Iron(III) Coordinated Polymeric Nanomaterial: A Next-Generation

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Iron (III) Coordinated Polymeric Nano-material: A Next Generation Theranostic Agent for High Resolution T1 weighted Magnetic Resonance Imaging and Anticancer Drug Delivery Diptendu Patra, Saikat Mukherjee, Ipsita Chakraborty, TAPAN KUMAR DASH, Shantibhusan Senapati, Rangeet Bhattacharyya, and Raja Shunmugam ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00294 • Publication Date (Web): 17 Apr 2018 Downloaded from http://pubs.acs.org on April 17, 2018

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ACS Biomaterials Science & Engineering

Iron (III) Coordinated Polymeric Nano-material: A Next Generation Theranostic Agent for High Resolution T1 weighted Magnetic Resonance Imaging and Anticancer Drug Delivery Diptendu Patra,1 Saikat Mukherjee,1 Ipsita Chakraborty,2 Tapan Kumar Dash,1 Shantibhusan Senapati,3 Rangeet Bhattacharyya,2 and Raja Shunmugam*1 1

2

Polymer Research Centre, Department of Chemical Sciences,

Department of Physical Sciences, Indian Institute of Science Education and Research Kolkata. Mohanpur-741246, West Bengal, India. 3

Tumor Microenvironment and Animal Models Laboratory, Institute of Life Sciences, Bhubaneswar, Odisha, India, *E-mail: [email protected]

Abstract Theranostic based nanomedicine plays a crucial role in the field of cancer therapy. This is due to having the capability to combine both therapy and diagnosis together in single system. Herein a new class of metal-ligand based nanocarrier in a norbornene backbone has been designed as a theranostic system. Fe+3-terpyridine complex (Fe-Tpy) has been used here as T1contrast agent for high resolution MR imaging and hydrazone linked doxorubicin is used for effective pH responsive delivery. Polyethylene glycol functionalized with folic acid (peg folate) motif is used to make whole system water dispersible for longer retention and site specific therapy respectively. All these specialty functional groups are anchored in a single system by using ring opening metathesis polymerization (ROMP) technique under the norbornene backbone.

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Relaxivity study and 1D image experiments have shown the utility of Fe-Tpy complex as an effective T1 contrast agent. In vitro studies are performed to confirm the promising potentiality of the nanocarrier as the efficient nanotheranostic system in prostate cancer. Keyword: Nanotheranostic, drug delivery, ROMP, MR imaging, 1D image. Introduction Recently, theranostic nanosystem provides huge benefits in the field of site specific cancer therapy by combining both therapy and diagnostic agent together in a single system to monitor the therapeutic response of cancer affected areas. 1-3 There are many systems that have been used in theranostics, depending on the materials used for imaging and therapy.

1, 4

Polymer based

theranostic system shows enormous potential not only due to site specific bio-distribution profiles through enhanced permeation and retention (EPR) effect but also for its applications in diagnosis by multiple imaging methods.

4-7

A polymer-derived site specific theranostic material

ideally have the following motifs: (i) a water soluble and biocompatible polymer (ii) an imaging chemical functionality, such as contrast agent, fluorescent moiety (iii) a drug molecule and (iv) site specific targeting moiety. Among different imaging techniques, MRI has emerged as a powerful technique in clinical disease diagnosis due to its non-invasive in nature as well as it provides a number of benefits, for example, spatiotemporal resolution, deep penetration into soft tissues, which gives clear insight about the affected area. 8-10 MRI detection deals with relaxation of water proton that is of tissuedependent, where the magnetic inorganic particle plays principle role.

11, 12

Depending upon the

magnetic particles, there are mainly two different kinds of water relaxation mechanisms are observed. The magnetic particle responsible for the diminishing of longitudinal time (T1) of water molecules known as T1 contrast agent and the MR image appears bright, whereas the


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particles cause for diminishing of transversal time (T2) of water molecules, known as T2 contrast agent which result dark contrast in the image.8-12 The superparamagnetic nanoparticle based T2 contrast agents are suffering from several disadvantages that limit their extensive clinical applications.

13, 14

The inflammatory lesions or

tumour labelled with T2 agents, can be easily confused with the hypointense area produced by various pathogenic conditions like bleeding, calcification or metal deposition. Thus intrinsic dark signal in the T2-weighted MR image can misguide clinical diagnosis at some instances. Moreover, the high magnetic susceptibility of superparamagnetic T2 contrast agents induces perturbation of local magnetic field on neighbouring normal tissues. This local distortion or so called ‘Blooming Effect’ blurs the MR image and demolishes the background around labelled area.13, 14 Thus in clinical diagnosis T1 contrast agents are more desirable than T2 contrast agents because of producing bright MR image. Paramagnetic compounds of Gd+3, Mn+2 and Fe+3 are very well known as T1 contrast agent due to having large number of unpaired valance electrons, which interacts with the proton of water molecules during MRI study.15-17 Though MRI is an essential diagnostic technique in clinic but it undergoes through a numbers of challenging problems like, low sensitivity, low specificity, and latent toxicity of contrast agent. Such as, the nonbiological Gd+3 provide a numbers of paramagnetic complexes with the various chelating ligands and is one of the frequently used T1 active contrast agent in clinic. But the free Gd+3, leached out from the chelating ligand confinement shows nephrotoxicity.

16, 18, 19

Because

of this, Food and Drug Administration (FDA) already warned of nephrogenic systemic fibrosis (NSF) in patients who received gadolinium based imaging agent.

20

Moreover, lack of targeting

ability, short blood circulating time due to rapid urinary excretion, makes a bottleneck of these



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complexes. Thus in case of Gd-complexes better T1 weighted MR image can be achieved by long scan time which is a serious concern. The higher spatial resolution and lesser acquisition time are in high demand in the field of MR imaging. This can be achieved by performing MRI experiment at higher magnetic field by increasing the signal to noise ratio. Thus the trend in MR imaging is increasingly moving to the higher magnetic field. Though majority of MRI scanner operates at magnetic field 1.5T and 3T in clinic, but installation of 7 T whole body scanner is increasing worldwide.

