Synthesis of doxorubicin-PLGA loaded chitosan stabilized (Mn, Zn)Fe2O4 nanoparticles: Biological activity and pH-responsive drug release

    Synthesis of doxorubicin-PLGA loading on chitosan stabilized (Mn, Zn)Fe 2 O4 nanoparticles: Biological activity and pH-responsive drug release studies Wararat Montha, Weerakanya Maneeprakorn, Nattha Buatong, I-Ming Tang, Weeraphat Pon-On PII: DOI: Reference:

S0928-4931(15)30424-0 doi: 10.1016/j.msec.2015.09.098 MSC 5810

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

14 June 2015 3 September 2015 28 September 2015

Please cite this article as: Wararat Montha, Weerakanya Maneeprakorn, Nattha Buatong, I-Ming Tang, Weeraphat Pon-On, Synthesis of doxorubicin-PLGA loading on chitosan stabilized (Mn, Zn)Fe2 O4 nanoparticles: Biological activity and pH-responsive drug release studies, Materials Science & Engineering C (2015), doi: 10.1016/j.msec.2015.09.098

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Synthesis of doxorubicin-PLGA loading on chitosan stabilized

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(Mn, Zn)Fe2O4 nanoparticles: Biological activity and

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pH-responsive drug release studies

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Wararat Montha a, Weerakanya Maneeprakornb, Nattha Buatonga, I-Ming Tangc,

a

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Weeraphat Pon-On a,*

Department of Physics, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.

c

National Nanotechnology Center, Pathum Thani 12120, Thailand.

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b

Department of Materials Science, Faculty of Science, Kasertsart University

*

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Bangkok10900, Thailand.

Corresponding author: [email protected] Tel.:662 562 5555 ext. 3008-3011; Fax: 662 942 8029

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Abstract We have synthesized Mn1-xZnxFe2O4 ((Mn, Zn) ferrite) magnetic nanoparticles (MNPs)

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having radius of 25 nm to act as platforms for delivering drugs. The Mn0.9Zn0.1Fe2O4 MNPs They were

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exhibits superparamagnetic behavior with large saturation magnetization (Ms).

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encapsulated in polymer in order for them to be developed into PLGA-coated chitosan stabilized (Mn,Zn) MNPs, i.e., DOX-PLGA@[email protected] as an effective carrier of the anti-

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cancer drug doxorubicin (DOX) whose release would be controlled by the pH in the environment

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surrounding the cancer tumor. The structure of the as-prepared particles is of a magnetic coreencapsulated by polymer shell layer of around 50 nm thick. At a pH of 4.0, the DOX release

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within the first 5 hr. is fast (around 57%). It becomes slower (around 46% over the next 25

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hours) when the pH is increased to 7.4. The DOX-PLGA@[email protected] (for concentrations lower than 125μgmL-1) showed lower toxicity against HeLa cells that using DOX

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only. When the DOX-PLGA@[email protected] is increased to 250μgmL-1, the DOXPLGA@[email protected] show greater anti-cancer activity and has satisfactory therapeutic effect. The slow sustained release of the DOX by the drug loaded particles while they are in the physiological pH environment (7.4) of normal tissues and mild toxicity of DOX against cancer cell at low concentration point to the DOX loaded PLGA@[email protected] being safely used for treating cancer. The higher dosage of DOX needed to kill the cancer cells will be released when the synthesized carriers are subject to the pH stimuli surrounding these cells.

Keywords: drug delivery systems; pH responsive; magnetic particles; controlled release

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1. Introduction The possible use of nano particles as platforms for delivery of therapeutic drugs has

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recently emerged [1-11]. Much of the research on these drug delivery systems (DDSs) have

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been for the treatment of cancer. The research has center on overcoming the main obstacle to the treatment of cancer by chemotherapy, i.e., the destruction of healthy tissue by the therapeutic

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agents. To overcome this, nanoparticle delivery systems have been developed which have a

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specific (size) distribution of anticancer agents and which have a mechanism to trigger the release of the therapeutic agents so that the drug release would only lead to an accumulation of

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the agents at the tumor sites. The typical materials in the platforms used primarily for drug delivery are either polymer or inorganic materials or combinations of these two materials. These

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materials are then mixed with the anti-cancer drug in a way such that they will be released in response to the physiological environment surrounding a tumor (internal stimuli).

