Preparation, characterization and pH responsive delivery of Lenalidomide conjugated Fe3O4 nanoparticles


Raja Sundararajan*Sri Anusha Mallina

Department of Pharmaceutical Analysis & Quality Assurance, GITAM Institute of Pharmacy, GITAM (Deemed to be University), Visakhapatnam, Andhra Pradesh, India-Pincode - 530 045.

*Corresponding Author E-mail:



Selective administration of cancer drugs in a specific manner with respect to the physiological need at the tumor site can increase the efficiency of drugs and reduce side effects. Herein, developed a formulation, comprising of anti-cancer drug Lenalidomide conjugated to chitosan (Chs) coated iron oxide nanoparticles (Fe3O4-Chs) via a pH sensitive imine bond formation which enables active topical drug delivery in response to intra cellular pH change was demonstrated for potential treatment of cancer. The particle size was found to be around 40 (mean± SD (n = 20)) nm. The structural characterization of the synthesized formulation was done by X-ray diffraction, thermo gravimetric analysis, differential scanning calorimetry, Fourier transform infrared spectroscopy, vibrating sample magnetometer and transmission electron microscopy techniques. Therapeutic efficacy of Fe3O4-Lnd nanoparticles (Nps) was carried out in human breast cancer cells (MCF7) and human ovarian cells (SK-OV-3) by MTT assay which showed improved therapeutic efficacy of Lnd with Fe3O4-Lnd nanoparticles compared to free Lnd


KEYWORDS: Iron oxide nanoparticles, Chitosan, pH dependent, conjugation, targeted drug delivery.




Cancer is one of the most devastating diseases threatening human life and chemotherapy is an important approach for cancer treatment currently. Chemotherapy is the general treatment for cancer. However, which is limited to the serious toxicity and poor water solubility of most anticancer drugs1. Recently, several kinds of multifunctional nanotechnology approaches have been developed as potential drug carriers to overcome theselimitations2-3. The hydrophobicity of anti-cancer drugs can be altered by incorporation of conjugation4-5 to nanoparticle. Furthermore, blood circulation time and passive absorption of drug in tumor site can be enhanced by enhanced permeability and retention effect (EPR effect)6


Among all of these nano-carriers magnetic iron-oxide nanoparticles have gained significant attention because of their low toxicity, good biocompatibility and biodegradability7. A super-paramagnetic iron oxide nanoparticle with appropriate functionalization is a powerful and illustrative nanoparticle platform for several biomedical applications like magnetic resonance imaging8, controlled drug delivery9-10, gene delivery11, protein separation12, hyperthermia13 and photodynamic therapy14.  


The synthesis conditions and functionalizing agents play a key role on the size and biocompatibility of the magnetite nanoparticles15. The stable dispersions and biocompatible surface modification of nanoparticles can be resulted by the use of natural biopolymers like as dextran, chitosan etc. The stabilizing agents amenable for modifications and conjugations are required for a range of biomedical applications. Chitosan stabilized nanoparticles are more appropriate for cancer therapy applications because of its biocompatibility and biodegradability16. In addition, its positive charge is an


advantage for interacting and internalizing within the cells.


Lenalidomide (Lnd) was chosen as a model drug and it was approved by the US Food and Drug Administration. It is a thalidomide analogue, is an immunomodulatory agent with antiangiogenic and antineoplastic properties17. pH dependent delivery systems are emergently used systems18. Usually pH-sensitive linkages include hydrazones, poly (ortho-esters), vinyl ethers, cisaconityls, acetals, polyketals, thiopropionates and silyl ethers19. The major advantage with pH sensitive nanoparticle conjugated drug delivery system is nanoparticles are stable and no or very less leaking of drug is observed in blood circulation (pH = 7.4). However, at the target tumor cells or acidic endosome/lysosome (pH =4-6) rapid release of drug takes place20. In this study, we designed an imine bond (-C=N-) based pH-sensitive delivery of nanoparticle system by simple chemical conjugation methods. Initially in-situ chitosan functionalized Fe3O4 nanoparticles are prepared by room temperature hydrolysis method. These synthesized nanoparticles were conjugated with Lnd via glutaraldehyde with the formation of pH cleavable imine bond.




