Multifunctional Magneto-Fluorescent Nanocarriers for Dual Mode

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Multifunctional Magneto-Fluorescent Nanocarriers for Dual Mode Imaging and Targeted Drug Delivery Ashish Tiwari, Ashutosh Singh, Ayan Debnath, Ankur Kaul, Neha Garg, Rashi Mathur, Anup Singh, and Jaspreet Kaur Randhawa ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.9b00421 • Publication Date (Web): 11 Apr 2019 Downloaded from http://pubs.acs.org on April 14, 2019

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ACS Applied Nano Materials

Multifunctional Magneto-Fluorescent Nanocarriers for Dual Mode Imaging and Targeted Drug Delivery Ashish Tiwariǂ, Ashutosh Singh ᵵ, Ayan Debnathᵵ, Ankur Kaulδ, Neha Garg*ᵵ, Rashi Mathur*δ, Anup Singh*ᵵ and Jaspreet K. Randhawa*ǂ

ǂSchool

of Engineering, Indian Institute of Technology Mandi, Mandi, Himachal Pradesh-

175005, India ᵵSchool

of Basic Sciences, Indian Institute of Technology Mandi, Mandi, Himachal

Pradesh-175005, India ᵵCentre

for Biomedical Engineering, Indian Institute of Technology Delhi, Hauz Khas, New Delhi-

110016, India δInstitute

of Nuclear Medicine & Allied Sciences, Defence Research and Development Organization,

Brig SK Mazumdar Road, New Delhi-110054, India KEYWORDS. Magneto-fluorescent nanocarriers, magnetic resonance imaging, optical imaging, targeted drug delivery, dual imaging probe and cancer theranostics.

ACS Paragon Plus Environment

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Abstract

The authors describe a novel multifunctional magneto-fluorescent MFCSNPs-FA-CHI5FU nanocarriers that consists of magneto-fluorescent nanoparticles (MFCSNPs) targeted with folic acid (FA), modified with chitosan (CHI) and loaded with a 5-flouoruracil (5-FU) in dual mode imaging and targeted drug delivery. Multifunctional magnetofluorescent nanocarriers show multicolor emission and superparamagnetic behavior which are advantageous for bioimaging and magnetic resonance (MR) imaging respectively. In-vitro drug release studies show a pH- activated drug release with 92% of loaded 5-FU released in 30 hours and MR imaging exhibiting excellent dose-dependent signal enhancement in T2-weighted images. This suggests that MFCSNPs-FA-CHI-5FU nanocarriers can be used as T2-weighted negative contrast agents in cancer diagnosis. In-vitro cytotoxicity and anticancer activity of the synthesized MFCSNPs-FA-CHI-5FU nanocarriers were evaluated on three cancer cell lines having different percentage of folate receptors viz. A375, MCF-7 and HeLa cells. The results show a very sensitive drug targeting response. Confocal laser scanning microscopy imaging (CLSM) display


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targeted cellular internalization of MFCSNPs-FA-CHI-5FU nanocarriers in cancer cells. The biocompatibility of MFCSNPs-FA-CHI-5FU nanocarriers was ascertained ex-vivo by hemolysis and serum stability studies. The in-vivo biodistribution studies were evaluated by radiolabeling MFCSNPs-FA-CHI-5FU nanocarriers with 99m Technetium (99mTc). Thus, the synthesized nanocarriers, a dual mode imaging probe, show great potential in targeted drug delivery to improve the existing cancer theranostics.

1. Introduction

The current requisite to advance the cancer theranostics is to target, guide and control the release of therapeutic1,2. Accordingly, much attention has been focused towards the designing of single platform for diagnostics and therapy2,3. Varieties of nanomaterials especially porous structures metal oxides nanoparticles, noble metal nanoparticles, quantum dots, carbon nanodots and organic fluorescent materials have been broadly studied as carriers in cancer therapy,4,5,6 but biological and technological barriers limit the site specific bioavailability of the administered therapeutics7,8. In addition, there are some concerns in terms of biodegradability, toxicity and targeting ability of these


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nanomaterials9,10. Thus, external stimuli responsive drug delivery systems that specifically respond to light, magnetic field, electric field, heat and ultrasound offer potential towards targeting and systemic administration 11.

Superparamagnetic iron oxide nanoparticles (SPIONs) hold great potential due to their exceptional magnetic properties, chemical stability, biocompatibility, targeting ability, thermal conversion efficiency and biological degradability12. SPIONs can also be used as T2-weighted MR imaging agent13. In addition, functionalized SPIONs with fluorescent tags are also established as biocompatible drug carriers and being used as dual modal i.e. optical and MR imaging agent14,15. Thus, explicitly multifunctional magnetic and fluorescent nanoparticles kindle a design not only to improve the therapeutic efficacy but also offer simultaneous possibilities for diagnosis and therapy. In recent years, significant advancements have been attained in targeted drug delivery combining magnetic and fluorescent nanoparticles with specific biomarkers as targeting agents16. For example, Rana et al. amalgamated folic acid conjugated magnetic nanoparticles for targeted drug delivery in tumor therapy17. Similarly, Sun et al. designed dual color magnetic and


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fluorescence probes for cellular bioimaging in cancer cells18. Further, Lin et al. established magnetic and fluorescent nanocomposites loaded with therapeutic molecule for targeted cancer therapy19. Ye et al. synthesized a nanocarrier with SPIONs and quantum dots into polymer nanoparticles for therapeutics and cell imaging20. In their work, He et al. prepared dual mode fluorescence and MR imaging nanoprobe synthesized by encapsulating CdSe@CdS and Fe3O4 in amphiphilic copolymer for drug delivery21. Li et al. designed multifunctional magnetized porous silica nanoparticles for pH stimuli drug release and MR imaging22. However, multistep synthesis of magneto-fluorescent nanoparticles, surface deterioration of fluorescent molecules and low biocompatibility limits their real time applications in multimodal bioimaging23,24. For this reason, a single step synthesis of biocompatible and photostable fluorescent SPIONs is desirable.

In our previous work, we have developed multifunctional core-shell fluorescent SPIONs coated with carbon (MFCSNPs) in a single step synthesis25. The core of MFCSNPs consists of small sized SPIONs nanocrystals and the shell is made of amorphous carbon. These MFCSNPs have practically been evaluated and showed excellent MR imaging


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ability, multicolor emission and high photostability. The core–shell nanoparticle exhibits superparamagnetic behavior at room temperature, thus emphasizing their potential in biomedical applications. Furthermore, the carbonyl and hydroxyl groups present in MFCSNPs also offer a platform for surface functionalization with cancer biomarkers for targeted drug delivery. The aforesaid characteristic properties of MFCSNPs prompted us to explore their use as a dual mode imaging agent in cancer theranostics.