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During the past ten years, great efforts had been devoted to synthesizing stimuli-sensitive nanoparticles that would release the agents when certain values of a stimulus based on some physiological parameters (internal stimuli) such as pH, glucose or enzyme etc [12-20] were encountered. Special attention has been paid to the development of pH responsive nano-particle materials since the pH surrounding different types of tissues are different. The pH in the environment surrounding cancer cells is around 6.5 while the value around acidic organelles is around 4.0 to 5.0 [12,18, 22]. As the drugs are released from the DDSs into the acidic region, the toxicity of the drug in the other parts of the body will be lessen. In addition to external stimuli, external stimuli such as light, magnetic field, heat and ultrasound have been employed to make the particles more useful for applications in the biomedical fields [11-13, 21]. Use of an externally applied magnetic field gradient to increase

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the accumulation of drug in the target region, one must have magnetic nanoparticles (MNPs). The magnetic materials for these DDSs must be biodegradable and be able to release the drugs in Such a material that is the most

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response to internal stimuli (endo/lysomal condition).

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commonly used material in biomedical applications, are the superparamagnetic iron oxide nanoparticles (SPIONs) [23-29]. In the present study, we have chosen Mn and Zn substituted

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ferrite (Mn1-xZnxFe2O4) nanoparticles since they can be easily made to have a small size and the

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high magnetization needed for them to respond to an external magnetic field [29, 30]. Furthermore, Li et al. [30] reported that Mn-Zn ferrite embedded in a polymer matrix exhibited

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low Tc (Curie temperature) close to the body temperature which would be useful for clinical viability and safety.

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To achieve the required pH sensitive controlled release properties, we have pick chitosan (CS) as the agent for triggering the release of the therapeutic agents. There is widespread

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interest for its potential application in DDSs [31-41] due to them being biodegradable and being a biocompatible cationic polymer with low toxicity. Javid et al [35], Unsoy et al [36], Deng et al [37] and Hu et al [38] reported that chitosan-coated super paramagnetic iron oxide and silica nanoparticles showed better doxorubicin (DOX) drug-loading, pH controlled release, enhanced cancer cell uptake and inhibition cancer cell growth after exposure to the doxorubicin. To improve the effectiveness of this drug, we have coated chitosan stabilized (Mn,Zn) MNPs with Poly D, L-lactide-co-glycolic acid (PLGA). PLGA is a FDA approved biocompatible and biodegradable polymer [42-44]. For PLGA application, Sivakumar et al. [45] studied on SPION and curcumin encapsulated HER2 targeted PLGA and found that they displayed promising potential anticancer activity by destroying pancreatic cancer cells. Functionalize PLGA by chitosan nanoparticles have been widely used for the delivery carrier of various chemo-therapeutic agents

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to the target site [39-44]. Utilizing the above interesting properties, we aimed to synthesis the anti-cancer drug doxorubicin (DOX) into the PLGA-coated chitosan stabilized (Mn, Zn) ferrite (DOX-PLGA@[email protected]).

We

have

characterized

the

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nanoparticles

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physicochemical properties and biological effects of the (DOX-PLGA@[email protected]) particles and compared them with those of the free drug. To further investigate the drug delivery

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application, we studied their pH stimuli controlled release to see whether they would be an

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effective doxorubicin (DOX) anti-cancer drug carrier, i.e., released the doxorubicin (DOX) at the target sites.

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2. Materials and methods 2.1 Materials

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Ferric chloride (FeCl3 anhydrous), Manganese nitrate (Mn (NO3)4H2O) and zinc nitrate ((Zn (NO3))2 6H2O) were obtained from UNIVAR (Australia). The sodium dodecyl sulfate

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(SDS), Na2SO4, citric acid, and ethylene glycol (EG) were obtained from Fluka (Switzerland). Glutaraldehyde (UNILAB) and Poly (lactic-co-glycolic acid) (PLGA) 85/15 with molecular weight of approximately 190,000-240,000, Chitosan (CS, deacetylation degree 90%, Mv 3.8x105) with 3-[4, 5-Dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT) assay and other biological reagents, were purchased from Sigma-Aldrich (USA). 2.2. Fabrication of the PLGA@CS@(Mn,Zn) ferrite nanoparticles. The PLGA-coated chitosan stabilized (Mn, Zn) ferrite nanoparticles were synthesized in a two-step process based on the electric attraction between the positively charged (–NH3+) ions on surface of the chitosan and the negatively charged carboxyl groups (–COO-) radicals of the PLGA. First, chitosan coated citric acid functionalized (Mn, Zn) ferrite nanoparticles are made. The chains on the chitosan are needed for the formation of the layer which would be sensitive to

