excellent physico-chemical properties, their application is limited due to reasons such as toxicity, nonbiodegradability and high cost. In order to address these issues, recently self fluorescent polymers are used to replace the toxic quantum dots and organic dyes to construct magneto-fluorescent nanocarriers. Wang etal reported magneto-fluorescent nanocarrier based on conjugated self fluorescent polymer poly(Fluorene-benzothiadiazole) (PFBT) and iron oxide nanoparticles for multimodal tumor imaging.20 However, the ionic side chains present in this polymer can lead to nonspecific interaction with cells and tissues. These issues faced by the existing magneto-fluorescent nanocarriers urge the development of safer and efficient nanocarriers capable of combining both magnetic and fluorescent characteristics. Polyethylene glycol is an FDA approved polymer extensively used for pharmaceutical formulations.21 PEGylation of iron oxide nanoparticles has been reported as an efficient strategy to increase the biocompatibility of iron oxide nanoparticles.22


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Recently, we have reported the intriguing intrinsic fluorescence characteristics of PEG based polyesters. The fluorescent nanogels synthesized from these PEGylated polymers have shown good theranostic potential.23-25 In the current study, we have explored a novel design of constructing a magneto fluorescent nanocarrier in which a self fluorescent PEGylated aliphatic polymer was used to impart both stability as well as fluorescence property to iron oxide nanoparticles. This simple and novel design avoids the usage of toxic quantum dots and organic dyes required for the construction of magnetofluorescent nanocarrier. There are no reports on the construction of magneto-fluorescent nanocarriers based on fluorescent PEG and iron oxide nanoparticles to the best of our knowledge. The novel magneto-fluorescent nanogels was based on photoluminescent comacromer [PEG-maleic acid-glycine], N, N Dimethyl amino ethylmethacrylate (DMEMA) and superparamagnetic iron oxide nanoparticles (SPION) endowed with both fluorescence and magnetic properties for potential theranostic applications. 2. Experimental section 2.1. Materials The following materials were obtained from Merck Limited (India): Maleic acid, polyethylene glycol (MW:1000), N,N dimethyl amino ethyl methacrylate (DMEMA), Ferrous chloride tretrahydrate, ferric chloride anh., and citric acid. Further the following reagents

were

purchased

from

Sigma-Aldrich

(India):

(NH3)2S2O8,

tetramethylethylenediamine, propidium iodide (PI), acridine orange-ethidium bromide (live/dead stain), and glycine. Sourcing from Invitrogen India yielded the following: dialysis polymer tubing (1000D MW limit), and Dulbecco’s modified eagle’s medium mixture supplemented with fetal bovine serum. A standard MTT assay kit was sourced from Hi Media (India).


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2.2 Synthesis of photoluminescent comacromer (PEG-M-GL) The PEG-M-GL was synthesized with modifications from the previously reported procedure.25 Briefly, a 3 neck round bottom flask charged with 12.5g PEG-1000 (conc. 0.1 M) and 1.5 g maleic acid (conc. 0.05 M). This followed with condensation taking place under N2 atmosphere at a temperature of 180 °C. After melting 1.13 g glycine (conc. 0.05 M) was added and the reaction was allowed to proceed for 6h. The molar ratio of PEG (diol) to glyine was taken as 1:0.5 to form mono-glycine-functionalized polymer. The reaction product solution was dissolved in DI water and dialysed for 3 days to purify by removing the unreacted starting material and, thus, isolate purified PEG-M-GL comacromer. A solution of isolated PEG-M-GL was lyophilised then maintained at 40C for storage. The functional group analysis of the PEG-M-GL product was accomplished by Fourier transform infrared (FT-IR) spectrometer (Jasco, FT/IR-4200, Easton, USA). Spectra were captured from 400 to 4000 cm-1 using resin smear method with KBr plates24. Nuclear magnetic resonance (NMR) spectra of the product was capture with a Bruker NMR spectrometer (Bruker Biospin, 500 MHz, Billerica, MA) in CDCl3. Analysis of the molecular weight distribution was performed using a gel permeation chromatography (GPC) system (Waters). Styragel columns were used and connected in a series configuration (HR-3, HR-4E and HR-5E, Waters) and employed THF as mobile phase at a flow rate of 1 mL/min. Calibration was provided by polystyrene (PS) standards (MW 3 – 2000 kDa). 2.3 Analysis of photoluminescence of PEG-M-GL Photoluminescence of the PEG-M-GL product in aqueous environment was determined by spectrofluorimeter (JASCO FP-8200) at ambient temperature. Solutions of PEG-M-GL (conc. 1 mg/ml) was added to a 10 mm path length quartz cuvette. Slit widths of