Herein, our objective was to design a target specific multifunctional magnetofluorescent drug delivery nanocarriers. To achieve this, MFCSNPs were functionalized with folic acid-chitosan (FA-CHI), conjugate and then loaded with the anticancer drug 5Fluorouracil (5FU) which is represented as MFCSNPs-FA-CHI-5FU nanocarriers (Scheme 1). The study includes a thorough and systematic evaluation of morphological, structural and functional properties of nanocarriers. In-vitro drug release experiments were done under acidic conditions (pH 5.5) to stimulate the tumor micro environment and compared with the release of 5-FU at the physiological pH (7.4). In addition, MFCSNPsFA-CHI-5FU nanocarriers demonstrated dose dependent contrast enhancement (signal


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darkening) in in-vitro MR imaging as T2-weighted contrast agents. In-vitro cytotoxicity studies tested on three cancer cell lines i.e. A375, MCF-7 and HeLa cell lines proved that MFCSNPs-FA-CHI-5FU nanocarriers are specific for folate receptor cancer cells. Confocal imaging displayed that nanocarriers can be internalized through accumulation in cell cytoplasm. The biocompatibility of the nanocarriers has been evaluated ex-vivo by hemolysis, and serum stability studies. The serum stability and in-vivo bio distribution in Balb/C mice was investigated by radiolabeling the MFSCNP and MFSCNP-FA-CHI-5FU nanocarriers with 99mTc.

2. Experimental section

2.1. Materials and Methods

All chemicals were purchased from Sigma Aldrich and the reagents were of analytical grades.

2.2. Material characterization


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X-ray diffraction (XRD) pattern were studied on a Rigaku Smart Lab diffractometer, using CuKα radiation from 5 to 80-degree 2θ with a scanning rate of 2 degrees/min. Particle size and elemental mapping were obtained on FEI Tecnai TEM (transmission electron microscopy) with STEM-HAADF (scanning transmission electron microscope high angle annular dark field) imaging operating at 200 kV. The surface morphology and energy dispersive x-ray analysis (EDAX) were performed using FESEM (field emission scanning electron microscopy-NOVA NanoSEM 450) at an accelerating voltage of 10 kV. Atomic force microscopy (AFM) imaging studies were performed in tapping mode at a scanning rate of 0.9 Hz using dimension ICON (Bruker) AFM. UV-Vis spectroscopy studies were undertaken using Shimadzu U-2450 (UV-Vis spectrophotometer) in the range of 800-200 nm. Fluorescence measurements were recorded on Horiba spectrophotometer in the range of 800 to 200 nm. FTIR spectroscopy was performed on Agilent Technologies Cary 6000 series from 400 to 4000 cm-1of wavelength. The thermal stability measurements were performed on Perkin Elmer Pyris thermogravimetric analyzer under a nitrogen atmosphere. Magnetic properties were analyzed through SQUID vibrating sample magnetometer (VSM) under the vibrating magnetic field of 4T at


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300 K. Raman measurements were performed on a Horiba LABRAM high-resolution Raman spectrophotometer. Raman measurements were done using 534 nm He-Ne laser. Hydrodynamic size and zeta potential were studied by dynamic light scattering (DLS) method. Brunauer–Emmett–Teller (BET) measurements were performed on Quanta chrome Autosorb 1C instrument to analyze the surface area and pore size distribution with nitrogen absorption and desorption isotherm at 77 K.

2.3. Synthesis of carbon coated core–shell multifunctional fluorescent SPIONs (MFCSNPs)

MFCSNPs were synthesized by our previously reported synthesis procedure.25 Initially, 0.2 gm of ferrocene was dissolved in 20 ml acetone and sonicated for 40 minutes. After intense sonication, 5 ml of H2O2 (30%) solution was slowly added to above solution under continuous stirring. Additionally stirring was continued for another 1 hour for completion. This was transferred to a 50 ml Teflon hydrothermal reactor at 220°C for 48 hours. Following the completion of the reaction, black precipitate was obtained. The precipitate was centrifuged, washed with acetone and dried at 40°C in a vacuum oven.


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2.4. Synthesis of folic acid and chitosan (FA-CHI) conjugate

Folic acid and chitosan conjugates were prepared by following a dehydration condensation reaction between the carboxyl groups of FA and amino groups of CHI.26 Typically, 20 mg FA was dissolved in 50 mL dimethyl sulfoxide (DMSO) and 20 mg of CHI dissolved in an acetic acid aqueous solution (0.1 M, pH 4.7). Both the solutions were mixed continuous stirring conditions. Following this, 40 mg N-(3-Dimethylaminopropyl)N-Ethylcarbodiimide hydrochloride (EDC) was added to the above solution under dark conditions and reaction was continued for 72 hours. After the reaction completion, pH of the solution was adjusted to 9.0 using 1 M NaOH solution. Then, the obtained FA-CHI conjugate were washed with DMSO and lyophilized to get purified conjugates.

2.5. 1H NMR spectroscopy studies of folic acid and chitosan (FA-CHI) conjugate

FA-CHI conjugate was characterized by

1H

NMR spectroscopy in D2O 3% v/v

CD3COOD solution for better solubility of the conjugates.


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2.6. Synthesis of multifunctional magneto-fluorescent nanocarriers (MFCSNPs-FACHI-5FU nanocarriers)

Typically, 0.1 g MFCSNPs, 0.1 g of 5-FU and 0.5 g FA-CHI conjugates were added to DI water followed by addition of 0.2 g of EDC under ultrasound for 30 minutes. The stirring was continued for 48 hours at room temperature in dark conditions. Finally, the reaction product was collected by centrifugation and washed with DI water followed by drying in vacuum oven at 25°C.

2.7. Drug loading estimation

The estimated concentration of 5-FU present in MFCSNPs-FA-CHI-5FU nanocarriers was determined by a standard calibration curve which was attained via analyzing different known concentration solutions of 5-FU at 265 nm. Drug loading capacity was calculated using the given equation:

Drug loading capacity (%) = ((m1 − m2)/m*100)


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Here, m1 denotes initial amount of 5-FU, m2 denotes amount of 5-FU present in supernatant solution and m denotes total amount of 5-FU.

2.8. In-vitro drug release study studies

In-vitro drug release of 5-FU from MFSCNP-FA-CHI-5FU nanocarriers was monitored at two different pH conditions (pH 7.4 and pH 5.5) using dialysis membrane method in PBS solution. To perform drug release studies, 2 mg/mL aliquot nanocarriers were placed in a dialysis bag and immersed in a 10 ml PBS at 37°C. At regular time interval, 2 mL of aliquot was replaced with an equal volume of PBS to maintain sink conditions. The collected aliquot was analyzed by UV-Vis spectra at 265 nm. [ref] Finally, the cumulative release of 5-FU at each time point was plotted against time and calculated as:

Percent cumulative release = (amount of released drug/amount of total drug) × (100%)

2.9. Phantom Preparation for magnetic resonance imaging (MRI) scanning


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MR imaging was performed following the reported literature27 using a base solution with 1.5% agarose gel. This base solution was put into a large (2L) cylindrical beaker as well as in nine 15 mm small test tubes. In small test tubes different iron concentrations were added to get concentration of nanocarriers ranging from 0.005, 0.01, 0.015, 0.02, 0.03, 0.05, 0.1, 0.25 and 0.5 mM respectively. These small phantoms 1 to 9 were inserted vertically (circumscribed) inside the large phantom.

2.10. MRI and relaxivity studies

MR imaging experiments were experimented at 3T whole-body MRI instrument (Ingenia, Philips Healthcare, The Netherlands) equipped with 16 channel receive only coil. Data for T2 map was acquired using vendor supplied T2 mapping pulse sequence with the option of multiple echoes. MRI protocol consisted of FOV=200*200 mm2, matrix size=256*256, number of slices=12, slice thickness=6 mm, TR =6000 ms and TE = 30, 60, 90, 120, 150, 180, 210 ms. MRI data for T1 map was also acquired using inversion recovery based pulse sequence. For this sequence we used TI = 100, 300, 500, 700, 1000, 1500, 2000, 2500, 3000 and 3500 ms.