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the pH stimuli. To stabilize surface and to increase the drug adsorption by the colloidal CS@(Mn,Zn) ferrite nanoparticles, poly (lactic-co-glycolic acid) (PLGA) was coated onto them

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to form the core-shell (PLGA@CS@(Mn,Zn) ferrite nanoparticles. The inclusion of PLGA

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assisted in making the outer shell biodegradable.

2.2a. Preparation of chitosan-stabilized (Mn, Zn)Fe3O4 nanoparticles.

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Magnetic Mn1-xZnxFe2O4 nanoparticles were made using the chemical co-precipitation

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method with x value adjusted from 0.1 to 0.9. Typically, 50 ml of mixed solution containing manganese nitrate (Mn(NO3) 4H2O), zinc nitrate ((Zn(NO3))2 6H2O) and ferric chloride in their

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respective stoichiometry were first prepared. 10 ml EG and 5 ml HCl (0.2M) was then added into the mixture under vigorous stirring. The solution was then stirred for an addition 30 min at room

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temperature. Then, in a 250 ml three-neck round-bottom flask equipped with a magnetic stirring bar, 3 g NaOH and 0.54 g SDS was dissolved in 25 ml of distilled water at 80°C for 30 min. The

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precursor solution was injected into the flask slowly and the stirring was continued for another 30 min under N2 at 80°C. The precipitate was collected by exposing the final solution to them to an external magnetic field. The concentrated precipitates were physically removed from the solutions and washed three times with ethanol and deionized water. They solutions were then freeze dried to obtain the nanoparticles. We used the MNPs having the highest magnetic moments as determined by our VSM measurement (Fig. 3). Our next step was the fabrication of the chitosan-stabilized (Mn, Zn)Fe2O4 particles (CS@Mn1-xZnxFe2O4) and the detailed procedures combined with previous reports [20, 30, 40].

We selected x = 0.1, i.e., Mn0.9Zn0.1Fe2O4 as the magnetic

precursor. Surface modification of these magnetic nanoparticles was carried out by first treating it with citric acid. Citric acid (CA) (0.05M) and the Mn0.9Zn0.1Fe2O4 NMPs were mixed together in

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a three-neck round-bottom flask having a magnetic stirring bar inside. The mixture was stirred for 1.5 h at 85°C under N2 atmosphere. At the end of reaction, the carboxylic functional magnetic

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particles were washed with deionized water for three times. The synthesized nanoparticles were

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freeze-dried and stored at -20°C for further studies.

The chitosan stabilization was achieved by dispersing the CA-modified Mn0.9Zn0.1Fe2O4

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nanoparticles into 15 ml of chitosan solution (CS) (1g of chitosan dissolved in 100ml of 2.5 wt%

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acetic acid). This was achieved by rapidly stirring the mixture using a magnetic stirrer for 30 min at room temperature. At the end of the mixing, 3ml glutaraldehyde solution (GD, 25wt %) was

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added in the solution and stirred for an addition 4 hr. Finally, the CS-stabilized magnetic nanoparticles were collected by magnetic bar and washed three times with ethanol and deionized

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water, respectively. The obtained products were by freeze dying and stored at -20°C for further studies.

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2.2b. Preparation of PLGA-coated chitosan stabilized (Mn, Zn)Fe3O4 nanoparticles and doxorubicin drug loading (DOX-PLGA@CS@(Mn,Zn) ferrite). 50 mg CS-modified Mn0.9Zn0.1Fe2O4 nanoparticles were added to a solution of PLGA (50mg in 0.5ml chloroform) and doxorubicin (1mg/ml). This was mixed with a magnetic stirrer for 4 hrs. The mixture was then stirred continuously overnight at 4°C so that the organic solvent (chloroform) would evaporate.