21

In addition

human imaging at high magnetic field 9.4 T performed successfully and also well documented.22 At high magnetic field scanner, the main obstacle for T1 active contrast agent is typical decreased in longitudinal relaxivity (r1) and static or increase in transversal relaxivity (r2) and thereby results overall increase in (r2/r1) ratio.15, 21, 23, 24 So, there is a pressing need to have biologically relevant paramagnetic metal ion to overcome disadvantages of Gd-complexes as well as the higher value of (r2/r1) parameter at high magnetic field. Strong paramagnetic Fe+3 have five unpaired electrons in its valance shell which can interact strongly with the water molecules and make it capable to show T1 contrast efficiency.23, 24

Furthermore owing to biocompatibility, iron based systems are well accepted contrast agent.23,

24

So the incorporation of Fe+3 with a judicious molecular design to polymer backbone is very

important for better imaging application. In this study, we have designed and synthesized a norbornene based magnetic copolymer (NrDx-PgFl-Tr-FeCl3) using ring opening metathesis polymerization (ROMP) for the purpose of theranostic application.25, 26 Herein DOX and Fe+3 has been attached to norbornene backbone by hydrazone link and terpyridine chelation respectively. PEG-folate was attached to prevent


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opsonisation and for site specific accumulation of macromolecule.7 The magnetic nature of nanocarrier is explored by vibrating sample magnetometer (VSM). Unlike many other iron based T1 contrast agent, Nr-Dx-PgFl-Tr-FeCl3 system has been showing high longitudinal relaxation (r1) as well as relatively low (r2/r1) ratio at strong magnetic field which lead the system to next generation for high resolution T1 weighted MR imaging. NMR 1D image experiment further confirmed the contrast efficiency. To the best of our knowledge, this is the first report where high spin Fe+3-terpyridine (Fe-Tpy) complex has been utilised as T1 contrast agent for the purpose of making nanotheranostic system.27, 28 The efficacy of the nanocarrier is evaluated in vitro by studies like MTT, fluorescence microscopy and flow cytometry. Cytotoxicty and cellular uptake studies show high amount of accumulation of doxorubicin inside the cell which distributed in both nucleus and cytoplasm. This nanocarrier (Nr-Dx-PgFl-Tr-FeCl3) is expected to have a greater application in the field of theranostics. Experimental Section: Preparation of Grubbs’ Third Generation (G-3) Catalyst: Grubbs’ third generation catalyst was prepared according to the previously reported literature procedure.3 The calculated amount of Grubbs’ second generation catalyst was taken in a glass vial and 2-bromopyridine was added to it inside the glove box under inert atmosphere of nitrogen and allowed to stir for 2 mins. A green colour compound was immediately formed which confirmed the formation of Grubbs’ third generation (G-3) catalyst which was precipitated in pentane. It was then collected and dried under nitrogen atmosphere inside the glove box. The fresh G-3 was immediately used in the ring opening metathesis polymerization (ROMP) to synthesis all the polymers.



Synthesis of Nr-Dx-PgFl-Nhs: The polymerization was done by following previously reported literature procedure.3 35 mg (0.04 mmol) of Mono 1, 15 mg (0.04 mmol) of Mono 2, and 170 mg (0.08 mmol) of Mono 3, were weighed separately and taken in a 15 mL glass vial and dissolved in minimum amount of 9:1 ratio mixture of dry DCM and dry MeOH and stirred for 10 mins to form the homogeneous reaction mixture at room temperature in dark (Figure 1b). 3.4 mg of Grubbs’ third generation catalyst (G-3) was weighed in a separate vial and minimum amount of dry DCM was added to it to make the homogeneous solution of the catalyst. The catalyst solution was then added to the 15 mL glass vial containing the monomer solution in an inert atmosphere inside the glove box (Figure 1b). An aliquot of sample was quenched with ethyle vinyl ether, at different time lag and precipitated in diethyl ether and taken for GPC analysis. Gel permeation chromatography (GPC) was done in tetrahydrofuran (flow rate = 1 mL/min). The molecular weight of the random copolymer was measured as Mn = 32000 Da (PDI = 1.12) by using poly(methyl methacrylate) standard (Figure 2b). Then the remaining reaction mixture was quenched with ethyl vinyl ether and the product was precipitated by the addition of diethyl ether. To remove the catalyst it was again dissolved in THF, and then passed through neutral alumina and precipitated again by the addition of diethyl ether to get Nr-Dx-PgFl-Nhs.


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Figure 1. a) A tool box comprising monomers (Mono 1-3), b) Synthesis scheme of Nr-DxPgFl-Nhs, Nr-Dx-PgFl-Tr and Nr-Dx-PgFl-Tr-FeCl3.