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2.11. Relaxivity mapping

MR images were processed using in-house written programs in MATLAB. Voxelwise T2 map was generated by fitting signal intensity data corresponding to different TEs to following mono-exponentially decaying function:

𝑆(𝑇𝐸) = 𝐴 × 𝑒

―

𝑇𝐸 𝑇2

[1]

ROIs were drawn over small phantoms and average T2 values were computed for each ROI. R2 = 1/T2 values were computed. Transverse Relaxivity (r2) of contrast agent is estimated using the following equation:

𝑅2 = 𝑅20 + 𝑟2 × 𝐶

[2]

where, R2 is concentration dependent relaxation time and R20 is relaxation time of base solution or agarose without contrast agent and C is the concentration of contrast agent.

2.12. In-vitro targeted MRI studies


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HeLa and A375 cells were cultured under experimental hypoxic conditions in 6-well plates for 24 hours. When the cells confluence reached 80%, the cell culture medium was than substituted with 3 mL of fresh medium containing six different concentrations ranging from 0 to 200 μg/mL of MFCSNPs-FA-CHI-5FU nanocarriers. After another 24 hours, both the cancer cells were washed with fresh medium, digested and collected by centrifugation (1,000 rpm for 10 min). On the other hand, a base solution was similarly prepared with 1.5% agarose. This base solution was put into a large (1L) cylindrical beaker as well as in six 15 mm small test tubes. In small test tubes, cancer cells incubated with different concentrations of nanocarriers (0 to 200 μg/mL) were added respectively. These small phantoms 1 to 6 were inserted vertically (circumscribed) inside large phantom as shown in Figure 6. Control experiments without using nanocarriers were performed in small phantom tube 1. In-vitro MR imaging was performed by placing large phantom in a 3 T whole-body MRI scanner. For T2 mapping, MRI protocol consisted of FOV=200*200 mm2, matrix size=256*256, number of slices =12, slice thickness=6 mm, TR =6000 ms and TE = 30, 60, 90, 120, 150, 180, 210 ms. MRI data for T1 map was also acquired using inversion recovery based pulse sequence. For this sequence we used


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TI = 100, 300, 500, 700, 1000, 1500, 2000, 2500, 3000 and 3500 ms. T2 and T1 maps were generated. Region of interests (ROIs) were selected to encompass cross sections of respective tubes and average T2 values were computed.

2.13. Cell culture studies and in-vitro cytotoxicity assays

Three cancer cells i.e. A375, MCF-7 and HeLa were purchased from the National Centre for Cell Science (NCCS), Pune, India. At the optimized confluency of the cells, it were seeded in 96 well plate (density of 5000 cells/well) and allowed to adhere overnight. Cells were then treated with various concentration (12.5, 25, 50, 100 and 200 µg/mL) of MFCSNPs,

MFCSNPs-FA-CHI-5FU

nanocarriers

and

5-FU

respectively.

The

concentration of 5-FU used for cell treatment was relative to the % loading of 5-FU in MFCSNPs-FA-CHI-5FU nanocarriers (loading percentage = 6.72 %). After 24 hours of incubation, 20 µl of 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was added and incubated for 3 hours. The cells were then dissolved in 100 µl of DMSO and absorbance was monitored at 570 nm with 650 nm as a reference in Tecan Infinite M200 PRO plate reader.


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2.14. Confocal laser scanning microscopy studies for cellular internalization

Cellular internalization studies were performed as earlier reported method.5 For confocal studies, HeLa cells were cultured on a cover slip in a 6 well plate and allowed to adhere for 24 hours. Then, cells were treated with 25 µg/mL of MFCSNPs and MFCSNPsFA-CHI-5FU nanocarriers for 3 hours and imaging was done using Nikon Eclipse Ti-U inverted confocal microscope.

2.15. In-vitro targeted cellular internalization

For in-vitro cell uptake studies A375 and HeLa cells were cultured as described in cellular internalization studies. A375 and HeLa cells were incubated with 25 µg/mL of MFCSNPs-FA-CHI-5FU nanocarriers for 3, 6 and 12 hours and imaging was done using Nikon Eclipse Ti-U inverted confocal microscope.

2.16. In-vitro hemolysis studies

A human blood sample (5 mL) was collected from a volunteer and taken in an Ethylenediamine tetraacetic acid (EDTA) glazed tube and used within an hour. A


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centrifugation was done at 1600 rpm for 10 min to distinct RBCs from blood sample. The separated RBCs were washed three times with PBS solution. A 4% suspension of prepared of RBCs solution in PBS was used for further experiments. In a typical experiment, a round bottom 96 well plate 100 µL containing RBCs suspension was treated with 100 µL of various concentrations of MFCSNPs and MFCSNPs-FA-CHI-5FU nanocarriers in the range of 12.5, 25, 50, 100, 200 µg/ml. All concentrations were taken in quadruple. Subsequently, plate was stirred gently and further incubated for 2 hours at 37 ºC. RBCs incubated in PBS were used as a negative control and RBCs with 1% SDS were used as a positive control. After the postulated incubations, all samples were again centrifuged at 1600 rpm for 10 min followed by transferring supernatant to a 96 well plate. The collected supernatant was determined at 414 nm in UV-Vis spectroscopy via Tecan Infinite M200 PRO plate reader. Hemolysis percentage was calculated using the given equation,

Hemolysis percentage = (At ‒ Ac)/ (Ax ‒ Ac)


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where, At is the absorbance of treated supernatant, Ac is the absorbance of negative control and Ax is the absorbance of positive control.

2.17. In-vitro serum stability studies

The in-vitro serum stability of MFSCNPs and MFSCNPs-FA-CHI-5FU nanocarriers was determined in serum samples by following the standard protocol.28 For serum stability experiments, 100 mL of

99mTc

labelled nanocarriers were incubated in 900 μL of the

serum (in duplicate) at 37oC. Then samples were analyzed to check for any dissociation by instant thin layer chromatography (ITLC) using silica gel strips in developing solvent (0.9% NaCl aqueous saline solution). The variation in labelling efficiency was examined for 24 hours.

2.18. Radiolabeling studies

An aqueous solution of MFSCNPs and MFSCNPs-FA-CHI-5FU nanocarriers (9.78 nM) was added in stannous chloride solution in a shielded vial by following the reported work.28 The solution pH was adjusted to 6.5–7.0 with NaHCO3 (0.5 M) and filtered through


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a 0.22 μm filter into a sterile vial. Freshly eluted

99mTcO 4

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- (93.7 MBq) was added in this

solution and incubated for 30 min at RT for optimum labelling yield. The estimation of labelling efficiency was determined using ITLC-SG as a stationary phase and 100% acetone as a mobile phase. The paper chromatography confirmed the absence of unlabeled Na99mTcO4.