The DOX encapsulated PLGA@[email protected] nano

particles were collected by centrifugation with the supernatant being collect so that the concentration of drug remaining in it could be determined. The resulting products were freezedried and stored in a refrigerator at 4°C for further investigations. 2.3

Physicochemical

characterization

(Mn, Zn)Fe3O4 nanoparticles

of

the

PLGA-coated

chitosan

stabilized

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The crystal structures of the synthesized powders were determined by powder X-ray diffraction (XRD) (Bruker diffractometer, Model D8 Advance) using the Cu Ka radiation and

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operating at 40 kV with 40 mA current. The XRD patterns were scanned from 2θ = 20°-70° at a

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scanning speed of 1 s per step with an increment of 0.037° per step. For the FT-IR absorption measurements of the with and without DOX-loaded PLGA@CS@(Mn,Zn) ferrite, the powders

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were mixed with KBr and pressed into pellets under a pressure of 10 tons for 1 min. The pellets

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were analyzed using a FT-IR spectrophotometer (Spectrum GX, Perkin Elmer) which performed 16 scans over the range 370-4,000 cm-1. The magnetic properties of PLGA@CS@(Mn,Zn)

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ferrite particles were measured using a room temperature VSM (vibrating sample magnetometer (Lakeshore, Model 4500)). Meanwhile, the core-shell particles were also examined by a

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transmission electron microscope (TEM) (JEOL model JEM-2010). Dynamic light scattering

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(DLS) with the concentration of 0.5 mg mL-1 was utilize to record hydrodynamic radius at 25°C

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using DLS, Zetasizer nanoseries model S4700 (Malvern instrument, UK)). The chemical composition of the PLGA@CS@(Mn,Zn) ferrite nanoparticles was analyzed by Raman spectroscopy (Senterra Raman microscope; Bruker Optics, Ettlingen, Germany) using an excitation wavelength of 532 nm at 5mW. Raman spectra were recorded in the range 450070 cm-1. 2.4

Cell culture and cytotoxicity analysis Human cervical cancer cells (HeLa cells) were cultured onto 25 ml flask using a culture

medium (RPMI supplemented with 10% FBS, 100 units/ml penicillin G and 100 mg/ml streptomycin). The cultured cells were kept at 37



C in a humidified CO2 incubator during

cultivation and during the experiments. Cytotoxicity analysis was conducted on HeLa cells. The cultured

cells

were

treated

with

different

concentrations

of

doxorubicin

loaded

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PLGA@[email protected] nanoparticles. Briefly, cells were seeded in 96 well plates and allowed to grow overnight at 37°C in a CO2 incubator. The culture medium was then replaced

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with fresh medium containing nanoparticles in the range of concentrations 0-50  g(Fe)/mL.

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The cells were then allowed to grow for a further 24 h at 37°C in a CO2 incubator. The wells

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were then washed with PBS to remove any excess nanoparticles and replaced with fresh medium containing the Presto Bluecell viability reagent (Life Technology) according to the

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manufacturer’s specifications. The treated cells were incubated for 1 h in the dark at room temperature. The fluorescence of each well was measured in a microplate reader at an excitation

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and emission of 560 and 590 nm, respectively. The fluorescence intensity is directly proportional to the number of viable cells in the well. Experiments were performed in triplicate and results

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were normalized to control conditions (cells that were not exposed to nanoparticles). A negative control where cells were exposed to 0.1% Triton-X-100 instead of the nanoparticles was used in

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each experiment.

2.5 In vitro release studies

The in vitro study of pH controlled DOX release from the PLGA@[email protected] nanoparticles was performed as follows: First, 1mg of DOX-PLGA@[email protected] nanoparticles were placed in 15 mL of phosphate buffer solution (PBS) (pH=7.4 and 4.0) with the

temperature

at

37°C.

The

lengths

of

time

that

the

DOX

released

PLGA@[email protected] nanoparticles were kept in the PBS solution were then varied (the time periods being 0.5, 1, 3, 5, 7, 15, 20 and 25 hrs). At the end of each time period, the supernatant was taken out. The concentration of the DOX was determined using a UV-vis spectrophotometer (Jenway, model 6405) operating at a wavelength of 480 nm. The drug content in supernatant is determined by comparing the measured absorbance to a standard curve for the

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DOX adsorption. The DOX release experiments were performed in triplicate and the amount of DOX released at each time point was determined as a mean value ± standard derivation (SD).

