Methotrexate anchored carbon dots as theranostic probes: digitonin conjugation enhances cellular uptake and cytotoxicity

Abstract

In recent years carbon dots (CDs) have been drawing increasing attention in the area of nano medicine. Their indubitable roles in cellular imaging, drug delivery and diagnosis are widely acknowledged. Digitonin (DG) has traditionally been known as a cell membrane permeabilizing agent. Based on this fact, we modified CDs with DG (CDDG) and further conjugated them with methotrexate (MX). This probe, CDDG conjugated MX (CDMX) was subjected to physico chemical characterization, cytotoxic evaluation via MTT assay and cellular uptake studies using confocal laser microscopy. The drug release study implied that at physiological pH, release is less reflecting maximum drug retention in the probe during circulation. The results which emerged have shown that DG is impacted in enhancing cellular uptake and cytotoxic potential of the drug carriers. The study indicates that theranostic probes with improved features can be generated from CDs by a judicious modification.

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1. Introduction

Fluorescent nano materials are emerging as potential agents in biomedical domains particularly in drug delivery and sensing applications. Multifunctional nano materials as theranostic carriers have opened up new paradigms for early detection and treatment of diseases.^1,2 Among various fluorescent nano materials, CDs have met ever increasing interest as carriers of therapeutic payloads for cancer treatment owing to their biocompatibility, rapid cellular uptake, and limited influence on drug activity.³ The notable features of CDs are excellent photoluminescence, inherent aqueous solubility, ease of surface modification and tuneable optical properties.^4,5 The non-toxic nature of CDs makes them ideal candidates for investigating biological systems particularly in cell imaging.⁶

Recent World Health Organisation report shows that cancer results in more deaths than coronary heart disease or stroke.^7,8 Over 20 million new cancer cases are also expected in low and middle income countries by 2025.⁹ In United States in 2015 alone, new cases and deaths due to cancer were estimated to be 658 370 and 589 430 respectively.¹⁰ Among various strategies designed, nano medicine has evolved as promising approach to tame cancer. Nanotechnologies to a great extent result in improved distribution and targeting of anticancer medication.¹¹

The potential of CDs as drug delivery vehicles has been explored by several groups. Sorbitol derived CDs were used for the folic acid mediated delivery of doxorubicin and bio imaging.¹² CDs within mesoporous silica followed by the capping of PEG were equipped with doxorubicin for drug release and cell imaging studies.¹³ Zheng et al. have developed CDs anchored with oxaliplatin for combining chemotherapy and bio imaging applications.¹⁴ Doxorubicin loaded hyaluronic acid and CDs based nano complex as theranostic carrier has recently been reported.¹⁵ Wang et al. have shown that CDs present in commercial beer is safe probe for the image guided cancer therapy.¹⁶ Zheng et al. have synthesised CDs from D-glucose and L-aspartic acid which showed high selectivity and targeting ability towards C6 glioma cells.¹⁷

DG is a glycoside obtained from Digitalis purpurea which is used in removing membrane proteins, precipitating cholesterol and permeabilizing cell membranes.¹⁸ We reasoned that incorporation of DG can significantly enhance cellular uptake of nano probes carrying drugs and thereby therapeutic potential can be improved. In this work, we chose MX as a model drug considering its wide usage in cancer treatment. Its structure is similar to folic acid and hence can enter cells via the transport system similar to folic acid.

Here in, we report the generation of DG conjugated CD anchored with MX (CDMX) for the simultaneous imaging and destruction of cancer cells. CDDG was conjugated with MX via DCC/DMAP reaction and purified by dialysis. The nano probes were subjected to physico-chemical characterizations, cytotoxic evaluation and cellular uptake studies.

2. Materials and methods

2.1. Materials

Citric acid anhydrate, poly(ethylene glycol) bis(3-aminopropyl) terminated (MW 1500), glycerine, adipic acid (AD), N-(3-dimethylaminopropyl)-N′ ethylcarbodiimide (EDC), digitonin, dicyclohexylcarbodimide (DCC), dimethylamino pyridine (DMAP) and methotrexate were obtained from sigma Aldrich, Bangalore. All other chemicals used were of analytical grade and obtained from Merck India Ltd, Mumbai, India.

2.2. Synthesis of CDs and modification with DG

Amine capped CDs were prepared as reported earlier¹⁹ using citric acid as the carbon precursor and PEG diamine as the capping agent. The resulting product was dialyzed against distilled water using a cellulose ester dialysis membrane [molecular weight cut off = 3500] for 2 days in order to remove any unreacted components. Conjugation of DG onto CDs was carried out as reported in our earlier work by DCC/DMAP reaction²⁰ (see ESI† for the synthetic steps).

