A promising Stimuli-Responsive nanocomposite as a Theranostic agent for Targeted Delivery

Document Type: Original Article

Authors

1 Division of Chemistry and Diseases, Advanced Technologies Research Group, Tehran, Iran

2 Biomaterial Group, Department of Biomedical Engineering, Amirkabir University of Technology, Tehran, Iran

Abstract

Introduction: A range of strategies for eliminate the cancer tissue have been used, consisting of medical operation, chemo and radiotherapy.

Objective: There is a significant issue about chemotherapy, the medications targeted delivery to object tissue cells is found challengeable because of the presence of numerous physical obstructions.

Materials and Methods: In the present study, we synthesized and characterized a novel and promising specific pH-stimuli-responsive tumor-targeted DDS. The synthesized magnetic nanoparticles (Nanocomposite) were fully characterized by FESEM, TEM, DLS, XRD and VSM.

Results: The morphology of Nanocomposite are acquired in semi-spherical shape with the specified diameters. The Capsaicin was successfully loaded Nanocomposite represented promising and admissible biocompatibility as well as showing higher toxicity versus SK-N-MC cells in comparison with control group.

Conclusion: Furthermore, uptake investigation proved that the effectively internalized Drug Delivery System (DDS). In addition, the in vitro assay represented that the synthesized Nanocomposite can act nearly fully pH stimuli-responsive and release the Capsaicin at controlled condition. MTT assay verified the higher toxicity of Capsaicin loaded into the Nanocomposite in comparison with the control group. MRI in vitro investigation demonstrated that the prepared Nanocomposite can utilized as MRI imaging agent.

Graphical Abstract

A promising Stimuli-Responsive nanocomposite as a Theranostic agent for Targeted Delivery

Keywords


 

  1. Introduction

In recent times, cancer actually turns into critical issues of Healthcare, as well as, medication procedure burden tremendous financial costs on people and authorities. By the record from Reliable References, in 2012, more than 8 million people with cancer passed away. It is normally anticipated which unfortunately trends of the number of cancer patients would definitely enhance to over 20 million by 2030 [1-7].

A range of strategies for eliminating the cancer tissue has been used, consisting of medical operation, chemo- and radiotherapy. There is a significant issue about chemotherapy; the medications targeted delivery to object tissue cells is found challengeable because of the presence of numerous physical obstructions [8-15].

Nanotechnology is a new field of science that takes advantage of the peculiar properties of matter at the nanoscale. The extremely high ratio of surface area to mass that is typical of nanoparticles, allows them to interact efficiently with their environment, but yet they can act as contained carriers for their constituent molecules as opposed to the same molecules in solution. Nanoparticles are therefore promising carriers for targeted delivery of therapeutic agents. The particle size (ranging from a few nanometers to the micron range) can directly influence cell uptake. Although there is no consensus on the optimum size, many studies suggest that a size which is less than 100 nm provides stability in body fluids, a higher ability to penetrate biological membranes, and a better capacity to accumulate inside cells. Different nanocarriers such as liposomes, micelles, polymeric nanomaterials, mesoporous silica, gold, and magnetic nanoparticles have improved biomedical applications including drug and gene delivery. On the other hand, suitable functionalization of the nanoparticle surface, not only can increase specific targeting by ligand-recognition or enzyme-responsiveness but can also enable the monitoring of drug delivery by attached imaging reporters. Moreover, nanoparticle drug delivery vehicles significantly decrease the side-effects of drugs, (particularly anticancer chemotherapy) by increasing their water solubility and therefore decreasing the required overall dose [6, 14, 16-22].

Several types of magnetic nanoparticles (Nanocomposite) can be found broadly used as nanocarriers as well as, targeted contrast agents. Different stimuli-responsive-based modifications on the surface of these Nanocomposites could enhance the biocompatibility of these nanosystems whereas led to decrease in the off-target result. Cobalt-based nanosystems have significant magnetic and catalytic features that have been utilized widely in different biomedical applications [23-28].