Lenalidomide, glutaraldehyde (25%), ferrous sulphate heptahydrate, chitosan (medium molecular weight). Disodium hydrogen phosphate, sodium dihydrogen phosphate, acetic acid, sodium acetate, sodium hydroxide, potassium nitrate, potassium chloride and sodium chloride were purchased from Sigma Aldrich Chemical Co. All the reagents were used as received without further purification.


Synthesis of chitosan functionalized Fe3O4 nanoparticles (Fe3O4-Chs)

Different amounts of chitosan stabilized Fe3O4 NP formulations were prepared by hydrolysis method at room temperature. Initially 10 mg/mL (w/v) chitosan stock solution was prepared by dissolving chitosan in 5% (v/v) acetic acid/water solution followed with overnight stirring to completely dissolve the chitosan. Then 2 mg/mL, 4 mg/ml, 6 mg/ml and 8 mg/ml of chitosan stock solutions were prepared by diluting with same composition of acetic acid/ water mixture. Chitosan functionalized Fe3O4 nanoparticles were prepared by following method (Scheme 1). Briefly, 700 mg of FeSO4.7H2O was dissolved in 100 mL of Milli-Q water, followed by the addition of 10 mL of (2.0 M) KNO3 and 10 mL of (1.0 M) NaOH and this solution was stirred at room temperature. After that 15, 25, 35 and 50 mg (2 mg/ml stock, dissolved in 5% acetic acid solution) of chitosan solution was added drop wise into different vials and then stirred for 2 h at room temperature under nitrogen environment to form Fe3O4-Chs nanoparticles. The formed Fe3O4-Chs nanoparticles were washed several times with water with the help of an external magnet. Among all the formulations, 10 mg of chitosan stabilized Fe3O4 nanoparticles thus synthesized were used for the intended biological applications.


Lenalidomide conjugation to Fe3O4-Chs nanoparticles through glutaraldehyde

Here the synthetic strategy is formation of pH sensitive imine bond. Based on this glutaraldehyde was used as cross linker to conjugate Lnd to the free amine groups of the chitosan functionalized Fe3O4 nanoparticles through a pH sensitive imine bond. For that, first glutaraldehyde was conjugated to the surface amine groups, by the following procedure; 1 ml (1mg/ml) of Fe3O4-Chs nanoparticles were dispersed in PBS and 25 μl of glutaraldehyde (25%) (62.5 mM) was added and stirred for two hours at room temperature. After completion of the reaction, the Fe3O4-Chs-glutaraldehyde nanoparticles (Scheme 1) were separated with a magnet and washed several times with water to remove the unbound reagents followed by conjugation with Lnd. This was followed by several washings and finally the Fe3O4-Chs-glutaraldehyde nanoparticles were re-dispersed in PBS to which 50 μl of Lnd (1mg/ml) was added and stirred for 6 h at room temperature. This was followed by separation with magnet and several washings with water and the supernatant was collected for estimating the encapsulation efficiency of Lnd (Fe3O4-Lnd).


Characterization of Fe3O4-Chs and Fe3O4-Lnd nanoparticles

Powder X-ray diffraction (XRD) patterns were recorded in reflection mode on a Seimens (Cheshire, UK) D5000 X-ray Diffractometer. Transmission electron microscope (TEM) (Philips Tecnai FEI F20, operating at 120 kV) was used to reveal the morphology and size of the particles. Magnetic studies were carried out on a Micro-sense Vibrating Sample Magnetometer (VSM) in the applied magnetic field, sweeping from −22 to 22 KOe. The zeta potential values of the NPs were recorded in phosphate buffered saline (150 mM NaCl, 10 mM sodium phosphate, buffer pH 7.2) at 25°C using a Malvern Zetasizer Nano S instrument (Malvern Instruments, Malvern, UK).