Synthesis of Nr-Dx-PgFl-Tr: The attachment of amine functionalized terpyridine ligand (Tr-NH2) to the Nr-Dx-PgFl-Nhs, was performed according to the literature procedure. 200 mg of Nr-Dx-PgFl-Nhs was weighed and dissolved in the 9:1 solvent mixture of dry DCM and MeOH respectively. 15 mg of Tr-NH2 (8) was added to it and stirred for overnight at room temperature (Figure 1b). The reaction was carried out under dark condition. After completion of reaction, precipitation in diethyl ether was done to get Nr-Dx-PgFl-Tr. It was dialysed in dark condition with a dialysis tube of Mw 3500 Da cut off for the removal of excess free terpyridine ligand in the copolymer. The pure terpyridine attached copolymer; Nr-Dx-PgFl-Tr was dried and immediately used for next step of ferric chloride attachment (Figure 1b). Synthesis of Nr-Dx-PgFl-Tr-FeCl3: The chelation of ferric chloride to the terpyridine unit of Nr-Dx-PgFl-Tr copolymer was performed according to literature procedure.27 150 mg of pure of Nr-Dx-PgFl-Tr was taken in a 15 mL glass vial and 1.5 mL dry THF was added to it. The reaction mixture was stirred for 5 mins at room temperature in dark to make the homogeneous solution. 7 mg of anhydrous FeCl3 dispersed in dry THF was added to the reaction mixture and stirred for 12 h at room temperature in dark (Figure 1b). After completion of the reaction, the solvent was evaporated and the solid mass was dissolved in dry DCM. The product was precipitated from diethyl ether and again dialysed with 3500 Da (Mw) cut off dialysis tube to get pure Nr-Dx-PgFl-Tr-FeCl3 copolymer. MRI Experiments: All relaxivity experiments and T1-weighted 1D-imaging experiments were performed using Bruker 500 MHz Avance III spectrometer at room temperature (25 ºC). Total five solutions of


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Nr-Dx-PgFl-Tr-FeCl3 at different concentrations (0.1-0.6 mM range) were prepared, in 2:3 mixture of H2O and D2O v/v ratio. The relaxation rates were measured on the water peak at 4.7 ppm. Longitudinal relaxation rate (R1) was measured using conventional inversion recovery experiment whereas CPMG (Car-Purcell-Meiboom-Gill) sequence was used to determine the transverse relaxation rate (R2). Every experiment was performed with 4 scans and durations of recycle delays were chosen as ~ 5T1. Two solutions of Nr-Dx-PgFl-Tr-FeCl3 copolymer at two different concentrations (0.1 mM and 0.2 mM) were chosen for T1-weighted 1D-imaging experiments. A simple inversion recovery type sequence i.e. 180º pulse followed by a variable delay (τ) and a spin-echo sequence (90º-τ'-180°) (Figure: 6a) were applied on water proton peak at 4.7 ppm. Fourier Transformation (FT) of acquired Free Induction Decay (FID) collected under linear gradient of strength 4 G/cm, yields a T1-weighted one-dimensional image of Nr-Dx-PgFlTr-FeCl3 solution for various delay owing to spatial encoding. The delay (τ) between 180º and 90º pulses has been varied from 50 ms to 450 ms in 8 steps and the delay of following spin-echo sequence (τ') was kept constant at 100 µs. We have chosen the delay values (τ) such a way that after FT, a series of inverted 1D- spectra decaying to null obtained. A set of spectra generated before null point was collected. A particular range of those spectra was selected for contour plotting. For a given concentration of Nr-Dx-PgFl-Tr-FeCl3 solution, the selected regions of resulting broad spectra are plotted using a contour diagram. The experimental raw data are processed using Julia®. For all experiments, radio frequency power was kept fixed at 17.8 kHz. Drug Content and Release Experiments: Drug content in Nr-Dx-PgFl-Tr-FeCl3 nanoaggregate was determined by fluorometric method. Fluorescence intensity of free DOX at five different concentration (10, 25, 50, 75 and 100 ng/mL) solutions in DMSO were measured and fitted in linear calibration curve. DOX content



in 100, 400 and 800 ng/mL solutions of Nr-Dx-PgFl-Tr-FeCl3 were calculated by interpolation of respective fluorescence intensities in the calibration curve. After determination of DOX content in newly designed Nr-Dx-PgFl-Tr-FeCl3 nanoaggregate, the pH responsive nature was tested by dialysis method (Figure 10a). 1 mg/mL solution of NrDx-PgFl-Tr-FeCl3 was loaded into a dialysis tube of MWCO 3500 Da, and dialyzed against 70 mL of acetate buffer solution of pH 5.5, in a completely covered beaker. The fluorescence emissions at 560 nm and 590 nm were recorded by exciting the aliquot of the sample at 510 nm in different time interval which was an indication of the release of free doxorubicin from the NrDx-PgFl-Tr-FeCl3 nanoaggregate. The aliquot was then poured back to the solution to maintain the constant volume of the solution. Cell culture: DU145 prostate cancer cells were cultured in DMEM containing 10 % FBS and 1% antibiotic solution (penicillin and streptomycin). The cells were incubated at 37˚C in a humidified atmosphere. Harvesting of cell was done using Trypsin-EDTA solution and passage was done 23 times a week. Uptake of synthesized prodrugs in DU145 prostate cancer cells: Identical to a previously reported procedure, 5x104 DU145 cells were seeded in to each well of a 24 well plate containing 13 mm cover slips.29 The cells were allowed to adhere firmly on the cover slips for 24 h. Then Nr-Dx-PgFl-Tr-FeCl3 nanoaggregate treatment was done in different concentrations (50, 200 µg/mL) and incubated in cell culture conditions for 4 h. The cells then washed twice with PBS to remove out cellular debris or loosely attached cells. Cells were fixed by incubating in 4% PFA for 20 min. The cells then washed with PBS and treated with 0.5