2.19. Biodistribution studies

To perform biodistribution studies, the Balb/C mice were administered with 100 μCi of 99mTc

labelled nanocarriers through the tail intravenously as described in previous

reported work.28 At subsequent time intervals of post injection (1, 2, 4 and 24 hours), animals (n = 3) were euthanized and by cardiac puncture, the blood was collected into pre-weighed tubes. Further, mice were dissected and various organs (heart, lungs, liver, spleen, kidneys, stomach, intestine, muscle) were eradicated and weighed. The radioactive counts were recorded with the help of gamma counter for all the removed organs. The quantitative uptake of radiolabeled nanocarriers into each organ was dignified per gram of the tissue/organ and expressed as the percentage injected dose per


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gram (% ID/gm) organ weight. The radioactivity residing in the tail (at injection point) was also quantified and used in calculating the radioactivity.

2.20. Animal handling and experiments for in-vivo studies

In-vivo animal experiments protocols were approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India, New Delhi. Animal experimentation and studies were performed following the guidelines of the Institutional Animal Ethics Committee. The Balb/c mice of age 6 to 8 weeks were given food, water, libtium and housed in 12/12 hours light and dark cycles respectively.

3. Results and Discussion

3.1. Morphological and structural analysis

Carbon coated core-shell multifunctional fluorescent SPIONs (MFCSNPs) were synthesized as described in our previous studies.25 TEM images of MFCSNPs as shown in Figure 1(a-d) reveal spherical assembly of small iron oxide nanocrystals covered with in an amorphous carbon shell with a mean particle size of 220±10 nm. High-resolution


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(HR) TEM imaging displays inter-planer distance i.e. d-spacing to be 0.25 nm (Figure 1 (e, f)). This value matches with the reflection of (311) plane of Fe3O4. However, the HRTEM image also reveals the absence of any crystalline ordering in the shell as shown (Figure S1 SI (supporting information)) which support that carbon is present in amorphous form. STEM-HAADF imaging (Figure 1 (g)) is comparable with this observation. Further, the TEM image of MFCSNPs-FA-CHI-5FU nanocarriers shown in Figure 1 (h) exhibits additional surface layer. This is attributed to the functionalized layer of FA-CHI. FESEM images of MFCSNPs reveal uniform spherical shell structure as shown in Figure S2 (a) (SI). The elemental mapping overlay image (b) successfully confirmed the presence of iron (c), oxygen (d) and carbon (e). This is supported with FESEM-EDS spectra (f) of MFCSNPs. Similarly, the FESEM image of MFCSNPs-FA-CHI-5FU nanocarriers shown in Figure 2 (a) display spherical morphology. The elemental mapping (Figure 2 (b-g)) and EDS spectra (h) subsequently confirmed the presence of iron (c), oxygen (d), carbon (e), nitrogen (f) and fluorine (g) which signify that MFCSNPs were conjugated with FA-CHI and loaded with 5-FU. Atomic force microscopy (AFM) image of MFCSNPs-FA-CHI-5FU nanocarriers in Figure 2 (i) showed spherical morphology. The height profile analysis


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(Figure 2 (j)) displayed similar topographies i.e. particle size and structural features as obtained in FESEM and TEM imaging respectively.

X-ray diffraction (XRD) pattern of MFCSNPs reveal the presence of crystalline magnetite phase as shown in Figure 3 (a). The sharp 2θ reflections at (111), (220), (311), (400), (422), (511), and (440) represent Fe3O4 in cubic crystal phase that matches with JCPDS card no. 01-1111. The broad peak with weak intensity at 24 degree is distinctive of amorphous graphitic carbon reflection (002). XRD spectra of MFCSNPs-FA-CHI-5FU nanocarriers was found quite similar to pure MFCSNPs without any additional peak which confirms that the presence of FA-CHI and 5-FU did not change the crystal structure of MFCSNPs. The vibration modes presents in MFCSNPs and MFCSNPs-FA-CHI-5FU nanocarriers were studied using Raman spectroscopy as shown in Figure 3 (b). The bands at 214, 283, 384, 479, 654 cm-1 are attributed to various binding modes of Fe3O4 present in MFCSNPs.29 In addition, the bands observed at 1331 and 1586 cm-1 correspond to D and G band of carbon respectively.30 Moreover, MFCSNPs-FA-CHI-5FU nanocarriers showed all the vibrational modes of Fe3O4 and carbon similarly present in


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MFCSNPs. A decrease in the intensity of G band was observed due to less prominent ratio of D and G band which may be ascribed to the presence of FA-CHI conjugates. However, no such characteristic features for FA-CHI surface coating were recognized. This observations align with the FTIR spectroscopy studies.

3.2. Formation of Folic acid and chitosan conjugate

An acid and amine coupling reaction was used to conjugate FA and CHI as specified in Scheme S1 (SI). The conjugation was confirmed via 1H NMR spectroscopy as shown in Figure S3 (a) SI. The peaks at 7.31, 7.83, and 8.69 δ ppm are due to the aromatic proton of FA. The peaks in between 3.44-3.74 δ ppm and 3.03 δ ppm corresponds to -CH of CHI and NH-C=O bond between FA and CHI respectively. The characteristic peaks of FA and CHI in FA-CHI conjugates establish the successful bonding of FA and CHI.26,31 The 1H NMR spectra of FA and CHI is shown in Figure S3 (b, c) SI respectively. FA-CHI conjugation was further confirmed by FTIR spectra as shown in 4 (a). The pristine folic acid displayed stretching vibrations of C=C observed at 1608 cm−1 while bands at 2921 and 2856 cm−1 were assigned to C-H vibrations. The broad band between 3500-3300


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cm−1 is attributed to N-H and O-H vibrations respectively and a sharp peak at 1384 cm−1 was assigned to symmetrical CH3 deformation mode. The stretching vibration of C=O was noticed at 1695 cm−1 in FA spectra. Similarly, FTIR spectrum of FA-CHI conjugates contained the characteristic bands of FA and CHI. However, the stretching vibrations of carboxyl groups of FA were absent in FA-CHI conjugate. The broad band appeared after conjugation at 3500 to 3300 cm−1 is from chitosan in FA-CHI spectra and the absence of stretching vibrations of carboxyl groups of FA in FA-CHI spectra is due to formation of amide bond between FA and CHI molecules which is in accordance with NMR results. Moreover, FA-CHI conjugate illustrated two new peaks at 1705 and 1565 cm−1 which are related to amide I and amide II bands through a reaction between the COOH groups of FA and NH2 of chitosan.32 This results persistent with 1H NMR spectroscopy and confirmed the formation of FA and CHI (Figure S3 (a) in SI).

3.3. Functional group, thermal stability and optical property studies

Subsequently, Figure 4 (b) shows the FTIR spectra of 5-FU, MFCSNPs, FA-CHI, MFCSNPs-FA-CHI and MFCSNPs-FA-CHI-5FU nanocarriers. In 5-FU spectra, the peaks


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at 1506 and 1427 cm−1 are attributed to symmetric stretching and wagging vibrations of C=O and N-H bonds respectively. The broad bands at 1637 and 1424 cm−1 are assigned to C=O and C=C vibration in MFCSNPs. In addition, Fe-O metal oxide peak was also spotted at 554 cm-1. An intense peak observed at 1558 cm-1 corresponded to amide band confirmed the conjugation of MFCSNPs and FA-CHI. FTIR spectra established the formation of amide bond between COOH of MFCSNPs and NH2 of chitosan. FTIR spectra of MFCSNPs-FA-CHI-5FU nanocarriers in Figure 4 (b) display all the noticeable peaks of MFCSNPs, 5-FU and FA-CHI stretching vibrations demonstrating the fabrication of MFCSNPs-FA-CHI-5FU nanocarriers. The peaks at 1656 and 1243 cm−1 are associated with 5-FU. While the peaks at 554 and 1565 cm−1 are related to MFCSNPs and FA-CHI, respectively. Parallel with FA-CHI and MFCSNPs, the peak around 3500 cm−1, derived from the N-H stretching vibration in MFCSNPs-FA-CHI-5FU nanocarriers, becomes narrowed and red-shifted. This established the formation of hydrogen bonds between MFCSNPs and FA-CHI conjugate with 5-FU.32,17 Moreover, the FTIR spectra of MFCSNPs-FA-CHI-5FU nanocarriers showed all the characteristic peaks of 5-FU,


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MFCSNPs and FA-CHI, which clearly validate the successful formulation of MFCSNPsFA-CHI-5FU nanocarriers.