Scheme 1: Preparation of Chitosan functionalized iron oxide nanoparticle


Lenalidomide (Lnd) encapsulation efficiency

After completion of reaction the Fe3O4-Lnd NPs were separated from unbound Lnd by magnetic decantation. The drug encapsulation efficiency was quantified by measuring the absorbance of the unbound drug in supernatant by UV spectroscopy at 315 nm. The estimated amounts were substituted in eq. 1 below to obtain the encapsulation efficiency.



Encapsulation Efficiency =

Amount of Lnd added(µg)-amount of Lnd in supernatant(µg)





Amount of Lnd added (µg)



Study of liquid chromatography (HPLC)

The HPLC study of Lnd was performed by maintaining reported conditions21. The chromatographic conditions were reverse phase, isocratic mode by a mobile phase consisting of 20% acetonitrile having 0.1% trifluoroacetic acid, flowrate: 1 mL/min; column: Agilent, Eclipse plus C8 -150 X 4.6 mm, 5 µm. The eluted products were detected by UV detector at 315 nm is shown in (Figure 1).



Figure 1: HPLC chromatogram of Lenalidomide

In vitro release of Lenalidomide

The pH dependent release of Lnd from Fe3O4-Lnd nanoparticles was studied under different pH conditions. For this study, the Fe3O4-Lnd nanoparticles (15 mg) were diluted in different pH solutions of 7.4, 6.5, 5.5 and 4.5 and incubated at 37°C. At every one hour interval, Fe3O4-Lnd nanoparticles were centrifuged, the supernatant is collected and the same volume of fresh buffer solution was replaced in the respective tubes. This sample collection procedure was continued up to 8 hrs and the released Lnd concentration in supernatants were estimated by using absorption spectroscopy and HPLC studies. Percentage of Lnd release was calculated and compared with standard Lenalidomide HPLC measurements.


Cell lines

The human ovarian cancer SKOV3 and human breast cancer MCF7 cell lines were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The cells were maintained at 37°C in a humidified 5% CO2 containing atmosphere and sub cultured biweekly to maintain sub confluent stocks.


Cell Viability and MTT Assay Protocol

The cell viability assay was carried out in two cancer cell lines, human ovarian cancer and breast cancer cell lines, SK-OV-3 and MCF7 respectively. The MCF7 and SKOV3 cells were seeded with 2×105 cells per well in a 96-well plate in DMEM complete medium and incubated at 37°C under 5% carbon dioxide (CO2) environment overnight. After cells reach 70-80% confluence in all the wells the medium was removed and 100 μl fresh DMEM complete medium was added into each well. Further Fe3O4-Lnd and free Lnd of different concentrations were correspondingly added in triplicates. Thereafter the plates were mixed well and again incubated at 37°C under 5% CO2 environment for 24 hours. After 24 hours of incubation the complete medium was removed and washed with 200 μl PBS per well twice to remove any unbound particles and serum. Then to each well 100 μl of MTT reagent prepared with DMEM medium (i.e. 25 μl of MTT reagent (stock conc. 2 mg/ml) + 75 μl of DMEM medium without FBS was added and incubated at 37°C for 3 h under 5% CO2 environment. After 3 h of incubation the MTT reagent was removed and 100 μl of DMSO/methanol (1:1 v/v) added into each well and incubated for 15 mins on shaker and the optical density at A540 nm excitation was noted.