Between several chemotherapeutic agents utilized with regards to cancer therapy, Capsaicin, trans-8-methyl-N-vanillyl-6-nonenamide, a natural compound that has been presented in red pepper demonstrated anti-carcinogenic, anti-mutagenic, and chemo-preventive in a large number of cancer, has gained significant attention due to the exclusive features. The scientists published several articles related to anti-carcinogenic, anti-mutagenic and chemo-preventive effect of Capsaicin by inducing apoptosis as well as anti-angiogenic and anti-metastatic activities [29-33].

In the present study, a novel CoFe2O4@ZSM-5 nanocomposite with freshly promising synthesis route, Egg-white (EW) which led to taking biocompatible nanocomposite has been revealed for controlled Capsaicin delivery along with monitoring by MRI study.

 

  1. Materials and Methods

2.1.   Materials

Dimethyl sulphoxide (DMSO), Cobalt (III) nitrate hexahydrate (Co(NO3)2.6H2O), iron nitrate (Fe(NO3)2.9H2O), Glutathione, Oleic Acid, sodium hydroxide, Capsaicin, ammonia solution (NH3·H2O, 28% of ammonia), absolute ethanol (EtOH) and N-hydroxy succinimide (NHS) were purchased from Sigma-Aldrich (Germany). SK-N-MC neuroblastoma cell line (as model cancerous cell line) was obtained from Pastor Institute Cell bank (Tehran, Iran).

 

2.2.   Characterizations

For the nanocomposite characterizations, Transmission electron microscopy (TEM, LEO 912AB electron microscope), Field emission scanning electron microscope (FESEM, MIRA3 TESCAN microscope), X-ray diffractometer (XRD), Vibrating sample magnetometer (VSM, Squid-VSM, Quantum Design, Germany) and Dynamic light scattering (DLS, Malvern Zeta Sizer ZS90, UK) was applied. For TEM, a fraction of the synthesized nanocomposite was fixed to the carbon film with a copper grid and dried. Morphology of the synthesized nanocomposite was investigated by applying SEM microscopy by dropping the diluted nanocomposite solution on glass slides and dried at room temperature. Hydrodynamic size of the nanocomposite was investigated by DLS evaluation. The structural investigation was investigated by X-ray diffraction, XRD patterns acquired with Cu Kα radiation at 40 keV and 30 mA, and scanning rate was set to 2°min-1 in the 2θ range from 10° to 80°. Ultraviolet-visible spectra of the nanocomposite were assessed by UV-Vis Jasco-530 spectrophotometer. The hysteresis loop of the nanocomposite was measured at room temperature through the VSM with a maximum magnetic field of 10 and accelerating voltage of 15 kV.

 

2.3.   Synthesis of CoFe2O4

Briefly, in the EW synthesis route, egg white was mixed with deionized water in 2:3 ratios. Afterward, the mixture had been strongly stirring at room temperature until a homogeneous solution was achieved. Then, a stoichiometry amount of Cobalt (III) and Iron (III) nitrate were separately dissolved in distilled water and poured into the first homogenous solution and stirred vigorously for 3 hours. In the next step, 25 mL of sodium hydroxide 3M was added and the final solution stirred up to pH = 12. Then, 1 mL Oleic acid as a chelating agent was added to the above suspension. The temperature of the suspension was raised to 80 oC with continued stirring for 2 hours. The product was cooled to room temperature, and the precipitate was separated by centrifuge, washed twice with distilled water and ethanol. The precipitate was dried at 65 oC (dark brown powder), calcined at 400 oC for 8 hours followed by recalcination at 800 oC for 4 hours and black powder was obtained.

 

2.4.   Preparation of the CoFe2O4@ZSM-5 nanocomposite

Briefly, we have three beakers, in the first beaker 0.5 g NaAl2O3 was dissolved in 45 ml H2O, in the second beaker 7.9 g Silicic acid was dissolved in 35 ml H2O and in the third beaker 0.17 g the CoFe2O4 NPs were dispersed in 10 ml H2O. After that, the solution of the first beaker added to the third beaker and vigorously stirred for 20min, following by that, the second beaker added to the mentioned solution under stirring vigorously. Eventually, the final mixture (suspension) inserted to a stainless Teflon and autoclaved at 200 oC for ̊48 hours. The final product was filtered twice, washed for triple times with ultra-pure water and dried at 65 oC.