Zeta potential and hydrodynamic diameter of nanocarriers were studied using DLS measurements (Figure S4 (SI)). Zeta potential of MFCSNPs and MFCSNPs-FA-CHI-5FU nanocarriers was found to be -4.18 and -15.6 mV respectively. This rise in negative zeta potential infers the presence of more carboxylic groups in MFCSNPs-FA-CHI-5FU nanocarriers. A small rise in mean hydrodynamic diameter was also evident from ca. 320 and 350 nm for MFCSNPs and MFCSNPs-FA-CHI-5FU nanocarriers respectively as shown in Figure S5 (SI). The stability of MFCSNPs-FA-CHI-5FU nanocarriers in aqueous solution was also studied by DLS measurements shown in Figure S6 (SI) and there is no significant change in hydrodynamic size which infers the stability of MFCSNPs-FA-CHI5FU nanocarriers. The pore volume and specific surface area measurement were studied using Brunauer–Emmett–Teller (BET) measurements as shown in Figure S7-S10 (SI). The graphs represent nitrogen adsorption and desorption isotherms and pore size distributions of MFCSNPs and MFCSNPs-FA-CHI-5FU nanocarriers. Surface area, pore


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volume and average pore size of MFCSNPs and MFCSNPs-FA-CHI-5FU nanocarriers were calculated to be 273.035 m2/g, 1.144 cc/g, 83.76 Å, 113.639 m2/g, 33.09 cc/g and 58.22 Å respectively. A decrease in surface area, pore size and pore volume suggests the conjugation of FA-CHI on the surface of MFCSNPs.

Optical properties of FA, CHI, FA-CHI, MFCSNPs and MFCSNPs-FA-CHI-5FU nanocarriers were also studied as displayed in Figure S11 (SI). Folic acid showed their characteristic absorption peaks at 285 and 368 nm while chitosan exhibited two broad absorption peaks at 290 and 360 nm. After conjugation of FA and CHI, the characteristic peak of FA was blue shifted and observed at 270 nm. This also confirms the interaction of FA and CHI conjugate. MFCSNPs spectra display two transitions, a broad absorption peak and a shoulder peak at 450 and 225 nm which corresponds to nπ* transitions of carboxylic groups and π-π* transitions of aromatic carbon in MFCSNPs respectively while in MFCSNPs-FA-CHI-5FU nanocarriers, the absorption peak was red shifted with an increase in the intensity. This is the unique absorption spectra of MFCSNPs-FA-CHI-5FU nanocarriers which could be employed in fluorescence


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imaging. The successful formation of MFCSNPs-FA-CHI-5FU nanocarriers was further confirmed by Thermogravimetric analysis (TGA) performed under inert atmosphere for MFCSNPs, FA, CHI, FA-CHI and MFCSNPs-FA-CHI-5FU nanocarriers. Figure S12 (SI) shows that the MFCSNPs were stable upto 900°C with ca. 25% of weight loss which can be attributed to carbon content present in sample. Thermal curves of FA and CHI were similar to the reported data in literatures and showed almost complete weight loss. However, in TGA curve of MFCSNPs-FA-CHI-5FU nanocarriers, a weight loss of ca. 12% estimated in between temperature range of 220 to 500°C corresponding to the mass loss of FA-CHI and 5-FU followed by second weight loss upto 900°C which corresponds to removal of carbon content present in sample respectively.

3.4. Fluorescence and magnetic properties studies

Figure S13 (SI) shows the multicolor emission behavior of MFCSNPs. Interestingly, an excitation dependent emission spectra was obtained ranging from 280 to 480 nm of excitation wavelength. Fluorescence intensity was red shifted on increasing excitation wavelength. This type of emission is an identical property of carbonaceous species


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present in MFCSNPs as confirmed by Raman and XPS spectroscopy.25 The prevailed fluorescence in MFCSNPs may be attributed to electronic transition of C=C cyclic aromatic hydrocarbons present in the frame of amorphous carbon shell. The excitation energy of incident photons stimulates π- π* electronic transitions from ground state to higher energy states. Due to energy dissipation of emission photons during relaxation, they are of lower energy and appears at higher wavelength in fluorescence spectra. Further, as evident from FTIR spectroscopy, the carbon shell of MFCSNPs have abundant functional groups such as C=O, C=C, C-O-C and C-O-H on their surface leading to presence of various surface states that enable series of emissive energy traps during the excitation phenomenon. When a particular light of certain excitation wavelength is imposed on MFCSNPs, these surface energy traps prevail the emission. On subsequent change in excitation wavelength of incident light, different energy traps present on carbon shell dominate and contribute in the emission. This may lead to a conclusion that various energy states due to presence of surface functional groups are responsible for multicolor emission behavior of carbon shell of MFCSNPs.33 Further, on comparing emission peaks of MFCSNPs and MFCSNPs-FA-CHI-5FU nanocarriers at


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excitation wavelength of 320 nm (Figure 5 (a)), no significant change in spectra was observed, this signifies that coating of FA-CHI on MFCSNPs merely modulated emission behavior. The advantage of carbon shell in MFCSNPs is to prevail the multicolor fluorescence to enable the optical imaging properties making the MFCSNPs itself optically active to avoid use of optical bioimaging agent. Remarkably, strong fluorescence of MFCSNPs-FA-CHI-5FU nanocarriers can be used for optical bioimaging. Figure 5 (b) displays the hysteresis (M-H) curve (measured at 300 K) for MFCSNPs and MFCSNPs-FA-CHI-5FU nanocarriers, which showed that both the MFCSNPs and MFCSNPs-FA-CHI-5FU nanocarriers displayed superparamagnetic behavior. The saturation magnetization value of MFCSNPs-FA-CHI-5FU nanocarriers was 24.57 emug−1, slightly lower than 29.04 emug−1 of MFCSNPs. This may be attributed to the shielding effect of FA-CHI coating and can be related to surface modification of nanoparticles leading to the disruption of long range magnetic ordering.34,35 This saturation magnetization value can fulfil the requirement of magnetic guiding of MFCSNPs-FA-CHI-5FU nanocarriers as shown in Figure S14 SI. MFSCNPsFA-CHI-5FU were dispersed in DI water and forms brown uniformly dispersed solution.


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After a span of time, the brown nanoparticles were drawn towards the glass wall on applying an external magnet. This clearly show that nanocarriers are strong enough to be magnetically guided in applied magnetic field during their circulation in blood plasma.