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3. Results and discussion

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3.1 Material characterization

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Figure 1 shows the powder XRD patterns of as-prepared magnetic Mn1-xZnxFe2O4 nanoparticles. The XRD patterns show that the (Mn, Zn)Fe2O4 nanoferrites have a single phase

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cubic spinel ferrite structure and do not exhibit any undesirable crystalline phase. The diffraction

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peaks at about 30.2, 35.6, 43.0, 57.1 and 62.5° corresponds to index of (220), (311), (400), (511) and (440), respectively, are clearly observed, which are the peaks of ferrite [29, 30], matching

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well with the JCPDS file of PDF 00-022-1012, 01-089-6609, 01-070-8731, 01-089-7556 and 01-

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086-0509 for Mn1-xZnxFe2O4 nanoparticles with x value adjusted from 0.1, 0.3, 0.5, 0.7 and 0.9, respectively. In addition, the variation of the lattice parameters of 8.46 Å (x=0.1), 8.44 Å

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(x=0.3), 8.45 Å (x=0.5), 8.44 Å (x=0.7) and 8.42 Å (x=0.9) are observed. A decrease in lattice parameter with further substitution of Zn atoms can be explained by substitution of bigger Mn2+ ions (0.91 Å) by smaller Zn2+ (0.74 Å) ions [29]. Transmission electron microscopy (TEM) was used to investigate the magnetic core and polymer shell of the samples. As seen in Fig 2(a), the as-synthesized magnetic nanoparticles show a monodispersed spherical morphology with a uniform size of 25 nm. In Fig 2(b), the coreshell structure of PLGA@[email protected] can clearly be seen. Dark particles of about ~20 nm surrounded by polymer shells of 25-30 nm thickness are clearly seen in each particle. On the basis of the images, most of the magnetic nanoparticles appear to have a double shell composed of a modified biodegradable external shell of PLGA and an internal pH responsive shell of CS on the surface of the (Mn, Zn)Fe2O4 nanoferrites. Next, the average diameter of particles measured

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by DLS was about 368 nm at 25°C. The hydrodynamic size of PLGA@[email protected] from DLS (Fig. s1) was larger than those determined from TEM. This could possible due to the

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shrinkage of the hydrate layer in the dry environment present during the TEM measurements.

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The room-temperature magnetization curves of (Mn, Zn)Fe2O3 and PLGA@CS@ferrite are

coercivity (Hc) or remanent magnetization (Mr).

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shown in Fig. 3. The magnetic hysteresis curves of the synthesized particles do not exhibit any They do however exhibit a saturation

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magnetization MS showing that that all the (Mn, Zn) ferrites possess super paramagnetic

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properties at room temperature. We see that the saturation magnetization MS of Mn1-xZnxFe2O4 systems depend on the Mn/Zn ratio. The Ms gradually decrease with the increase of the Zn2+

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dopants (a non-magnetic ion) (Figure inset). The change in Ms is due to the influence of the

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cationic stoichiometry and occupancy of cations in specific sites. The Ms of Mn0.9Zn0.1Fe2O4 (56.1 emug-1) nanoparticle is higher than that of PLGA@[email protected] (13.2 emug-1)

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nanoparticles (Fig. 4). The cause of this is the presence of the polymers shell in the latter nanoparticles. Even though the magnetizations of the PLGA@[email protected] would be less than that of the uncoated substrate, the PLGA@[email protected], the nano particles still have good magnetic responsiveness (Fig. 4 inset). This means that these coated nano particles can still be affected by an external magnetic field, opening up the possibility of them being targeted to sites by applying an external magnetic field to locations where the pH is not the physiological neutral 7.0. The

FTIR

spectra

of

PLGA@[email protected]

and

DOX-

PLGA@[email protected] are shown in Fig 5. The characteristic peak at around 3500 cm-1 corresponded to the O-H stretching band [39]. The characteristic peaks of chitosan in the region around 3431cm-1 ascribed to the N-H group bounded to the O-H group are also seen [40]. The

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peaks at 1632 cm-1 and 1560 cm-1 are due to the amide I and II bonds, respectively [40]. The characteristic peak at 1761 cm-1 corresponds to the C=O stretching vibration of lactone in PLGA

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[39, 40]. With respect to the PLGA@[email protected], the amide I and II bands of the absorbed CS are shifted to higher wave numbers (1635 cm-1 and 1569 cm-1). The two new peaks

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appearing at 1652 cm-1 and 1586 cm-1 account from the electrostatic interaction between the two

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polymers (CS and PLGA) [39, 40] and are the characteristic peaks due to the presences of the –

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NH3+ and –COO- radicals. The FTIR analyses were performed in order to account for the DOX loading efficiency on PLGA@[email protected]. The spectra of DOX alone shows multiple

and 1071 cm-1 (C-O) [35].