 

2.5.   Loading of Capsaicin on CoFe2O4@ZSM-5 nanocomposite

Loading procedure of Capsaicin into the nanocomposite proceeded by injecting 300 μL of 15 mg Capsaicin in acetone to 5 mL dispersion of the 10 mg of CoFe2O4@ZSM-5. The mixture was then stirred at 600 rpm for 24 h. The product was separated from free Capsaicin by magnetic separation and several times washing with deionized water. Afterward, the purified Capsaicin loaded CoFe2O4@ZSM-5 were freeze dried. Loading efficiency of Capsaicin was measured using UV-Vis spectrophotometer at 280 nm.

 

2.6.   In vitro drug release

To evaluate the drug release of Capsaicin loaded CoFe2O4@ZSM-5, 4 mg of the CoFe2O4@ZSM-5 was poured into a 10 mL tube containing 6 mL phosphate buffer saline (PBS) of pH=5.5 and pH=7.4 and 0.1% (w/v) Tween-80. The solutions were shaken at 37 °C at 200 rpm in an orbital shaker. The samples were centrifuged at 12,000 rpm for 15 minutes, and the supernatants were collected and stored at 4 °C. All samples were replaced by equivalent volumes of fresh medium. The obtained supernatants were measured for absorbance at 280 nm using a UV-Vis spectrometer. An identical standard of Capsaicin (0–10 μg/mL) was prepared, and absorbance was measured at 280 nm using the UV-Vis spectrometer to determine the amount of Capsaicin released from CoFe2O4@ZSM-5.

 

2.7.   In vitrocytotoxicity

MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazoliumbromide) cell proliferation assay was employed to assess in vitro cytotoxicity of Capsaicin and Capsaicin loaded CoFe2O4@ZSM-5. For this assay, SK-N-MC cell line, at an initial density of 2×103 cells per well, were seeded into 96-well plates in a volume of 200 μL DMEM (Dulbecco’s Modified Eagle’s Medium-High Glucose) and incubated overnight (100 IU/mL penicillin and 100 μg/mL streptomycin sulfate). Then, the medium was replaced with a fresh medium, containing PBS (as a control agent), Capsaicin and Capsaicin loaded CoFe2O4@ZSM-5 and followed by incubation for 48 hours at 37 oC. Afterward, 10 μL of the MTT labeling reagent was transferred into each well. After incubation (4 hours), 100 μL DMSO was added to each well to dissolve the insoluble formazan crystals. Finally, after incubation (overnight at 37 oC), the color intensity of solubilized formazan was assessed at 570 nm wavelength using a microplate reader (BioMate 3 UV-Vis spectrophotometer).

 

2.8.   In vitro Magnetic Resonance Imaging

In order to evaluate in vitro MRI properties, synthesized CoFe2O4@ZSM-5 were dispersed in 3% (w/v) agarose solution. Formulations were placed into the MRI scanner and followed by running a number of spin-echo sequences to determine T2 relaxation time. MRI solutions were scanned under a 3-Tesla clinical MRI scanner (Imam Khomeini Hospital, Tehran) at room temperature. The increase of relaxation rate (S-1, 1/T2) with increasing the metal concentration was analyzed through linear regression analysis. The concentration of iron oxide used in each formula was modulated to be within 1.0-50 mg Fe/mL. MRI parameters involved to acquire images of each sample, were as below: echo time (TE= 15-120 ms), repetition time (TR= 1000 ms), number of echoes (NE= 8), number of excitations (NEX= 4), matrix size of 128 × 128, and field of view (15 mm × 15 mm). To measure of signal intensity, a manually drawn region of interest was used for each sample by DICOM Viewer software. Furthermore, relaxation rates (r2=1/T2) were extracted with mono exponential curve fitting the signal intensities versus echo time at different concentrations for each of the samples. For further analysis, T2 relaxivities and r2 (mM-1 S-1) were calculated from the slope of T2 values (relaxation rate) vs the metal concentration according to the equation:

r2= (A + C) × exp (-r2×TE)

2.9.   Cellular uptake of CoFe2O4@ZSM-5 nanocomposite

The internalization of Capsaicin loaded CoFe2O4@ZSM-5 by SK-N-MC cells was analyzed through flow cytometry method. 4×105 cells were plated in 6-well dishes in 2 mL medium (DMEM) under standard culture conditions and incubated for 24 h. Afterward, media was replaced with 2 mL of serum-free medium containing Capsaicin or Capsaicin loaded CoFe2O4@ZSM-5 (40-100 μg/mL). After 12 hours of cell treatment, the cells were washed three times with cold PBS (pH= 7.4). Finally, the fluorescence intensity of Capsaicin per cell was quantified using a flow cytometer (FACScan, LYSIS II, Becton Dickinson) at the excitation wavelength of 280 nm and an emission wavelength of 316 nm.