3.5. Drug loading and release studies

To estimate the amount of 5-FU present in nanocarriers, drug loading capacity (DLC) was calculated using standard procedure as described in experimental section appropriately. A ca. 6.72% of 5-FU loading was achieved practically in MFCSNPs-FACHI-5FU nanocarriers. To estimate the drug release from MFCSNPs-FA-CHI-5FU nanocarriers, a standard calibration curve was plotted as shown in Figure S15 and S16 (SI). The calibration curve shows proportionality between absorbance and concentration ranging from 2 to 40 μg/mL. A linear fitting of calibration curve estimated the standard equation to figure out the concentration of released 5-FU in drug release studies. The standard equation could be figured out as follows (y = 0.02841x + 0.06176, R2 = 0.98406), where y is absorbance, x is the concentration of 5-FU and R is regression coefficient. The concentration of released 5-FU from nanocarriers was estimated from the calibration


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curve. Drug release experiments were performed in two PBS solutions i.e. pH 5.5 and pH 7.4. Percentage cumulative drug release as a function of time is expressed related to the proportion of drug released displayed in Figure 5 (c). The release profile of 5-FU follows two steps: initial fast and subsequent slow drug release. A rational ca. 70 and 30% of drug release was observed at initial 4 hours of time span followed by subsequent slow drug release of ca. 94 and 68% over 30 hours in pH 5.5 and pH 7.4 respectively. This implies that 5-FU entrapped into the nanocarriers was released faster in acidic conditions compare to physiological conditions. This may be due to the degradation of chitosan molecules in acidic pH which ensure fast release of 5-FU from nanocarriers in acidic conditions. This results demonstrate that MFCSNPs-FA-CHI-5FU nanocarriers are promising candidate for pH stimuli responsive release of 5-FU in drug delivery.

3.6. Magnetic resonance (MR) imaging studies

MR imaging of MFCSNPs-FA-CHI-5FU nanocarriers was performed to evaluate their use as a contrast agent. It is established that magnetic nanoparticles are able to decrease the MR signal intensity by dropping the value of transverse relaxation time. From the T2-


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weighted MR images shown in Figure 6 (a), it was perceived that MFCSNPs-FA-CHI-5FU nanocarriers decreased MR signal intensity on increasing Fe concentration. MR phantom images observed from various MFCSNPs-FA-CHI-5FU nanocarriers formulations showed better negative contrast (signal darkening) improvement than pure agarose gel. In Figure 6 (b), a color bar from red to blue shows a significant decrease in MR signal intensity. The plot of relaxation rate (1/T2) as a function of Fe concentration (0.005 to 0.30 mM) determined the relaxivity (r2) of MFCSNPs-FA-CHI-5FU nanocarriers to be 26.80 mM−1s−1 (Figure 6 (c)). The observed relaxivity (r2) for MFCSNPs-FA-CHI-5FU nanocarriers is comparable to the reported commercial MR contrast agents such as MIONs and Sinerem as reported SPIONs.36,37 This supports the use of MFCSNPs-FACHI-5FU as an excellent T2-weighted negative contrast agent in cancer diagnosis.

3.7. In-vitro targeted MR imaging studies

Target specific labelling of cancer cells with nanocarriers enhances MR contrast and enable the in-vitro performance in MR imaging. To evaluate the targeting capability of MFCSNPs-FA-CHI-5FU in cancer cells, the T2 weighted MR imaging of A375 and HeLa


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cells treated with MFCSNPs-FA-CHI-5FU nanocarriers were measured by a 3.0 T MR scanner. Typically, A375 and HeLa cells were cultured with various concentrations of MFCSNPs-FA-CHI-5FU nanocarriers (10, 25, 50, 100 and 200 μg/mL). Figure 6 (d, e), shows that the T2 weighted images gets darker with the increased concentration of MFCSNPs-FA-CHI-5FU nanocarriers respectively. Figure 6 (f) shows a plot between the internalized concentration of nanocarriers in HeLa cells and A375 cells against the concentration of nanocarriers incubated. Based on the relaxivity value of MFCSNPs, the internalized concentration of nanocarriers in HeLa and A375 cells was estimated to be 0.11 and 0.054 mM (at 50 μg/mL) respectively. This show that uptake of nanocarriers was high in HeLa cells due to presence of folate receptors. From the in-vitro MR imaging results, we proved that the MFCSNPs-FA-CHI-5FU nanocarriers can specifically target HeLa cells which have high folate receptors than A375 cells. This results indicated target specific internalization of MFCSNPs-FA-CHI-5FU nanocarriers in cancer cell and their potential as efficient T2-weighted contrast agent in in-vitro MR imaging.

3.8. In-vitro cytotoxicity studies


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A drug carrier should have the characteristic property of biocompatibility, non-toxicity and unique cellular uptake. To study these properties, MFCSNPs, MFCSNPs-FA-CHI-5FU nanocarriers and free 5-fluorouracil (5-FU) were screened on three different cell lines. Figure 7 (a-c) represent the cytotoxicity data for A375 (a), MCF-5 (b) and HeLa (c) cancer cells respectively. As presented in Figure 7, MFCSNPs did not show any cytotoxicity against all three cancer cell lines even at higher concentration. The percent cell viability of A375, MCF-7 and HeLa cells against 200 µg/mL concentration of MFCSNPs were found to be 87.5, 83.11 and 89.42% respectively. These results show that MFCSNPs are non-toxic for the tested cell lines and can be used as an efficient drug carrier. The cell viability data demonstrated that free 5-FU is showing appreciable cytotoxicity against A375 cells in a dose dependent manner, while the cytotoxic potential of free 5-FU got decreased against HeLa and MCF-7 cells. The observed cell cytotoxicity of free 5-FU toward A375, HeLa and MCF-7 at highest tested relative concentration of 13.4 µg/mL was found to be 59.13, 51.87 and 26.79 % respectively. The anticancer activity of free 5-FU obtained in the present study is in harmonious with the previous reports.38 On the other side, MFCSNPs-FA-CHI-5FU nanocarriers did not show any appreciable cytotoxicity


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against A375 and MCF-7 cells after 24 hours of treatment and obtained cell viability of MFCSNPs-FA-CHI-5FU nanocarriers at 200 µg/mL was found to be 70.73 and 69.12 % against A375 and MCF-7 cells respectively. On contrary to this, MFCSNPs-FA-CHI-5FU nanocarriers showed a noteworthy cell anti-proliferative potential toward HeLa (51.54 % cell viability at 200 µg/mL) and the activity was also found to be comparable with the cytotoxicity of free 5-FU toward the HeLa cell line. A375 cells are the folate receptor (FR) negative cell line39 and always used as a FR negative control, while the FR expression on the cell surface of MCF-7 is moderate as compared to the HeLa cells which have highly expressed FR markers.40,41 Therefore in the present study, synthesized MFCSNPs-FA-CHI-5FU nanocarriers that are folic acid conjugated show considerable cell cytotoxicity against HeLa cells. The obtained cytotoxicity of the MFCSNPs-FA-CHI5FU nanocarriers is almost same in the A375 and MCF-7 because though the MCF-7 cells show moderate FR expression but the IC50 of 5-FU against MCF-7 cell line is at higher range.