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µg/mL of DAPI for 10 min at RT. The free DAPI then washed out twice using PBS and cover slips were mounted on a drop of mounting media placed on a sterile glass slide. The slides then allowed to dry at RT for 24 h and observed under blue and red fluorescence channel of a microscope for uptake of DAPI and DOX. Reference imagers were captured and presented along with bright field image to analyze the uptake pattern Quantitative uptake and uptake kinetics: Uptake analysis was done as reported previously.30,31 10 x104 DU145 cells were seeded into each well of a 24 well plate and allowed to adhere for 24h. Finely adherent cells were treated with different concentrations (10-200 µg/mL) of Nr-Dx-PgFl-Tr-FeCl3 nanoaggregate for 4 h. The cells then washed twice with PBS and resuspened in sheath fluid containing 1% PFA that kept at 4 ºC until acquisition. 10x103 gated cells were acquired in a flow cytometer (BD, FACS Calibur) and DOX fluorescence was measured in fluorescent channel-3. Mean fluorescence intensity was presented in comparison to untreated cells. The aggregated system of prodrugs occasionally acts a barrier for endocytic uptake. Thus to determine the optimal time for unaggregation and uptake we performed a kinetic of uptake of NrDx-PgFl-Tr-FeCl3 nanoaggregate. For the above objective, similarly seeded cells were treated

with 200 µg/mL of Nr-Dx-PgFl-Tr-FeCl3 nanoaggregate. Treated cells along with untreated control were incubated at 37˚C under normal culture conditions and harvested different time points i.e. 2 h, 4 h, 8 h and 24 h post treatment. Harvested cells were washed with PBS, processed and analyzed for fluorescence intensity in flow cytometry as described previously. The uptake of DOX at different time points is presented considering the uptake at 24 h to be 100%.



Retention of polymeric prodrugs: Unconjugaed DOX is a well known substrate of cellular efflux pump and is generally expelled out of the cells by and energy dependent manner.31 However polymeric prodrug of DOX is not expected to have such limitations as their mode of cellular transport is endocytosis rather than passive diffusion. Thus Nr-Dx-PgFl-Tr-FeCl3 nanoaggregates were treated to ensure the increased duration of retention, we analyzed the cellular retention after treatment of 50-100 µg/mL of prodrug. After 6 h of treatment, supernatant was replaced with fresh media and incubated further. Harvesting of cells was done after 6 h of Nr-Dx-PgFl-Tr-FeCl3 nanoaggregates treatment as well as at 6 h and 10 h of fresh media replacement. The harvested cells were washed with PBS, resuspended in sheath fluid containing 1% PFA and kept at 4 ˚C until acquisition. The mean fluorescent intensities of 10000 gated cells were comparatively presented at different time points considering uptake at 6 h of treatment as 100%. Cyto-toxic effect of drug conjugated systems: a) MTT assay: 5x103 DU145 cells were seeded in to each well of a 96 well plate. After 24 h, different concentration of Nr-Dx-PgFl-Tr-FeCl3 nanoaggregates were treated in triplicate and incubated in normal culture conditions. After 72 h of treatment, 100 µL of 5 mg/mL of MTT solution was added in to each well. Reduction of MTT was allowed to happen for 4 h at 37 ˚C and formation of formazan crystals were confirmed under microscope. Then supernatant then discarded carefully by not disturbing the cell layer. 100 µL of DMSO was added and mixed for 5-10 min to solubilize the crystals formed. The optical density in each well was quantified in multimode


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reader at a wavelength of 570 nm. Percentage viability of each treatment group was quantified compared to untreated control. b) Cell viability assay through crystal violate staining: Briefly, 20x104 DU145 cells were sown in to each well of a 24-multiwell plate and allowed to adhere for 24 h. Then macromolecule of different concentration ranging 0.1 µg/mL-10 µg/mL were treated and incubated in a humidified atmosphere at 37 ˚C for 72 h. Treated cells along with untreated control were washed twice with PBS and incubated with 0.05% crystal violet for 30 min at room temperature. Further the wells were washed thoroughly with tap water after discarding the supernatant and allowed to dry at room temperature for 4 h. Then images of individual wells were captured and 200 µL of methanol were added to each well. The contents were mixed well for dissolving the crystal violet retained by cells. Then 50 µL from each well was aliquot in to each well of a 96 well plate twice and the optical density of crystal violet was measured at 580 nm. Percentage viability of each treatment group was calculated considering optical density of untreated wells as 100%. Result and Discussion Towards the goal of making site specific nanotheranostic agent, three different monomers (Mono 1, Mono 2 and Mono 3) and a chelating ligand system (Tr-NH2) for the attachment of Fe(III) have been synthesized and thoroughly characterized by NMR, IR and mass spectroscopy (ESI-MS and MALDI-MS) techniques (Figure 1a, S1-S19, S23-27). The six steps process involved to make Mono 1 has been started from the commercially purchased furan and maleic anhydride (SI Scheme 1). A simple room temperature stirring the reaction mixture gave Diels Alder adduct in high yield. The three characteristic peaks at 6.5, 5.2



and 3.0 ppm in 1H NMR spectroscopy technique confirmed the formation of the product (Figure S1). Also 13C NMR spectroscopy technique was used to confirm the formation of 1 (Figure S2). After successful synthesis of 1, it was treated with p-aminobenzoic acid to give exclusively 3 in high yield (SI Scheme 1). The formation of 3 was a two step process, first through open ring intermediate followed by closing the ring after treatment with acetic anhydride in presence of sodium acetate (SI Scheme 1). Compounds 2 and 3 were successfully characterized by 1H and 13

C NMR spectroscopy techniques (Figure S3-6). After successfully synthesizing 3, tertiary

butyl carbazate was reacted to it in presence of DCC and DMAP to get the BOC protected norbornene functionalized hydrazine (4) (SI Scheme 1). The successful formation of 4 was monitored by 1H and 13C NMR spectroscopy techniques (Figure S7 and S8). After successfully synthesized the BOC protected 4, deprotection of BOC group was carried out in presence of TFA (SI Scheme 1). The disappearence of 1.26 ppm peak in 1H NMR spectrum corresponds to BOC group which confirmed the formation of 5 (Figure S9).

Figure 2. a) Mn vs. M/I plot for three different monomers (Mono 1-3), b) GPC chromatogram of Nr-Dx-PgFl-Nhs copolymer (Mn = 32,000 Da, PDI= 1.12).