2.3. Conjugation of MX onto CDDG

The formation MX conjugated CDDG (CDMX) is depicted in Scheme 1. 1 mL of MX solution (5 mg mL⁻¹) was added to CDDG (10 mL, 1 mg mL⁻¹) in presence of DCC/DMAP for 3 h at room temperature and the resulting solution (CDMX) was dialyzed against DMSO alone followed by DMSO/H₂O mixture and distilled water alone using cellulose ester dialysis membrane [MWCO = 3500].

Scheme 1 Conjugation of methotrexate onto CDDG.

2.4. Conjugation of MX onto CD to form CM

This conjugate was synthesised by directly conjugating drug onto CD to compare the cellular uptake of the conjugate with that of the drug conjugated onto digitonin modified CD. The details of synthesis are depicted in ESI.†

2.5. Drug loading efficiency

12 ml of the reaction mixture was dialyzed against water–DMSO mixture overnight. The concentration of the unbound drug was measured spectrophotometrically at 303 nm and the calculation was made using the calibration plot generated with various concentrations of methotrexate. Drug loading efficiency (DLE) was calculated as per eqn (1),

2.6. In vitro drug release study

Release study was carried out using an aliquot of the sample (CDMX, 5 mL) placed in a dialysis bag (molecular weight cut off 3500) and suspended in 10 mL of PBS solution (pH 5.0) at 37 °C under mild stirring. Periodically, 2 mL of the release medium was withdrawn and replaced with the same volume of fresh solution. The amount of drug released was determined using a spectrophotometer at 303 nm. The same experiment was repeated in PBS at pH 7.4 to evaluate the release profile under physiological pH.

2.7. In vitro cytotoxicity study by MTT assay

The in vitro cytotoxicity of CM (probe obtained by conjugating MX onto CDs), CDDG and CDMX were evaluated using a standard 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The C6 glioma cells were trypsinized and seeded into 96 well plates and incubated for 24 h at 37 °C in 5% CO₂. Cells were then incubated with each sample (1 mg mL⁻¹) along with 1% Triton X-100 as the positive control and medium as the negative control in triplicates at 37 °C for 24 h in 5% CO₂. After 24 h incubation, samples were removed, MTT reagent was added to each well and cells were further incubated for 3 h. The reagent was then removed and dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals formed by the reduction of MTT and the absorbance was measured at 570 nm using an automated microplate reader (Infinite M200, TECAN). Cell viability was expressed as mean percentage sample absorbance relative to the absorbance of control as per eqn (2).

Cell viability = (absorbance of sample ÷ absorbance of control) × 100 (2)

2.8. Cellular uptake studies

For cellular uptake studies, C6 glioma cells were trypsinized and seeded into four well plates at a concentration of 1 × 10⁵ cells per well and incubated for 24 h at 37 °C in 5% CO₂. Samples were prepared at concentration of 1 mg mL⁻¹ of which 100 μL was added to the cell and incubated for 3 h in the F12HAM medium containing 10% FBS at 37 °C. After incubation, cells were washed with PBS, pH 7.4 and fixed in 1% formaldehyde in PBS. The uptake of nanoparticles was visualized and photographed using laser scanning confocal microscopy.

2.9. Characterization techniques

Fourier Transform Infra Red (FTIR) spectra of DG, CDDG, methotrexate and CDMX were collected in the range 600–4000 cm⁻¹ on a Nicolet 5700 FTIR spectrometer, Nicolet Inc, Madison, USA using a diamond ATR accessory.

Proton Nuclear Magnetic Resonance (¹H NMR) spectra of CDMX was recorded using 500 MHz Bruker AV 500 NMR spectrometer.

Transmission Electron Microscopic (TEM) image of CDDG was obtained on a Hitachi, H 7650 microscope, Hitachi, Tokyo, Japan. The colloidal solution (CDDG) was deposited onto a 200 mesh copper grid coated with a formvar film and dried overnight.

Size of CDDG and zeta potential values of CD, CDDG and CDMX were determined using Dynamic Light Scattering (DLS) (Malvern Instruments Ltd, UK).

UV-visible absorption spectrum was taken using a UV-visible spectrophotometer, Varian, Cary 100 Bio, Melbourne, Australia.

The fluorescence intensity of the same were measured using a spectrofluorimeter, Varian, Cary Eclipse model EL 0507, Melbourne, Australia.