X-ray diffraction studies

After synthesis of nanoparticles (Nps) was first confirmed with X-ray diffraction and the crystallinity is confirmed after conjugation of Lnd.  The crystalline planes of Fe3O4-Chs and Fe3O4-Lnd Nps were observed at 2theta values of 30.4, 35.4, 43.2, 53.4, 57.2 and 62.7 deg corresponding to the planes (220), (311), (400), (422), (511) and (440) respectively. Fe3O4-Chs and Fe3O4-Lnd Nps are represented in (Figure 2). Moreover, the 100% peak which was observed at 35.4 2theta corresponding to the (311) plane that perfectly represents pure magnetite plane of standard JCPDS data (Card No.19-0629). The pH sensitive NPs show a similar pattern with Fe3O4-Chs nanoparticles which indicates that the synthetic steps do not have any effect on the crystalline structure of Fe3O4 nanoparticles and the initial crystalline structure is preserved.


Figure 2:  X-Ray Diffraction pattern of chitosan stabilized magnetite NPs and Lenalidomide conjugated NP



Figure 3A and Figure 3B represent the SEM and TEM micrographs of the Fe3O4-Chs nanoparticles. The particle size of the nanoparticles was observed as around 40 nm.


Figure 3: FE-SEM (a) images and TEM (b) images of Fe3O4-Chs nanoparticles


Dynamic Light Scattering (DLS)

The studies from TEM and SEM have shown that particles are in the size range of 40-45 nm. As expected the hydrodynamic diameter of the particles determined by DLS, was found to be 60 ± 10 nm (Figure 4). The higher size obtained from DLS is due to the hydration shell present on the surface of the nanoparticles in DLS measurements, which was absent when analyzed in a dry state using TEM or SEM. The polydispersity index (PDI) value was found to be 0.245 ± 0.02.



Figure 4: Dynamic Light Scattering study Fe3O4-Chs nanoparticles


Zeta potential

The surface charge on the Fe3O4-Chs nanoparticles was measured by Zeta Potential measurements. Here the samples were dispersed in PBS with NaCl. Due to presence of surface free amine groups on Fe3O4-Chs Nps, the particles show Zeta Potentials value of +14.89 mV. However, the Zeta Potentials value of Fe3O4-Lnd is seen at +7.04 as shown in (Figure 5). The decrease in surface charge confirms the conjugation of Lnd to the positively charged amines of the chitosan on Fe3O4-Cs NPs.


 Figure 5: Zeta Potential studies of synthesized nanoparticles




Chitosan absorption and further Lnd conjugation on Fe3O4 NPs were studied by thermo gravimetric analysis (TGA). (Figure 6) shows the weight loss vs temperature curves of free Lnd, Fe3O4-Chs and Fe3O4-Lnd NPs. As shown in (Figure 6a), the adsorbed residual water molecules are removed along with other impurities between 0-200 oC, after that the chitosan degradation starts at 150 oC and continues up to 450 oC corresponding to the major breakdown of the chitosan chain covering the Fe3O4 nanoparticles. Significant weight loss was observed between 280-700 oC and thereafter up to 600 oC mild degradation occurred. In case of free Lnd a sharp weight loss at 240 0C (Figure 6b) indicates the degradation of Lnd. (Figure 6c) represents the Fe3O4-Lnd NPs, here the significant weight losses at 250 oC and 450 oC represents the chitosan and Lnd degradation respectively.


Figure 6: Thermo gravimetric analysis of Fe3O4-Chs nanoparticles Free Lnd (b) Fe3O4-chs (c) and Fe3O4-Lnd NPs


Vibrating Sample Magnetometer

The study of Fe3O4-Chs and Fe3O4-Lnd nanoparticles at room temperature using vibrating sample magnetometer (VSM) is presented (Figure 7). The low values of coercivity and remanence are attributed to the nanosized magnetic particles that are superparamagnetic in nature22. The similarity in the plots indicates that chitosan does not alter the magnetic character of the Fe3O4 NPs. An observed slight decrease in the saturation magnetization (Ms) of chitosan coated Fe3O4 nanoparticles after functionalization with Lnd, indicates a shielding effect. The significant decrease in the Ms may be attributed to surface effects caused by surface functionalization23, 24. In this study, the observed Ms value from (Figure 7) show that the Fe3O4-Chs NPs shows a Ms value of 50.98 emu/g and this value decreases to 40.46 for Fe3O4-Lnd NPs. This decrease can be attributed to the shielding effect by the Lnd conjugated on the Fe3O4-Chs NPs.