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The formation of product was also confirmed by 13C NMR spectroscopy technique (Figure S10). After confirming the successful formation of the norbornene functionalized hydrazine, doxorubicin was treated to it in mild acidic condition to synthesize pH responsive hydrazone linker (SI Scheme 1). The reaction was carried out in dark for 48 h with continuous stirring at room temperature followed by precipitation in acetonitrile to give pure Mono 1 (SI Scheme 1). The formation of Mono 1 was confirmed by 1H NMR spectroscopy and ESI-MS techniques (Figure S11 and S24). In ESI-MS analysis the observed protonated peak at 825.27(m/z) confirmed the successful synthesis of Mono 1 (Figure S24). The two monomers, Mono 1 and Mono 2 were synthesized for the purpose of therapy and MRI contrast agent respectively (SI Scheme 1). For that cis-5-norbornene-exo-2,3-dicarboxylic anhydride was heated to reflux at 120 °C for 12 h in presence of γ-aminobutyric acid, dry toluene was used here as solvent (SI Scheme 1). The successful synthesis of 6 was confirmed by 1H,

13

C NMR spectroscopy

techniques (Figure S12-S13). The appearance of a broad peak at 12.1 ppm was responsible for carboxylic acid in 1H NMR spectroscopy along with the characteristic norbornene olefinic peak at 6.2 ppm confirmed the formation of 6 (Figure S12). N-hydroxysuccinamide (NHS) was treated to 6 in presence of DCC and DMAP to synthesize Mono 2 (SI Scheme 1). The disappearances of 12.1 ppm peak along with the appearance of new peak at 2.8 ppm in 1H NMR spectroscopy technique were reason for NHS group, confirmed the successful formation of Mono 2 (Figure S14). The formation of Mono 2 was also successfully confirmed by 13C NMR spectroscopy and ESI-MS techniques (Figure S15 and S25). The observed sodiated peak at 369.11(m/z) confirmed the successful synthesis of Mono 2 (Figure S25). After successful synthesis of Mono 1 and Mono 2 the synthesis of Mono 3 was carried out to introduce the water solubility and site specific therapy (SI Scheme 1). The formation of Mono 3 was involved in



two steps process, starting from the functionalization of PEG to the folate moiety in presence of DCC and DMAP (SI Scheme 1). The formation of 7 was confirmed by 1H NMR spectroscopy (Figure S16). After successful synthesis of PEG functionalized folate moiety (7), exo-5norbornenecarboxylic acid was functionalized to the other end of PEG moiety (SI Scheme 1). The appearance of the folate and peg signals along with the characteristic 6.2 ppm signal which was responsible for the norbornene olefinic proton in 1H NMR spectrum successfully confirmed the formation of Mono 3 (Figure S17). Also MALDI-MS was used to confirm the formation of Mono 3 (Figure S26). Reaction between 4′-Chloro-2,2′:6′,2′′-terpyridine and ethanolamine in presence of KOH in a single step produced the amine functionalized ligand system, Tr-NH2 (8) as shown in SI Scheme 2. The formation of 8 was successfully characterized by 1H, 13C NMR, IR spectroscopy and ESIMS techniques (Figure S18-19, S23, S27). The appearance of signals at 4.21 and 3.1 ppm in 1H NMR spectroscopy corresponds to two methylene group of ethanolamine along with the appearance of aromatic signals of terpyridine confirmed the formation of 8 (Figure S18). The formation of 8 was also confirmed by

13

C NMR and IR spectroscopic techniques (Figure S19

and S23). The characteristic frequencies of N-H stretching (3412 cm-1), aromatic C=C stretching (1584-1566 cm-1, two bands), and the more important symmetric stretching (1022 cm1

) as well as antisymmetric stretching (1205 cm-1) of the newly formed ether bond between the

terpyridine and the alcoholic oxygen of ethanolamine (Ar-O-R) confirmed the formation of the amine functionalised terpyridine (Figure S23). The observed protonated peak at 293.14 (m/z) in ESI-MS analysis further confirmed the formation of 8 (Figure S27). After the successful synthesis of all monomers (Mono 1-3) and the amine functionalized ligand system, Tr-NH2 (8), the homopolymerization conditions of the monomers were explored by


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following previously reported literature.7,

8

A calculated amount of Grubbs’ third generation

catalyst was added to each different vials already contained three different norbornene monomers (Mono 1-3) with different M/I ratios to evaluate the livingness of all the monomers (Mono 1-3) (Figure 2a). It was observed that polymerizations were well controlled resulting in narrow PDI. The molecular weights of polymers were calculated by using gel permeation chromatography technique (GPC), PMMA as internal standard. After stabilization of homopolymerization conditions for all three monomers (Mono 1-3), we were encouraged to copolymerize them using Grubbs’ third generation catalyst in DCM and MeOH (9:1) under anhydrous conditions. (Figure 1b). A calculated amount of Grubbs’ third generation catalyst was added to the vial containing three monomers dissolved in the solvent mixture. The entire polymerization was carried out under inert nitrogen atmosphere for overnight in dark (Figure 1b). The GPC traces confirmed the formation of copolymer with Mn = 32000 with PDI = 1.12 (Figure 2b). The formation of copolymer was further confirmed by 1H NMR spectroscopy technique (Figure S21). After successful synthesis of Nr-Dx-PgFl-Nhs, the primary amine of Tr-NH2 was allowed to replace the NHS group to form Nr-Dx-PgFl-Tr (Figure 1b). 1H NMR and IR spectroscopic techniques were used to characterize the successful synthesis of Nr-Dx-PgFl-Tr (Figure S22 and 3a). The appearance of new aromatic peaks corresponding to the terpyridine protons in 1H NMR spectrum, confirmed the ligand (Tr-NH2) attachment to Nr-Dx-PgFl-Nhs, (Figure S22). It was further supported by the IR spectroscopic technique (Figure 3a). Just like Nr-Tr, two similar peaks at 1646 cm-1 and 1604 cm-1 in the IR spectra for Nr-Dx-PgFl-Tr was appeared which correspond to the C=O stretching frequency and N-H bending frequency of the newly formed amide bond (Figure 3a).