Cellular uptake studies of CDDG, CM (MX conjugated to CDs containing no DG) and CDMX were carried out using confocal laser microscope, NIKON A1R.

3. Results and discussion

We relied on FTIR analysis for getting insight onto the formation of CD modified with DG and MX. A comparison of the spectra depicted in Fig. S1 (ESI†) strongly indicate the conjugation of DG onto CD. The strong peak at 1011 cm⁻¹ due to C–O–C bond of DG apparently suggests the conjugation of DG onto CD through esterification to form CDDG.

The conjugation of MX onto CDDG was confirmed by comparing their FTIR spectrum given in Fig. S2 in the ESI.† In MX, the peaks at 1687 cm⁻¹ corresponds to the C=O stretching of the COOH group.^21,22 The peaks at 1641 cm⁻¹ and 1598 cm⁻¹ are assigned to the C=O stretching and N–H bending of the amide groups in MX.²³ Thus in CDMX, the peak at 1700 cm⁻¹ is due to the C=O stretching of COOH group and the peak at 1650 cm⁻¹ attributes to the C=O stretching of the amide group in MX. The sharp peak at 1104 cm⁻¹ indicates the C–O–C band due to esterification reaction. The peaks at 2916 cm⁻¹ and 2869 cm⁻¹ correspond to the characteristic peaks of CDs due to CH stretching as reported in our previous works.^24,25

¹H NMR spectrum of CDMX (Fig. S3, ESI†) showed signals at 3.0–3.3 ppm assigned to the aliphatic protons of MX. The peak at 3.6 ppm corresponds to the methylene group of PEG diamine.²⁵ Peaks at 6.8 ppm, 7.7 ppm and 8.6 ppm correspond to the benzoyl group and pteridine ring of MX.^23,26 Peak at 2.6 ppm is due to the methylene groups and that at 1.2 ppm is attributed to the aliphatic hydrogen atoms of DG.²⁰ Thus the above results clearly indicate the conjugation of DG and MX onto CDs.

The TEM micrograph of CDDG depicted in Fig. 1 indicates that it possesses a spherical morphology with an average size of ∼10 nm. In our previous studies, HRTEM images of CD indicated that they possess spherical morphology with an average size of 4 nm^24,25 and the size determined by DLS analysis was around 10 nm.²⁵ Conjugation of CDs with DG resulted in an increase in size to 10 nm as reflected in Fig. 1. The increase in size might be due to the interaction among the functional groups facilitating the formation of aggregates. To substantiate this view, their size was determined by DLS. Size of CDDG from DLS measurement was found to be 147 nm (Table S1, ESI†). It is a known fact that that DLS measurement records higher value for particle size since the light is scattered by the core as well as the layers formed on the surface of the particles.²⁷ Further, from TEM analysis the number-average particle size is obtained whereas DLS gives the z-average particle size.²⁸ Similar observations have been reported earlier in other studies also.²⁹ Apparently the increase in size confirms the conjugation of DG onto CDs.

Fig. 1 TEM micrograph of CDDG.

Table S2 (ESI†) displays the zeta potential measurements of CD, CDDG and CDMX. Change in the zeta potential value from −20.90 mV to −4.99 mV is a further reflection of conjugation of DG onto CD. DG is anchored onto CD through esterification reaction between –COOH groups on CD and –OH in DG. The significant reduction in the –COOH groups could be attributed to change in zeta potential from −20.90 to −4.99 mV. Though the zeta potential value is dropped, no aggregation was observed (which is evident from Fig. 1), as there was enough repulsive force for preventing the same. On conjugation with MX increase in charge was observed i.e. −4.99 mV to 0.57 mV which may be assigned to the –NH₂ functionalities in the system resulted from the conjugation of MX.

The conjugation of MX is further substantiated by UV-visible spectrum of CDMX (Fig. S4, ESI†). The peaks at 258 nm, 303 nm and a broad peak at 370 nm due to MX were observed apart from the absorbance peak at 360 nm due to CDs.

Fluorescence spectrum of CDMX is shown in Fig. 2. CDMX showed emission at 455 nm when excited at 360 nm. The photographic images given in the inset of the figure affirm that the fluorescence of CD is intact after the conjugation of MX onto CDDG.

Fig. 2 Fluorescence emission spectrum of CDMX. [In the inset are the photographic images of CDMX (A) in daylight (B) under UV lamp at 365 nm.]

Drug loading efficiency of CDMX calculated as per the eqn (1) was found to be 94%.