Figure 7: Vibrating sample magnetometer measurement plots of chitosan stabilized Fe3O4 nanoparticles (open circle) and Lenilodamide conjugated Fe3O4 NPs (dark circle) at room temperature


Lenilodamide encapsulation efficiency

The encapsulation efficiency of Lenilodamide on Fe3O4 nanoparticles surface was measured by recording the absorbance in DMSO.  Incorporating the parameters like initial amount of drug added and the amount of drug unbound, which were estimated from absorbance studies in equation.1 the encapsulation efficiency of Lnd was calculated to be 70%.


pH dependent drug release

In vitro drug release study of the Fe3O4-Lnd was carried out in different pH buffer solutions to evaluate the effect of pH on the cleavage of the imine bond of Fe3O4-Lnd linkage, at room temperature. The percentage of Lnd release was estimated at different time periods by HPLC and absorbance techniques. Fe3O4-Lnd was incubated in PBS at different pH solutions to examine drug release under conditions likely to be encountered following NP tumor uptake and intracellular suspension. The percentages of drug release in 8, 18, 40 and 70% PBS solutions at different pH conditions of 7.4, 6.5, 5.5 and 4.5 respectively after 8 hours incubation are represented in (Figure 8). Improvement in Lnd release at acidic pH indicates that Lnd is released preferentially in the environment that mimics endosomal/lysosomal compartment of the cell where it is protected from drug efflux 25.



Figure 8: In vitro drug delivery study at different pH (PBS) solutions of (a) 7.4 (open circle) (b) 6.5 (dark circle) (c) 5.5 (dark square) and (d) 4.5 (open square) with time variation.


Biocompatibility study of Fe3O4-Chs nanoparticles

Before going to study cytotoxic effect of the designed pH dependent Np formulations, we have studied the effect of Fe3O4-Chs nanoparticles on the cells at very high concentrations. As the effectiveness of the developed nanoparticle based drug delivery formulation also relies on the stability in physiological conditions. We studied and confirmed the stability in physiological conditions. Fe3O4-Lnd was incubated in DMEM medium with serum (1 mg/mL) for 24 h at 37oC. At every four hours interval, particles were taken and centrifuged, the supernatant collected and the same volume of fresh medium replaced. This sample collection procedure was continued up to 24 hours of incubation and the released Lnd concentration in supernatants were estimated by using HPLC. As shown in (Figure 9) the nonspecific release of Lnd was not observed even up to 24 h of incubation. Thus, the results imply that the designed nanoparticle delivery system could ensure the high stability of conjugated drug and be deemed suitable for further application.


Figure 9: Available Lenilodamide in Fe3O4-Lnd nanoparticles after incubated in 10% serum / DMEM

In Vitro cytotoxicity assay in SK-OV-3 and MCF7cell line

To investigate the cytotoxicity of the developed Fe3O4-Lnd nanoparticles in comparison with free Lnd, two cancer lines SKOV3 and MCF 7 were chosen. (Figure 10) represents the cell viability of SKOV3 and MCF 7cells, as determined by MTT assay with different concentrations of Lnd. The cell viability decreased with increasing concentration of Lnd with Fe3O4-Lnd nanoparticles and free Lnd in both SKOV3 and MCF 7cell lines. Both the cell lines show similar trend after 24 hours of incubation with free Lnd and Fe3O4-Lnd as represented in (Figure 10A & 10B). Interestingly, Fe3O4-Lnd particles were found to be more effective in killing the cancer cells at the same Lnd concentrations of the free drug under the same assay conditions in SKOV3 and MCF 7 cell lines.