Figure 3. a) IR spectra of Mono 2, Tr-NH2, Nr-Tr, and Nr-Dx-PgFl-Tr, b) UV_VIS spectra of Nr-Tr-FeCl3 and Nr-Dx-PgFl-Tr-FeCl3, c) TGA analysis of Nr-Dx-PgFl-Tr and Nr-Dx-PgFlTr-FeCl3.


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The absence of C=O stretching of NHS (1820 cm-1) and the activated ester (1740 cm-1) along with N-O stretching (1210 cm-1) and C-O stretching (1070 cm-1) clearly indicate the successful substitution of NHS group by the primary amine of Tr-NH2 in order to form Nr-Tr and Nr-DxPgFl-Tr (Figure 3a). This confirmed the attachment of terpyridine ligand to the copolymer backbone. The C=O stretching frequency of imide bond in the five-member cyclic system in the norbornene unit was observed at 1695 cm-1 in IR spectra (Figure 3a). Towards the goal of designing Nr-Dx-PgFl-Tr-FeCl3 as MRI active theranostic agent, anhydrous ferric chloride (FeCl3) was complexed to the terpyridine (Tr) unit of Nr-Dx-PgFl-Tr (Figure 1b). To confirm the attachment of Fe(III) to the terpyridine unit of Nr-Dx-PgFl-Tr, another control molecule (Nr-Tr-FeCl3) has been synthesized and its UV-VIS absorption was compared to Nr-Dx-PgFl-Tr-FeCl3 (Figure 3b). The UV-VIS absorption study of Nr-Dx-PgFlTr-FeCl3 showed two similar absorption peaks at 272 nm and 316 nm as that of the Nr-TrFeCl3 which clearly supported the attachment of ferric ion to the polymer backbone via post polymer modification (Figure 3b). The thermogravimetric analysis (TGA) was performed for both the copolymers, Nr-Dx-PgFl-Tr and Nr-Dx-PgFl-Tr-FeCl3. A sudden decrease in the weight percent at around 400 ºC temperature for Nr-Dx-PgFl-Tr and Nr-Dx-PgFl-Tr-FeCl3 were due to the norbornene backbone degradation. The weight percent of the attached FeCl3 in Nr-Dx-PgFl-Tr-FeCl3 was about 5, calculated by measuring the residual weight (%) at 600 ºC temperature from TGA analysis (Figure 3c).



Figure 4. a) Particle size distribution of Nr-Dx-PgFl-Tr-FeCl3 nanoaggregates, b) SEM image of Nr-Dx-PgFl-Tr-FeCl3 nanoaggregates, c) EDX analysis of Nr-Dx-PgFl-Tr-FeCl3 nanoaggregate. Self-assembly and Energy Dispersive X-ray (EDX) Study: After confirming the formation of Nr-Dx-PgFl-Tr-FeCl3, the self-assembly behavior of the coplymer was explored in water. 1 mg of Nr-Dx-PgFl-Tr-FeCl3 was dissolved in 1 mL water and stirred to get the clear solution for dynamic light scattering (DLS) study. The particle size was measured around 175 nm with 0.309 PDI by using DLS (Figure 4a). The same solutions


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was drop casted on silicon wafers and dried properly and then the morphology of the aggregate was determined by scanning electron microscopy (SEM) (Figure 4b). From SEM analysis, the shape of the Nr-Dx-PgFl-Tr-FeCl3 nanoaggregate was observed as spherical with the diameter of 150-165 nm, which was also in good agreement with DLS measurement (Figure 4b). To prove the presence of Fe(III) in the nanoaggregates, energy dispersive X-ray (EDX) study was performed which provided the elemental details (Figure 4c). The characteristic peaks for Fe and Cl in the EDX spectrum of Nr-Dx-PgFl-Tr-FeCl3 nanoaggregate also confirmed the attachment of ferric chloride to the terpyridine modified copolymer (Figure 4c). The presence of 1:3 elemental ratios of Fe and Cl in the EDX study further confirmed the successful attachment of ferric chloride (Figure 4c).



Figure 5. a) Vibrating sample magnetometer (VSM) study of Nr-Dx-PgFl-Tr-FeCl3, b) The relaxation rates (R1) of water peak are shown against concentrations of Nr-Dx-PgFl-Tr-FeCl3 solutions. c) The contour diagrams represent decay of signal intensity at 0.1 mM and d) at 0.2 mM concentrations of Nr-Dx-PgFl-Tr-FeCl3 during time span: 0.05-0.45 s. The yellow region and bluish regions represents the high and low signal intensity respectively. Magnetism and Relaxivity Study: Vibrating sample magnetometer (VSM) was done to check the magnetic property of the Nr-DxPgFl-Tr-FeCl3 nanoaggregates. It was observed from VSM study that, in absence of external magnetic field (G), the magnetic moment (emu/g) of Nr-Dx-PgFl-Tr-FeCl3 nanoaggregate was zero, which was due to the random arrangement of atomic moments hence cancelled each other (Figure 5a). However, in presence of applied magnetic field (G), each atomic moment tended to align with the field direction hence produced the net magnetic moment (emu/g), of Nr-Dx-PgFlTr-FeCl3 nanoaggregate (Figure 5a). The direct proportionality relation between net magnetic moment (emu/g) and applied magnetic field (G) confirmed the paramagnetic nature of the NrDx-PgFl-Tr-FeCl3 nanoaggregate (Figure 5a). Now this paramagnetic material in water creates a fluctuating magnetic field around the water molecule which can affect the relaxation behaviour of water protons (H) in the presence of a static magnetic field. Here we mainly focus on longitudinal (T1) relaxation property of the water protons in presence of paramagnetic polymeric nano-material. T1 relaxation is such a process where water protons (H) exchange their energy through the surroundings. A series of FT-NMR signal were obtained by varying delay time (τ) in the applied pulse sequence (Figure 6a). These resulting signals indicate how fast the longitudinal magnetization build up and become 63% of its saturation value at time T1 (Figure 6b-c). In presence of Nr-Dx-


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PgFl-Tr-FeCl3, the growing rate of longitudinal magnetization, Mz is enhanced as a function of

concentration, due to increase in spin-lattice relaxation process (Figure 6b-c). As all protons do not return to its initial energy state at the same time, thus the recovery of longitudinal magnetization is taken place exponentially with the time, producing the inversion recovery curve or simply T1 curve (Figure 6c).