3.9. Cellular internalization studies (CLSM studies)


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As evident from fluorescence studies that MFCSNPs show strong emission spectra which can be used for cellular internalization studies using confocal microscopy. Herein, we have studied the cellular internalization of MFCSNPs and MFCSNPs-FA-CHI-5FU nanocarriers against HeLa cells after 3 hours of incubation. Figure 8 clearly shows blue, green, yellow and red emission from cell cytoplasm which confirm that both MFCSNPs and MFCSNPs-FA-CHI-5FU nanocarriers are able to internalize inside HeLa cells and exhibit excitation wavelength dependent emission in cellular environment. The results conclude that the synthesized MFCSNPs-FA-CHI-5FU nanocarriers can be used as dual modal agent in targeted drug delivery.

3.10. In-vitro targeted cellular internalization studies on cancer cells

To further evaluate the targeted specific nature of nanocarriers, we also studied the cellular internalization of MFCSNPs-FA-CHI-5FU nanocarriers against two cancer cells having differential expression of folate receptors i.e. A375 and HeLa cells for 3, 6 and 12 hours of incubation time subsequently. As shown in Figure 9 nanocarriers were internalized in both the cells i.e. A375 (Figure 9 (a)) and Hela cells (Figure 9 (b)) in time


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dependent manner (for high incubation time high internalization was observed). The fluorescence gained form Hela cells which bears high folate receptors was prominent for all three time points, showing high cellular uptake due to targeting ability of nanocarriers. A comparison of mean fluorescence intensity (MFI) for A375 and HeLa cells at all three time points as displayed in Figure 9 (c), which was higher in HeLa cells than A375 (c.a. four times for 12 hours of incubation) justified that the folate receptors present on cancer cells surface enabled targeted cellular internalization of MFCSNPs-FA-CHI-5FU nanocarriers.

3.11. In-vitro hemolysis studies

The biocompatibility of the nanocarriers was tested by blood hemolysis experiments. MFCSNPs and MFCSNPs-FA-CHI-5FU nanocarriers (0-200 µg/mL) were incubated with red blood cells for 2 hours and the released hemoglobin was spectroscopically measured via Tecan Infinite M200 PRO plate reader. The hemolytic potency of nanocarriers was shown in Figure S17 (SI) along with 1% SDS (positive control). MFCSNPs and MFCSNPs-FA-CHI-5FU nanocarriers showed 5.7 and 5.4% hemolysis at highest tested


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concentration (200 µg/mL) after 2 hours of incubation respectively. The hemolysis experiment displayed that both the samples are highly biocompatible with least hemolytic activity even at higher concentration.

3.12. Serum stability studies

The serum stability was done after radiolabeling the nanocarrier with

99mTc

and

incubated with the serum at 37oC for a period of 24 hours. Figure 10 (a) shows the labeling efficiency of MFSCNPs and MFSCNPs-FA-CHI-5FU was found to be >97% over a period of 24 hours and samples were stable upto 24 hours in serum (labeling efficiency > 98%). MFSCNPs and MFSCNPs-FA-CHI-5FU show a 98% stability in serum over a period of 24 hours which demonstrates that there is no degradation of the nanocarrier due to the interaction with the serum proteins.

3.13. In-vivo biodistribution studies

After the hemocompatibility experiments, the in-vivo biodistribution of the MFSCSNPs and the MFCSNPs-FA-CHI-5FU nanocarriers over a period of 24 hours was evaluated.


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Figure 10 shows the in-vivo biodistribution, which was studied by radiolabeling the nanocarriers with technetium-99m (99mTc) radionuclide. The radiolabeling of nanocarriers was done with 99mTc to measure their biodistribution quantitatively. This is considered to be the best method to monitor the time dependent uptake of nanocarriers in in-vivo experiments.

In-vivo biodistribution of the MFCSNPs and MFCSNPs-FA-CHI-5FU nanocarriers show a hepatobiliary and renal route of clearance. After 4 hours of injection, the MFSCNPs in the circulation is less than 10% in most of the organs and so in the case with the MFSCNPs-FA-CHI-5FU nanocarriers except in liver which shows that most of the nanocarrier is metabolized in the liver. As displayed in Figure 10 (b), only 0.46% and 1.60% MFSCNPs and MFSCNPs-FA-CHI-5FU nanocarriers were present in blood after 24 hours respectively. This further corroborates our assumption that the nanocarrier will not have any significant toxicity in the blood. In case of the tumor microenvironment, the specific uptake would be higher due to the presence of folate receptors which would


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further impact the amount present in circulation. Hence, we conclude that the nanocarrier system would be really stable and biocompatible in in-vivo.

3.14. In-vivo animal evaluation studies

In-vivo antitumor efficacy of different concentration of nanocarriers were investigated on Balb/c mice. The Balb/C mice were administered with 100 μCi of

99mTc

labelled

nanocarriers through the tail intravenously as described in experimental section. Figure 10 (b) shows that MFCSNPs-FA-CHI-5FU nanocarriers were accumulated in liver maximally compare to other organs. The dose given to animals with respect to subsequent time is plotted and the accumulation of samples in various organs was estimated through radiolabeling. This accumulation was almost eliminated from the liver within 24 hour of post injection. A time dependent biodistribution in various body organs after IV administration (Figure 10 (b)) show that the nanocarriers are initially accumulated in liver and subsequently their presence was detected in kidney. After 24 hours of IV injection, low accumulation in liver showed possible clearance of nanocarriers. However, no significant accumulation of nanocarriers upto 24 hours in blood, heart, lung, stomach


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and intestine also minimizes any kind of the adverse effect and deliberated the biocompatibility in these organs. A low concentration in spleen also favors that nanocarriers were not being recognized as foreign particles by macrophages and could bypass this barrier. Similarly, as example, iron oxide nanoparticles, colloidal gold and silica nanoparticles demonstrated high accumulation in liver and spleen with minimum quantity in the blood.42,43 In conclusion, nanocarriers demonstrated extensive biodistribution in liver and kidney with minimum concentration in other organs. The biodistribution study of nanocarriers experimented through radiolabeling showed the invivo targeted ability of nanocarriers for efficient targeted drug delivery.

4. Conclusion

In summary, folic acid and chitosan (FA-CHI) targeted and 5-Flourouracil (5FU) loaded carbon coated core-shell multifunctional SPIONs (MFCSNPs) based drug delivery nanocarriers (MFCSNPs-FA-CHI-5FU nanocarriers) have been prepared in this work. MFCSNPs-FA-CHI-5FU nanocarriers show pH responsive release of 5-FU from nanocarriers and further, due to the presence of folic acid it is used as a site specific drug


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carrier. The potential of nanocarriers in MR imaging was also determined and a contrast enhancement (signal darkening) in MR imaging demonstrated that MFCSNPs-FA-CHI5FU nanocarriers show potential as T2-weighted MR contrast agent. In-vitro cytotoxicity and confocal microscopy imaging results displayed that MFCSNPs-FA-CHI-5FU nanocarriers are specific for folate receptor positive cancer cells and can be internalized through accumulation in cell cytoplasm. The preliminary biological evaluation also reinforces the hemocompatibility and the biocompatibility of the nanocarrier making the use of these multifunctional magnetic fluorescent nanoparticles as new targeted theranostic system. Additionally, this work provides an effective way to explore multifunctional magneto-fluorescent nanoparticles as new targeted drug delivery systems. Strong magnetic and bright fluorescence properties of nanocarriers are more advantageous for cancer theranostics.