Drug release profile in Fig. 3 indicates that 20% of drug was released from CDMX at physiological pH (7.4) whereas 81% release was observed at pH 5.0 over a period of 6 h. More amount of drug is released at pH 5.0 which is beneficial considering the lower pH inside cancer cells. Drug molecules (MX) are conjugated onto CDDG through esterification reaction between –COOH groups on MX and –OH groups in DG. It is well known that ester groups are susceptible to hydrolysis at a lower pH which could be causative for the enhanced drug release at pH 5. The release profile apparently suggests that drug is retained in the matrix at physiological pH. One of the essential criteria of a drug delivery vehicle is its ability to minimise drug loss during circulation. In that sense, the matrix highlighted in the present study has the potential to carry the drug safely to the predetermined site.

Fig. 3 Drug release profile of MX from CDMX.

Cytotoxic evaluation was carried out by MTT assay against C6 glioma cells. Fig. 4 shows the cytotoxic activity of CDMX, CDDG and MX conjugated to CDs without DG (CM). CDDG was found to be cell friendly at all chosen concentrations (93.03%, 90.67%, 88.68% and 81.99% for 12.5 μg mL⁻¹, 25 μg mL⁻¹, 37.5 μg mL⁻¹ and 50 μg mL⁻¹ respectively). CDMX showed enhanced cytotoxic response when compared to CM. CM showed cell viability of 92.01% at the lowest concentration (12.5 μg mL⁻¹) whereas only 57.40% cell viability was observed for the same concentration of CDMX. It can be seen that as the concentration increases the percentage of cell viability decreases. Quantitative assessment of the cytotoxicity to cells on contact with 12.5 μg mL⁻¹, 25 μg mL⁻¹, 37.5 μg mL⁻¹ and 50 μg mL⁻¹ of the free drug (MX) showed 81.43%, 78.64%, 77.01% and 71.63% metabolic activity respectively. Whereas for the same concentrations of CDMX showed 57.40%, 56.99%, 56.67% and 51.49% cell viability respectively. Thus CDMX exhibited higher cytotoxicity when compared to the free drug. Improved cytotoxic response of CDMX might be due to the cell permeabilizing effect of DG in CDMX. This observation can be assigned to enhanced uptake of CDMX by the cells which is in fact facilitates the transport of more drugs into the cells. A similar observation has been made in an earlier study using MX loaded chitosan nano particles.²³ The action of the free drug at the site is based on the passive diffusion mechanism and may not be effective in inflicting instant cellular damage.²⁷

Fig. 4 MTT assay of CDDG, CM, CDMX and free methotrexate.

To further annotate MTT assay results, we studied the internalisation of CDDG, CM and CDMX by incubating C6 glioma cells for 3 h and the uptake was visualised by confocal laser microscope. Fluorescent images in Fig. 5 and 6 indicate the effective internalisation of CDDG and CDMX by C6 glioma cells. The fluorescence was mainly observed in the cytoplasm area as it is a known fact that CDs get distributed in cytoplasm. The uptake of CM was also carried out (Fig. 7) and it is evident that the fluorescence intensity of the cells is less when compared to those incubated with CDMX (Fig. 6). This data obviously suggest that the enhanced cytotoxicity of CDMX comparing to CM as reflected in MTT assay is due to the improved internalisation of CDMX facilitated by DG.

Fig. 5 Confocal laser microscopic images under bright field of (a) cells alone (b) CDDG (c) merged images.

Fig. 6 Confocal laser microscopic images under bright field of (a) cells alone (b) CDMX (c) merged images.

Fig. 7 Confocal laser microscopic images under bright field of (a) cells alone (b) CM (c) merged images.

4. Conclusions

We have successfully modified CDs with DG as a nano carrier for loading the anticancer drug, MX. In vitro drug release profile of MX from the system ensured the safe delivery of the drug under physiological conditions thereby meeting the criteria of an efficient drug delivery carrier. The probe CDDG showed negligible cytotoxicity to C6 glioma cells but CDMX showed enhanced cytotoxicity when compared to the free drug. Confocal images of the cells incubated with CDDG and CDMX confirmed that they are efficiently taken up by the cells. Our results suggest that DG can assist in better internalization of drug carriers and thus significantly can increase the therapeutic potential of the drug. Additionally it seems that potential theranostic probes can be created from CD by less complex chemical approaches.

Acknowledgements

Authors wish to thank DBT, New Delhi for funding. Authors are also grateful to NIIST and RGCB, Trivandrum for providing technical assistance in getting ¹H NMR and confocal laser microscopic images respectively.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra11660d

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