Figure 6. Graphical representation of a) inversion recovery followed by spin echo sequence. b) Recovery of longitudinal magnetization (Mz) and c) respective T1 curves for Nr-Dx-PgFl-TrFeCl3 nanoaggregate solution (red coloured) and blank solution (blue coloured) at time t.

Further the T1 relaxation time of water molecules was measured in presence of Nr-Dx-PgFl-TrFeCl3 nanoaggregates with different concentrations (Figure 5b). It was observed that the



longitudinal relaxation rates were found to be increasing with increase of the concentrations of Nr-Dx-PgFl-Tr-FeCl3 nanoaggregate (Figure 5b). The longitudinal relaxivity values (R1) were plotted in the graph with respect to Nr-Dx-PgFl-Tr-FeCl3 nanoaggregate concentrations which showed a linear dependency fitted with straight line. The corresponding equation is R1 = (R1)0 + r1.[c], where R1 and (R1)0 are the longitudinal relaxation rates of sample solution and blank (in the absence of solute) respectively, [c] indicates concentration of the Nr-Dx-PgFl-Tr-FeCl3 nanoaggregate solution and r1 is the specific relaxivity constants which was measured from the slope of the straight line (Figure 5b). Similarly r2 was determined to calculate (r2/r1) ratio which is an important parameter to estimate the efficiency as a T1 contrast agent. As it is very much challenging to have high r1 and low (r2/r1) ratio at very high external magnetic field, thus the relaxation time was measured at 11.7 T magnetic resonance spectrometer to examine the feasibility of Nr-Dx-PgFl-Tr-FeCl3 nano aggregate as a high resolution T1 weighted magnetic resonance imaging theranostic agent. The r1 relaxivity of 18.12 mM-1S-1 for Nr-Dx-PgFl-Tr-FeCl3 nanoaggregate is a relatively high value among the reported relaxivities of many other T1 contrast agents. Again the (r2/r1) ratio of 5.38 comes for the sake of weak magnetic inhomogeneity around the Nr-Dx-PgFl-Tr-FeCl3 nano aggregate, which demonstrated the potentiality as an efficient T1 constrat theranostic agent. 1D Imaging Experiment: The absolute intensities of the acquired spectra of the water proton have been plotted on a descending color-scale of yellow to blue (Figure 5c-d) and it has been scaled from 1.0 to 0. The contour diagrams show how rapidly the signal decays from high intensity (yellow region) to low intensity (bluish region). For the solution of concentration 0.2 mM the signal nearly vanishes near at 450 ms (most dark blue shade) whereas for 0.1 mM solution the intensity decays to nearly


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40% of its maximum at 450 ms, hence it does not converge to the darkest shade of the blue region within the same time limit (Figure 5c-d). Thus, the contour diagram clearly depicts that the rate of decay of the acquired signal intensity is enhanced with the increase in the concentration of Nr-Dx-PgFl-Tr-FeCl3 nanoaggregate. The non-parallel segments of the curves result from the non-uniform image profile owing to rf inhomogeneity and non-linear gradient profile across the sample. However, this does not prevent us to draw the above conclusion that the higher concentration leads to faster decay of signal intensity and thus it can be concluded that the Nr-Dx-PgFl-Tr-FeCl3 nanoaggregate is a T1-contrast theranostic agent.



Figure 7. a) Calibration curve of DOX. b) Drug release profile of Nr-Dx-PgFl-Tr-FeCl3 at pH 5.2 and 7.4. Drug Content and Release Study: The drug content in Nr-Dx-PgFl-Tr-FeCl3 has been determined from interpolation of fluorescence intensity of DOX, in the linearly fitted calibration curve of free DOX (Figure 7a). In the calibration curve, Nr-Dx-PgFl-Tr-FeCl3 solutions with concentrations 100, 400 and 800 ng/mL correspond to equivalent intensity with free DOX solution at concentrations 13, 45 and 92 ng/mL. Thus about 11.5% DOX was loaded in Nr-Dx-PgFl-Tr-FeCl3 nano aggregate (Figure 7a). In the release study, ~ 80 % of DOX was released within 24 h at pH 5.2, whereas less than 5 % released was observed at pH 7.4 (Figure 7b). Due to acidic nature, cancerous environment can get sustain drug accumulation for long period of time. However in normal environment, the nanoaggregates will not be disturbed in physiological condition, which will help to reduce the side effects. Further presence of site specific folate group can guide the nanoaggregates specifically to cancer cells, which will also minimise the side effects of DOX (Figure 8).


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Figure 8. Cartoon representation of mode of action on cellular surface of Nr-Dx-PgFl-Tr-FeCl3 nanoaggregate. Biological Studies: Uptake of Nr-Dx-PgFl-Tr-FeCl3 in DU-145 cells: Uptake of Nr-Dx-PgFl-Tr-FeCl3 into cells is expected to occur by folate receptor mediated endocytosis.

The uptake pattern of Nr-Dx-PgFl-Tr-FeCl3 nanoaggregate at two different

concentrations (50 and 200 µg/mL) has been observed in prostate cancer cells (Figure9). The merged images of DAPI and DOX fluorescence indicate the DOX colocalization at the nucleus. On the other instance, presence of cytoplasmic fluorescence (marked by arrow) indicates gradual release in cytoplasm supporting the conception of endocytic uptake (Figure9).