Furthermore, the IC50 (half maximal inhibitory concentration) of Fe3O4-Lnd is around at 10 µM while in the case of free Lnd it is 15.89 µM, which indicates effective cytotoxicity with Fe3O4-Lnd than free Lnd. These results indicate that the Lnd activity is not affected by Fe3O4-Chs nanoparticles conjugation and that the nanoparticles facilitate drug internalization, which enhances drug efficacy.  These observations suggest that intracellular concentration of Lnd delivered through Fe3O4-Lnd nanoparticles is higher than that of free Lnd and that the Lnd-loaded nanoparticles were able to enter the cells and exhibit a positive pharmacological effect on the cancer cells.




Figure 10A: In vitro cell viability studies in human ovarian cancer cell line (SKOV3) (a) Fe3O4 - Lenilodamide (dark circle)

(b) free Lenilodamide (open circle)



Figure 10B: In vitro cell viability studies in human breast cancer cell line (MCF 7) (a) Fe3O4 - Lenilodamide (dark circle)

(b) free Lenilodamide (open circle).


The biomedical applications of these nanoparticles were demonstrated by using the Fe3O4-Chs nanoparticles as carriers for drug delivery specifically to the cancer cells in two cell lines viz. human breast cancer cells (MCF 7) and human ovarian cancer cells (SK-OV-3). In the study of pH sensitive delivery of Lnd using nanocarriers, the carbonyl group of Lnd was utilized for making imine bond26. In the present study the amine groups of Lnd were conjugated with carbonyl groups of glutaraldehyde to form imine bond (-C=N-) with encapsulation efficiency of 70 %. The advantage of imine bond is that it selectively cleaves at lower pH conditions and based on this strategy the present drug delivery application has been designed. As represented in (Figure 8) it is seen that after 8 hours around 80% of the drug is released at pH 4.5 and 10% at pH 7.4. Additionally, to substantiate the imine bond destabilization in lower pH, the releasing profile was also studied with control particles in which the imine was reduced. The above result illustrates that the designed imine linkage is specifically triggered at lower pH and ensures the drug release at the intracellular components in the cancer cells. The Fe3O4-Lnd nanoparticles confirmed the cell death by MTT assay.  In both, the SK-OV-3 and MCF 7 cell lines, after 24 h treatment, higher cell death was observed with Fe3O4-Lnd nanoparticles than free Lnd for equivalent concentration of Lnd. These results clearly indicate that the Fe3O4-Lnd nanoparticles showed increased therapeutic efficiency in the cancer cell lines when compared to the effect of free Lnd and believe that this system would be useful in reducing toxicity to normal tissues while simultaneously increasing the therapeutic efficiency to cancer tissues. 





The chitosan stabilized magnetite NPs were in-situ synthesized by a simple aqueous medium at room temperature. An anti-cancer drug lenilodamide was conjugated to the magnetite NPs through glutaraldehyde cross linker with imine bond, which is labile at pH of the intracellular components, endosomes and lysosomes of the cancer cell. The synthesized lenilodamide conjugated magnetite NPs, were used for the endosomes and lysosomes pH drug release studies in different pH buffer solutions. In vitro drug release (cell viability) experiments in human breast cancer (MCF7) and human ovarian (SK-OV-3) cell lines establish that the magnetic nanoparticle conjugated Lenalidomide showed an enhanced therapeutic effect when compared to corresponding concentrations of free drug along with the added advantage of reduced toxicity to the normal cells because of the targeting capability.



The authors declare no conflict of interest.



We express our sincere gratitude to the management of GITAM (Deemed to be) University, Visakhapatnam, Andhra Pradesh, India for supporting our work.



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Received on 14.08.2018          Modified on 11.09.2018

Accepted on 12.10.2018        © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(10): 4605-4612.

DOI: 10.5958/0974-360X.2018.00843.0