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peaks at 2932 cm-1 (C-H), 1730 cm-1 (C-O), 1618 cm-1 and 1577 cm-1 (N-H), 1414 cm-1 (C-C) These peaks are also present in the FTIR spectrum of DOX-

results

indicate

that

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PLGA@[email protected] , i.e., 2929 cm-1 (C-H) and 1717 cm-1 (C-O), respectively. These the

DOX

had

been

successfully

loaded

onto

the

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PLGA@[email protected] nanoparticles. In addition, the composition of synthesized particles was investigated by Raman spectroscopy (Fig s2.). The PLGA spectrum of due to the C-O stretching vibrations show peaks at 1771.5cm-1, 1147cm-1 and 1044cm-1. Furthermore, the C-H bending and stretching vibrations are observed at 1453 cm-1and 870 cm-1, respectively. In the spectrum of CS, the strong band corresponding to stretching vibrations of amide bond is confirmed at 1380 cm-1 [46]. 3.2 Drug release The in vitro drug release of DOX from the loaded PLGA@[email protected] nanoparticle was carried out at pH values of 7.4 and 4.0 over 25 h at 37°C. As a control, different pH values were used to simulate the drug release of pH-sensitive nano carriers in normal tissues and tumor tissues conditions, respectively. Figure 6 shows the DOX release rate is pH dependent,

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increasing as the of pH values decrease. The two behaviors are observed as: an initial small burst release during the first five hour. This is followed by sustained release after fifth hour. The initial

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burst release of DOX is attributed to the release of DOX molecules attached to the surface of

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particles. As seen for the case of pH = 7.4, the drug release rate is relatively slow (the initial burst release is only about 34.26 %.) and then it slow down after 5h. The accumulated release amount

4.0

is

much

faster

and

more

complete.

The

burst

release

from

the

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is

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is less than 46.60% after 25h. The release of the nanocarriers when the pH of the buffer solutions

PLGA@[email protected] particles during the first five hours is about 57.18%. The faster

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drug release rate in lower pH medium can be attributed to two factors: one is the loose nanoparticle structure which causes the stronger protonation of the free amino groups of chitosan

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and the enhancement of PLGA degradation when the pH is low; the other is the higher solubility of DOX at lower pH [35, 36]. The drug release rate from nanoparticles is reduced with the

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increase in the pH value of dissolution medium. It is usually assumed that the release of the drug in given by some power law dependence. In the Peppas’s model): [47], the release is given by

Mt  kt n M0

(1)

where M t and M o are the concentration of drug released into a medium up to time t and the initial concentration of drug on the particles. M t M 0 is the fractional concentration of the release drug. We have applied a regression analysis to fit Peppas’s model to the data given on Fig. 6 to Eqn, (1). The result fitting shown have correlation coefficients Rc of 0.938 and 0.947 when the pH value of medium is 7.4 or 4.0, respectively. The important parameter obtained from power law is the value of n which provides information on the nature of the transport

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mechanism. The regression analyses give the values of n of 0.14 and 0.09 for the two pH values of medium, respectively. Values of n being between 0 and 0.5 indicate that that the transport

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process for the DOX release is a Fickian diffusion [47]. The values obtained indicated that at

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both pHs, the DOX release from PLGA@[email protected] is controlled by the diffusion

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process. This means that the DOX inside the particles would be stable with very little leakage into the blood when they are circulating in normal blood, thus reducing its toxicity to normal

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tissue. Upon reaching the targeted tumor site which has a weakly acidic environment, the DOXPLGA@[email protected] MCPs would be immobilized by the magnetic field gradient

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superimposed there and be concentrated about the cancer tumor. Some DOX would be released because of the low pH of the environment but even more DOX would be released as these As a

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particles reach the lower pH regions inside the endosome/lysosome compartment. consequence, more cell death would occur.