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Figure 1. (a, b) Transmission electron microscopy images of MFCSNPs (Scale Bar (a)-100 nm and (b)-200 nm), High resolution (HR-TEM) image (Scale Bar (c)-20 nm and (d)-10 nm) (c-f) confirming the presence of Fe3O4 nanocrystals similarly matched with XRD spectra. (g) Scanning transmission electron microscope


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high angle annular dark field (STEM-HAADF) image shows core-shell structure of MFCSNPs (Scale Bar200 nm) and (h) TEM image of MFCSNPs-FA-CHI-5FU nanocarriers respectively (Scale Bar-50 nm).

Figure 2. Field Emission Scanning Electron Microscopy image of MFCSNPs-FA-CHI-5FU nanocarriers (a), overlay image of elemental mapping (b), elemental mapping images for iron (c), oxygen (d), carbon (e), nitrogen (f), fluorine (g) and EDS pattern (h) respectively. Scale Bar (10 µm). Atomic force microscopy (AFM) image (i) and 3D height profile (j) of MFCSNPs-FA-CHI-5FU nanocarriers. (Scale Bar-1µm)


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Figure 3. (a) X-ray diffraction spectra of MFCSNPs and MFCSNPs-FA-CHI-5FU nanocarriers showing the reflections of diffraction peaks. The standard card of Fe3O4 is referenced (JCPDS card no. 01-1111). The diffraction peak noted with arrows was indexed to amorphous graphitic carbon. (b) Raman spectra of MFCSNPs and MFCSNPs-FA-CHI-5FU nanocarriers showing different bending modes of Fe3O4 and amorphous graphitic carbon respectively.

Figure 4. (a) Fourier transform infrared spectra of folic acid, chitosan and folic acid-chitosan conjugates. (b) FTIR spectra of 5-fluorouracil, MFCSNPs, MFCSNPs-FA-CHI and MFCSNPs-FA-CHI-5FU nanocarriers respectively.


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Figure 5. (a) Fluorescence emission spectra of MFCSNPs and MFCSNPs-FA-CHI-5FU nanocarriers obtained in aqueous solution at excitation wavelength of 320 nm. (b) Magnetic hysteresis (M-H) analysis curve of MFCSNPs and MFCSNPs-FA-CHI-5FU nanocarriers obtained at 300 K. (c) In-vitro drug release profile of 5-fluorouracil in PBS solution at acidic and physiological conditions.

Figure 6. (a) T2-weighted and (b) color overlaid T2 map (ms) images respectively. Numbers (1 to 9) in (a) indicate regions corresponding to small phantom tubes with different concentration of MFCSNPs-FA-CHI5FU nanocarriers. Phantoms were prepared using different concentrations of MFCSNPs in Agarose (1.5%) as described in experimental section. (c) Plot of R2 vs MFCSNPs-FA-CHI-5FU nanocarriers with Fe concentration (red line represents linear fit). Color overlaid MR images of (d) A375 and (e) HeLa cells respectively. The numbers indicate regions corresponding to small phantom tubes with different


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concentration of MFCSNPs-FA-CHI-5FU nanocarriers (1 correspond to only cells and 2-6 corresponds to various concentrations of nanocarriers (0-200 µg/mL) incubated with A375 and HeLa cells in Agarose (1.5%). (f) Plot of concentration of nanocarriers incubated with cancer cells (x-axis) and estimated concentration of internalized nanocarriers (y-axis) calculated with the relaxivity value of nanocarriers.

Figure 7. Cytotoxicity studies of MFCSNPs, MFCSNPs-FA-CHI-5FU nanocarriers and 5-fluorouracil (5-FU) on (a) A375, (b) MCF-7 and (c) HeLa cells. Concentration values marked on x-axis are of MFCSNPs and MFCSNPs-FA-CHI-5FU nanocarriers. The studied concentration of 5-FU was relative to the loading percentage of 5-FU in MFCSNPs-FA-CHI-5FU nanocarriers.


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Figure 8. Confocal laser scanning microscopic images. (A) Negative control, (B) MFCSNPs and (C) MFCSNPs-FA-CHI-5FU nanocarriers obtained on HeLa cells after 3 hours of incubation time. Left panel shows fluorescent images of overlay images of blue, green, yellow and red, middle panels show different color images and the right one represents images of bright field.


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Figure 9. Confocal laser scanning microscopic images of (a) A375 cells and (b) HeLa cells incubated with MFCSNPs-FA-CHI-5FU nanocarriers after 3, 6 and 12 hours of incubation time respectively. Left panel shows fluorescent images of overlay images of blue, green, yellow and red, middle panels show different color images respectively. Figure 9 (c) below shows plot between the mean fluorescence intensity (MFI) obtained from A375 and HeLa cells with subsequent incubation time upto 12 hours respectively.


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Figure 10. In-vivo serum stability and in-vivo biodistribution studies of MFCSNPs and MFCSNPs-FA-CHI5FU nanocarriers. (a) Serum stability of MFCSNPs and MFCSNPs-FA-CHI-5FU nanocarriers with subsequent time up to 24 hours analyzed by ITLC. All the data is presented with mean standard deviation (n = 3). (b) In-vivo biodistribution studies of MFCSNPs and MFCSNPs-FA-CHI-5FU nanocarriers in various organs up to 24 hours with time intervals. Figure 10 (b) show the distribution of nanocarriers in various organs of tumor bearing mouse after IV injection from the tail.


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Scheme 1. Schematic diagram for the formation of MFCSNPs-FA-CHI-5FU nanocarriers.

ASSOCIATED CONTENT


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Supporting Information. Supporting information for “Multifunctional Magneto-Fluorescent Nanocarriers for Dual Mode Imaging and Targeted Drug Delivery”. HR-TEM images of MFCSNPs, FESEM elemental mapping images of MFCSNPs-FA-CHI-5FU nanocarriers, formation of FA-CHI, 1H NMR spectra of folic acid, chitosan and FA-CHI conjugate, Zeta potential and DLS graphs, BET and pore size analysis graphs, UV-Vis spectra, TGA graphs, emission spectra, magnetic separation images, drug release calibration graphs, DLS graph, hemolysis measurements, DLS graph for stability of MFCSNPs-FA-CHI-5FU nanocarriers. AUTHOR INFORMATION

Corresponding Author *Dr. Jaspreet Kaur Randhawa School of Engineering, Indian Institute of Technology Mandi A1 building, Kamand campus, Mandi, Himachal Pradesh -175005, India. Telephone No. +91 1905-267056 Email: [email protected]

Author Contributions


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A.T. performed synthesis and characterization experiments and wrote the manuscript text. A.S. performed cytotoxicity and confocal experiments. A. D. performed MR imaging experiments. A. K. performed the in-vivo animal experiments and biodistribution studies. J.K.R., A.S., R. M., and N.G. gave conceptual advice and edited the manuscript. All authors reviewed the manuscript.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT

Authors are thankful to IIT Mandi for providing research facilities to carry out the work. Authors thank Dr. R K Gupta from Fortis memorial Research Institute for the support in MR scanning. Dr. Anup Singh acknowledges funding support from SERB project, grant number YSS/2014/000092 for MR imaging. Dr. Neha Garg is grateful to Ramanujan SERB grant, India (SB/S2/RJN-072/2015). Mr. Ashish Tiwari acknowledges MHRD for


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the award of senior research fellowship. Mr. Ashutosh Singh acknowledges to UGC for the award of senior research fellowship.

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Multifunctional Magneto-Fluorescent Nanocarriers for Dual Mode

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