Figure 9. Cellular uptake of Nr-Dx-PgFl-Tr-FeCl3 nanoaggregate in DU-145 cells. Quantitative uptake of Nr-Dx-PgFl-Tr-FeCl3 in DU-145 cells: Accumulation of Nr-Dx-PgFl-Tr-FeCl3 in DU-145 cell was analyzed in flow cytometry using inherent fluorescence of DOX (Figure 10). Here DOX uptake was increased in a concentration dependent manner (Figure 10a-c). Further, in a study to determine the optimal incubation time for macromolecules at 8 h after treatment almost 80% of maximal uptake was obtained (Figure 10b-c).


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Figure 10. Quantitative uptake of Nr-Dx-PgFl-Tr-FeCl3 in DU-145 cells a) concentration and b) time dependent uptake study c) uptake data table. Respective concentration, time and uptake have been represented in colour code. Retention Study of Nr-Dx-PgFl-Tr-FeCl3 in DU-145 after treatment: Retention of DOX or Nr-Dx-PgFl-Tr-FeCl3 nanoaggregate is essential to have its anticancer activity. Small molecule or free drug effluxes out of the cell quickly by the cellular efflux pumps. On the other hand macromolecular conjugated system size is too big to be a substrate of efflux pumps and prevent efflux of drug by facilitating slower release inside the cell. Thus improves cellular retention and release in a bimodal way. To evaluate the retention, following treatment with Nr-Dx-PgFl-Tr-FeCl3, cells were nourished with fresh media. Zero hour time point in figure 11 represents time point of replacement with fresh media after 4 h of Nr-Dx-PgFl-TrFeCl3 treatment. As demonstrated in figure10d, in all treatment concentration >70% of DOX was retained in cell after 6 h and > 65% DOX was remained after 10 h of fresh media replacement. Thus Nr-Dx-PgFl-Tr-FeCl3 possessed a longer retention period in the cells. Colour code representation has been used to indicate respective concentration, time and DOX retention (Figure 11).



Figure 11. Cellular retention of Nr-Dx-PgFl-Tr-FeCl3 in DU-145 cells. a) retention of Nr-DxPgFl-Tr-FeCl3 (5 µg/mL and 10 µg/mL) after two different time interval (6 h and 10 h). b) retention data plot with varying time and concentration, and c) retention data table. Cytotoxicity of Nr-Dx-PgFl-Tr-FeCl3 in DU-145 cells Cytotoxiciy was evaluated by crystal violate staining and MTT based cell viability assay. From the results, it is observed that in both the methods Nr-Dx-PgFl-Tr-FeCl3 exerted concentration dependent cytotoxicity in DU 145 cells. In the cell viability assay done through crystal violate staining , crystal violet stained surface decreases as treatment concentration increases (Figure


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12a) and the same is represented in quantitative analysis of crystal violet concentration (Figure 12b).

From the dose response curve of MTT based cell viability assay, the IC50 of

macromolecule is determined to be 1 µg/mL (Figure 12c).

Figure 12. Cytotoxicity of Nr-Dx-PgFl-Tr-FeCl3 nanoaggregate on DU-145 cells. a) concentration dependent effect on the viability of DU-145 cells (stained with crystal violate), and cell viability data plots for b) crystal violate staining assay and c) MTT assay.



Conclusion: Norbornene based copolymer Nr-Dx-PgFl-Tr-FeCl3 has been designed and successfully synthesized using ROMP technique. This smart nanoaggregate can function as dual imaging drug cargo through function of red fluorescence of doxorubicin and T1 weighted MR imaging by high spin Fe(III)-terpyridine (Fe-Tpy) units. Nr-Dx-PgFl-Tr-FeCl3 nano aggregates result high r1 relaxivity of ˃ 18 mM-1S-1 and low (r2/r1) ratio of ˂ 5.4 even at 11.7 T magnetic field which signifies our hypothesis that it can be used as an efficient high resolution T1 active MRI contrast agent. This copolymer showed sustained release of doxorubicin at acidic condition rather due to presence of acid sensitive hydrazone linker. The presence of PEG-folate motif makes the nanoaggregate water soluble and as well as site-specific. The cellular uptake pattern, uptake kinetics and retention were evaluated using fluorescence microscopy and flow cytometry in DU145 prostate cancer cells. Uptake pattern was concentration dependent as well as gradual where in cytoplasmic fluorescence indicates distribution of prodrug in the cytoplasm followed by nucleus. The anticancer efficacy of the nanoaggregate was studied in DU-145 cells using MTT and crystal violate staining cell viability assays. The nanoaggregate inhibited tumor cell growth at low concentration in vitro. This newly designed theranostic agent is expected to open up a new avenue for more effective cancer therapy through well-informed decision making. Acknowledgement: D.P. thanks IISER Kolkata for research fellowship, S.M. and I.C. thank CSIR, New Delhi, for research fellowship, T.K.D thanks DST for SERB-NPDF, R.S. thanks the Department of Science and Technology, New Delhi, for a Ramanujan Fellowship and the DBT for funding. R.S., R.B., and thank IISER Kolkata for providing the infrastructure and start-up funding.


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Iron (III) Coordinated Polymeric Nano-material: A Next Generation Theranostic Agent for High Resolution T1 weighted Magnetic Resonance Imaging and Anticancer Drug Delivery Diptendu Patra,1 Saikat Mukherjee,1 Ipsita Chakraborty,2 Tapan Kumar Dash,1 Shantibhusan Senapati,3 Rangeet Bhattacharyya,2 and Raja Shunmugam*1 *E-mail: [email protected] Table of Contents (TOC):


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Iron(III) Coordinated Polymeric Nanomaterial: A Next-Generation

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