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1.5 nm were used for both excitation and emission. The photostability measurements of the PEG-M-GL were conducted with prolonged excitation at 420 nm in a spectrofluorometer and monitoring any changes in emission at 525 nm. The fluorescence lifetime measurements were evaluated by IBH (Horiba) nanosecond single-photon counter in both the visible and NIR domain by employing both 440 and 670 nm Nano-LED excitation sources and with C487802 micro channel plate detector (Hamamatsu). 2.4 Synthesis of citrate capped superparamagnetic iron oxide nanoparticles (SPION) Citrate capped superparamagnetic iron oxide nanoparticles were prepared. Initially SPION was synthesized as reported by cheraghipour.26 Briefly, 50ml of ferrous and ferric salts (1:2 molar equivalent) were dissolved in 50 ml deionised water in two beakers. Further, the salt solutions were transferred in to 3 neck round bottom flask equipped with nitrogen atmosphere. Vigorous stirring of the reaction mixture was taken place at 800 C to avoid the chances of formation of large polycrystalline particles. Afterwards ammonium hydroxide (25 % w/w) was introduced with slow drop-wise addtion to the flask with continuous monitoring of reaction pH. Once the pH value reached 12, formation of black precipitate has been taken place indicating the formation of SPION. The reaction mixture was cooled in to 25 °C. Afterwards the particles were centrifuged at 2000 rpm for 5 times to completely remove the impurities. Further, the particles were subjected for magnetic separation to get the purified SPION particles The synthesized SPION was modified to get SPION using citric acid as follows, briefly SPION were treated with 0.5 mg/ml of citric acid and the reaction was held at 900C for 1h under nitrogen atmosphere. The precipitate was formed after cooling the reaction mixture in to 25 °C. Afterwards centrifugation of the particles at 2000 rpm (5X repetition) to


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completely remove the impurities. Further, the particles were subjected for magnetic separation to get the purified SPION particles. 2.5 Synthesis of magneto-fluorescent nanogel (SPION-NG) SPION-NG was prepared by crosslinking the PEG-M-GL and DMEMA along with SPION. The molar ratio of PEG-M-GL and DMEMA used was 1: 0.25. 40 mg of PEG-M-GL was dissolved in 40 ml DI water in a round bottom flask with stirring (15 min under N2 atmosphere). The flask was charged with SPION (15 mg) and stirred followed by the addition of 10 µL of DMEMA, (NH3)2S2O8 and tetramethylethylenediamine (30 µL, conc. 0.001 M). The stirring continued for further 15 min to complete the crosslinking and formation of the nanogel. The crosslinking and formation of the nanogel was noted by slight turbid appearance in the reaction mixture. The mixture was sonicated for 30 min to disperse the synthesized nanogels. The unentrapped SPION were separated from the SPION-NG by dialyzing the crosslinked nanogel against DI water for 3 days and subsequently lyophilized. A semisolid product with a brown color was isolated and maintained at 40C. 2.6. Analyses of morphology and size of SPION-NG The size and morphology of the SPION and SPION-NG was determined by employing the techniques of dynamic light scattering (DLS) and transmission electron microscopy (TEM). SPION and SPION-NG were initially dispersed in distilled water at pH 7.4 and at 25 °C using a bath sonicator (a concentration of 1mg/ml was used for the experiment) and was transferred in to a cuvette and placed inside the zeta sizer for measurement. The hydrodynamic size distribution and surface potential was measured using Zetasizer (Malvern). TEM analysis (Philips, Netherlands), the SPION and SPION-NG was added to the copper grid and was desiccated for 2 days prior to measurement.