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3.3 In Vitro Cell Assay

The investigation of the cytotoxicity of the Nan carrier materials is the most important task since the entire purpose of the drug is to kill the cancer cells. Figure 7 shows the cell viabilities of

HeLa

cells

incubated

with

different

amounts

of

DOX

and

DOX-

PLGA@[email protected] for 24 hrs as determined by MTT assay. All the cell viabilities decrease with the increase in the concentration, demonstrating the dose-dependent cytotoxicity of the DOX by itself and as part of the drug carrier, PLGA@[email protected] . With its small molecular structure, DOX can diffuse into the cells more rapidly, which induces a clear cytotoxicity

of

free

DOX

at

low

concentration.

The

cytotoxicity

of

DOX-

PLGA@[email protected] is due to the DOX released particles after its endocentric uptake by the cancer cells. At higher concentration (250 μgmL–1), the uptake of these nanocomposites

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by the HeLa cells by means of endocytosis, followed by the acid-enhanced release of the DOX inside the endosomes, improves the effect of the drug on the cancer cells and causes the similar

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cytotoxicity (3.31% ) to that of free DOX.

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Conclusion

In this study, we have synthesized and investigated the possible therapeutic efficiency of

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DOX-PLGA@[email protected] nanoparticles. The nanoparticles clearly showed a magnetic The releasing

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core coated with polymer shell and have a good magnetic responsiveness.

efficiencies of DOX by the nanocarriers were found to depend on the pH (pH-responsive). As

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seen for the case of physiological pH=7.4, the drug release rate is relatively slow. When the pH of the buffer solutions is 4.0, the release is much faster and more complete. In vitro biological

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activity shows that the DOX-loaded synthesized nanoparticles exhibit the dose-dependent

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cytotoxicity. At concentrations less than 125μgmL-1, the DOX-PLGA@[email protected]

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particles showed lower toxicity against HeLa cells in comparison to that when only DOX was used. At higher concentration of 250μgmL–1, the DOX- PLGA@[email protected] showed clearer anti-cancer activity to HeLa cells than that seen when using free DOX. On the basis of the above investigation, i.e. pH-responsive controlled release and biological activity give the synthesized nanoparticles potential application in effective drug delivery system. Acknowledgments The authors would like to acknowledge the financial support from Thailand Research Fund (TRF)-Office of the Higher Education Commission-Kasetsart University (TRG5780269, to WP), the Departments of Physics and Materials science, Faculty of Science, Kasetsart University (to WM,

NB,

(to WM).

WP

and

IMT),

and

the

National

Nanotechnology

Center,

Thailand.

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Figure 1. XRD patterns of patterns of as-prepared magnetic Mn1-xZnxFe2O4 nanoparticles.

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Figure 2. TEM images of Mn0.9Zn0.1Fe2O4 (a) and (b) PLGA@[email protected], respectively.

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Figure 3. Magnetization curves of the Mn1-xZnxFe2O4 nanoparticles.

Figure 4. Magnetization curves of the as-synthesized Mn0.9Zn0.1Fe2O4 and PLGA@[email protected]

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MNPs. Photograph of PLGA@[email protected] attracted by an external magnet (inset).

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Figure 5. FTIR spectra of PLGA@[email protected] (a) and DOX- PLGA@[email protected] (b).

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analysis to fit Peppas’s model [43].

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Figure 6. Drug release of DOX from PLGA@[email protected] at pH 7.4 and 4.0 and a regression

Figure 7. Cell viability of HeLa cells with different amounts of DOX and DOX- PLGA@[email protected]

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for 24 hrs determined by MTT assay.

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Graphical Abstract

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Synthesis of doxorubicin-PLGA loading on chitosan stabilized

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(Mn,Zn)Fe2O4 nanoparticles: Biological activity and pH-responsive

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drug release studies

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Research highlights

Synthesis of PLGA-coated chitosan stabilized (Mn,Zn) MCPs as an effective carriers.

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The synthesized nanocarriers were found that the releasing efficiencies of DOX depend on pH

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In vitro biological activity revealed that the DOX-loaded synthesized nanoparticles

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demonstrating the dose-dependent cytotoxicity.

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(pH-responsive).