 

  1. Results and Discussion

3.1.   Characterization of the nanocomposite

 

 

Figure 1: SEM images of the (A) CoFe2O4 and (B) CoFe2O4@ZSM-5 nanocomposite. (C) TEM image of the CoFe2O4@ZSM-5 nanocomposite.

 

 

 


Surface morphology, size, and shape of the nanocomposite were investigated by FESEM, TEM and DLS analysis. The FESEM images approved the spherical morphology of the prepared Nanocomposite with negligible aggregation. Same as the FESEM images, The TEM images approved the formation of the Nanocomposite and spherical morphology of them (Figures 1 A-C).

 

 

Figure 2: DLS of the CoFe2O4@ZSM-5 nanocomposite

 

 

In addition, the DLS was applied to determine the average diameter of CoFe2O4@ZSM-5 Nanocomposite, which the data's were specified average diameter of CoFe2O4@ZSM-5 Nanocomposite about 24 nm (Figures 2). Furthermore, increasing in the average size of the Nanocomposite modified by ZSM-5 had been predicted.

 

Evaluation the surface charge of the Nanocomposite had been proceeded by surface zeta potential. Zeta potential of CoFe2O4@ZSM-5 Nanocomposite were acquire 19.2 mV. It has been proved that loading of the Capsaicin led to neutralization of the remained positive surface charge of these Nanocomposite through the carbonyl and hydroxyl functional groups on the structure of Capsaicin which have a negative charge, and following led to decreasing the surface charge of the Nanocomposite.

XRD analysis was applied to investigate the crystalline structure of CoFe2O4@ZSM-5 Nanocomposite (Figure 3). All of the significant peaks of the crystalline structure of the nanocomposite have seen and marked by a star on the XRD pattern. Slightly broad peaks (while no shift is observed) can be able to prove the small crystalline size of the Nanocomposite.

 

 

Figure 3: XRD pattern of the CoFe2O4@ZSM-5 nanocomposite.


VSM analysis was applied to investigating the magnetic properties of CoFe2O4@ZSM-5 Nanocomposite. The hysteresis curves of CoFe2O4@ZSM-5 Nanocomposite are depicted in Figure 4. Saturation magnetization value of CoFe2O4@ZSM-5 Nanocomposite was found 46 emu/g. However, these saturation magnetization of modified Nanocomposite is suitable to be applied as MRI imaging agents.

 

 

3.2.   Loading efficacy and in vitro release of Capsaicin

Loading of the Capsaicin into the CoFe2O4@ZSM-5 Nanocomposite was evaluated 153 μg Capsaicin per mg of CoFe2O4@ZSM-5 Nanocomposite with the drug loading efficiency of 28%. The results represented that Capsaicin was successfully loaded into the CoFe2O4@ZSM-5 Nanocomposite. The in vitro release profile of Capsaicin from Capsaicin loaded synthesized CoFe2O4@ZSM-5 Nanocomposite are depicted in Figure 5. It has been shown that the release profile of the CoFe2O4@ZSM-5 Nanocomposite in acidic medium (pH = 5.5) represented a significant release (55%, at first hour) whilst in neutral medium (pH = 7.4), initial release was 11%. The total amount of Capsaicin release in acidic medium (pH = 5.5) is nearly 50% superior in

 

Figure 5: in vitro release profile of capsaicin from capsaicin CoFe2O4@ZSM-5 at different pH's.

 

 

 

 


comparison with the neutral medium (pH = 7.4). The in vitro drug release profile investigation successfully

 

Figure 4. VSM analysis of CoFe2O4@ZSM-5 nanocomposite.