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2.7 Determination of cytocompatibility of the SPION-NG The cytocompatibility of SPION-NG was determined using MTT assay, live/dead staining assay, and cell cycle analysis. a. MTT assay Assays were conducted following previously reported procedures25. Seeding density at 1 x 105 cells/well was used and the cells were incubated with varying concentrations of SPION-NG (250-4000 µg/ml). After 24 h incubation, MTT assay was accomplished by reading absorbance at 570 nm. The cell viability was given using the equation given following:

Cell

Viability

=

[Abs] sample [Abs] Control

× 100

(1) b. Live dead cell assay Similar to previously reported live/dead assays cells were seeding density of 1 x 105 cells/well occurred. SPION-NG dissolved in media (500 and 1000 μg) was added and further incubated for 24 h at 37 °C. Analysis was accomplished via fluorescent microscope (optica, ITALY). c. Cell cycle analysis As with our previously reported experiment cell seeding density of 1 x 105 cells/well was employed25. SPION-NG dissolved in media (500 and 1000 μg) was added to Hela cells and further incubated. After they were analysed with a Flow Cytometer. Gating was accomplished using the reference to untreated control and samples were examined. 2.8 Cellular uptake studies of the SPION-NG


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Cellular uptake of the SPION-NG was determined by using HeLa cell line through Prussian blue staining. Briefly, the cells seeding at a density of 1 x 105 cells/well and cultured at the following conditions: 37 °C with a 5% CO2 atmosphere for 24 h. Different concentrations of SPION-NG (500 and 1000 μg) were added to the media and incubated for 24 h. Further, 500 μL ice cold methanol were added for fixing and incubated for 5 min. Methanol and added with Prussian blue reagent (1ml) and incubated for another 20 min. 1 ml of nuclear red solution to counter stain the nucleus and incubated for another 20 min. The cells were viewed with a light microscope. The cancer cell imaging capability of the SPION-NG was evaluated using previous methods25. 2 wt % of SPION-NG in PBS was added; and, the cellular uptake was viewed under a fluorescent microscope (optica, ITALY). 2.9 Magnetic Hyperthermia Experiments Hyperthermia studies were done in accordance with a previously published protocol.27 The Easy Heat induction system was employed and temperature change measured by noncontact thermometer. The SPION-NG (2 mg & 4 mg) was dispersed in 1ml distilled water in a 1.5 mL centrifuge tube insulated with ceramic wool. The entire assembly was placed in the centre of a water- cooled copper coil and the temperature change was monitored for 15 min. The heat generation of SPION-NG was studied using different alternating magnetic field strengths and concentrations. Distilled water was used as control for hyperthermia measurements. The dissipated energy has been determined and expressed as specific loss of power (SLP) was calculated. 𝑆𝐿𝑃 =

𝐶𝑉𝑠𝑑𝑇 𝑚 𝑑𝑡

(2)


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The magnetic hyperthermia experiments on Hela cells were carried out as previously reported27. Using similar cell protocols as used for cellular uptake studies the seeding density was 1 x 105 cell/well. SPION-NG suspended in media (1 mg) was added to the cells in the copper coil. Observation of the cells was conducted with a fluorescent microscope (optica, ITALY). 2.10 Murine in vivo bioimaging The in vivo bioimaging and bio-distribution studies were carried according to the previous report.25 A volume of 0.25 ml of SPION-NG in PBS with concentration 2 mg/ml was prepared for subcutaneous injection. Imaging was done with Xenogen IVIS spectrum system. Different excitations (640, 675, and 710 nm) were used and emissions were collected (780,800,820 and 840nm) using different filters. The same concentrations were used for biodistribution studies, and the Xenogen IVIS system was also used (675/800 nm respectively). Histopathological studies of isolated organs including heart, liver, and kidney followed as previously reported27. Animal experiments were conducted with proper permission from Institutional Animal Ethics Committee (IAEC). 2.11 Statistical significance analysis All collected data were analyzed for significance via Student’s t tests to evaluate the differences between the sub groups presented and any p values

New Magneto-Fluorescent Hybrid Polymer Nanogel for Theranostic

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