 

 

 


proved that CoFe2O4@ZSM-5 Nanocomposite is pH sensitive platform that can release the sensitizer/drug payload through the cancer cells microenvironment.

 

3.3.   In vitrocytotoxicity

The CoFe2O4@ZSM-5 Nanocomposite and Capsaicin loaded CoFe2O4@ZSM-5 Nanocomposite were evaluated in terms of in vitro cytotoxicity which are illustrated in Figure 6. The IC50 of CoFe2O4@ZSM-5 Nanocomposite and free Capsaicin were specified to be 65 and 32.1 μg/mL, respectively. The concentration effect of the Capsaicin loaded CoFe2O4@ZSM-5 Nanocomposite on cell viability after incubation represented that by increasing the concentration of the Capsaicin loaded CoFe2O4@ZSM-5 Nanocomposite from 20 to 100 μg/mL, the cell viability dropped down nearly 20%, while there was no considerable toxicity for unloaded CoFe2O4@ZSM-5 Nanocomposite. Therefore, the low toxicity of the Capsaicin loaded CoFe2O4@ZSM-5 Nanocomposite to cells was proof to demonstrate the potential medicinal and biological application of the CoFe2O4@ZSM-5 Nanocomposite.

 

 

 

Figure 6: Cell viability of CoFe2O4@ZSM-5 and CoFe2O4@ZSM-5-capsaicin

 

 

 

 

 

Figure 7: T2 relaxation curves of CoFe2O4@ZSM-5-Capsaicin MNPs in different concentrations as well as in 3% phantom agar gel (0.78–40 μg/mL) scanned under a MRI scanner

 

 

 

 



3.4.   In vitromagnetic resonance imaging

Nanocomposite has unique and considerable magnetic properties. Therefore, they can be able to apply as MRT contrast agent. This technique could improve and increase the differentiations between the target tissue [17]. In this section, in vitro MRI feature of Capsaicin loaded into CoFe2O4@ZSM-5 Nanocomposite was investigated. By enhancing the concentration of the nanoparticles, the T2-weighted signals will decrease. Furthermore, by enhancing the concentration of the nanoparticles, the transverse relaxation times will decrease too. In accordance with the outputs, the linear equation in terms of increasing order of transverse relaxation rate r2 (1/T2) versus the metal concentration represented. T2 relaxivity (r2) was evaluated 260.36 s-1 μg-1mL for CoFe2O4@ZSM-5-Capsaicin Nanocomposite (Figure 7, Figure 8). It should be noted that, based on the results, the prepared CoFe2O4@ZSM-5-Capsaicin Nanocomposite is an appropriate choice for MRI contrast agent application, hence, in vivo analysis is needed to prove this concept.

 

 

 

 

Figure 8: Plot of 1/T2 versus metal concentration of the CoFe2O4@ZSM-5 and its corresponding linear regression.

 

 

 

 

 

3.5.   Cellular uptake investigation

Flow cytometry analysis applied to investigate the cellular uptake of Capsaicin loaded into the Nanocomposite. For this purpose, the negative control is the cells without any Capsaicin, which shown auto fluoresce. Significant Capsaicin fluorescence discovered while the SK-N-MC cells were incubated with Capsaicin loaded CoFe2O4@ZSM-5 Nanocomposite. Also, the mean fluorescence intensity of the Capsaicin for both Capsaicin and CoFe2O4@ZSM-5-Capsaicin Nanocomposite had been above the negative control cells.

 

  1. Conclusion

To conclude, we synthesized and characterized a novel and promising specific pH-stimuli-responsive tumor-targeted DDS. Analysis of in vitro release profile demonstrated a pH sensitive biphasic release. This system can lead to a rebate of side-effects. The Capsaicin loaded Nanocomposite represented promising and admissible biocompatibility as well as showing higher toxicity versus SK-N-MC cells in comparison with control group.

Furthermore, uptake investigation proved that the effectively internalized DDS. With another perspective, the outputs of the in vitro MRI investigation demonstrated that the final Nanocomposite, has a great potential to be utilized as MRI contrast agent. Eventually, encapsulation of the Capsaicin to the multilayer Nanocomposite had considerable uptake and would be promising and effectively choice for specific pH-stimuli-responsive tumor-targeted DDS and other theranostic applications.

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