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Temperature and pH sensitive multi-functional magnetic nanocomposite for the controlled delivery of 5-fluorouracil, an anticancer drug
Journal Pre-proof Temperature and pH sensitive multi-functional magnetic nanocomposite for the controlled delivery of 5-fluorouracil, an anticancer drug T.S. Anirudhan, J. Christa PII:
S1773-2247(19)30651-3
DOI:
https://doi.org/10.1016/j.jddst.2019.101476
Reference:
JDDST 101476
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 8 May 2019 Revised Date:
14 December 2019
Accepted Date: 22 December 2019
Please cite this article as: T.S. Anirudhan, J. Christa, Temperature and pH sensitive multi-functional magnetic nanocomposite for the controlled delivery of 5-fluorouracil, an anticancer drug, Journal of Drug Delivery Science and Technology (2020), doi: https://doi.org/10.1016/j.jddst.2019.101476. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Graphical Abstract
Diagrammatic representation of release of 5-FU from the 5-FU loaded Fe3O4-Dex-MA-gP(NVI/NVCL).
Temperature and pH Sensitive Multi-Functional Magnetic Nanocomposite for the Controlled Delivery of 5-Fluorouracil, an Anticancer Drug T.S. Anirudhan*, J. Christa Department of Chemistry, School of Physical and Mathematical Sciences, University of Kerala, Kariavattom, Trivandrum -695 581, India
*Corresponding author: Tel: 0471-2308682 Email Address: [email protected]
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Abstract With the purpose of avoiding leakage of drug during blood circulation and burst release of the drug at the tumor site, a functionalized magnetic Fe3O4 drug delivery system (DDS) with pH and temperature sensitivity was developed for the controlled delivery of 5-fluorouracil (5FU). Fe3O4 nanoparticles were coated with glycidyl methacrylate grafted dextran and then N-vinyl caprolactam and N-vinylimidazole monomers were used for further modification so as to introduce temperature and pH sensitivity respectively. The drug release profiles were assessed at different pH values which mimic blood circulation pH and tumor pH. The pH sensitivity of the carrier helps in modulating the drug release by decreasing the drug release percentage at the blood circulation pH and facilitating the drug release at acidic pH. Drug release profiles at different temperatures indicated the temperature sensitivity of the material. Analysis of drug release kinetic data at pH 5.0, with Korsmeyer-Peppas model pointed out both swelling and diffusion controlled drug release. In vitro cell viability studies of 5FU loaded DDS on MCF-7 breast cancer cells substantiated the cytotoxicity of the drug loaded carrier towards cancer cells. Keywords: Magnetic nanocomposite; Controlled Delivery; 5-Fluorouracil; N-vinylcaprolactam; N-vinylimidazole
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1. Introduction Cancer is one of the life threatening diseases that affect humanity worldwide. Chemotherapy is largely adopted for treating various types of cancers. But direct administration of chemotherapeutic drug leads to several drawbacks such as poor biodistribution, lack of selectivity, accumulation of drug at the unwanted site etc [1]. Different modalities have been reported in literature to overcome these shortcomings. Encapsulation of drug in stimuli responsive materials is one of such strategy which could enhance the therapeutic efficacy of the drug by delivering the drug to the tumor cells only. The stimuli responsive polymers are able to change their conformation, solubility, transition between hydrophilic and hydrophobic behavior as per the external stimuli such as pH and temperature [2]. Neoplastic cells are characterized by their acidic microenvironment and leaky vasculature. The tumor extracellular pH is ~6.5, pH of endosome and lysosome is further low, ~5.0-5.5 while normal cells having a pH ~7.4. Temperature sensitive polymers show either a lower critical solution temperature (LCST) or an upper critical solution temperature, which causes change in their properties like dissolution/precipitation, hydrophilic/ hydrophobic surface change etc [3, 4]. Hence pH and temperature sensitive polymers play an important role in anticancer drug delivery systems (DDS) [5, 6]. Due to rapid growth and multiplication, tumour tissues demand more blood supply. Therefore new blood vessels are formed which are typified by discontinous endothelium with large fenestrations in the size range 200-780 nm [7]. Particles less than 500 nm are usually accumulated in tumour tissue due to enhanced permeability and retention effect offered by the leaky vasculature [8].
Moreover, magnetic nanoparticles embedded in polymeric
nanosphere drug carrier can be easily controlled by applying an external magnetic field [9].
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Magnetic nanoparticles can be passively targeted to the tumour site via applying an external magnetic field. They can be coated with stimuli responsive polymers, which enhance their colloidal stability and blood circulation time. The easy synthesis, stability towards oxidation and biocompatibility make Fe3O4 nanoparticle an accepted material to introduce magnetism [10,11]. These properties make iron oxide based hybrid systems suitable for magnetic field guided localized cancer therapy and controlled anticancer drug delivery [12, 13]. However it is difficult to target tumors present at deep tissues using an applied magnetic field. In such cases, magnetic implants are another alternative which can be used to locate the DDS to the tumor site [14-16]. Dextran is a hydrophilic, biocompatible natural polymer produced by microorganism. Structurally dextran is a soluble branched homo polysaccharide of glucose with α -1,6-linked Dglucopyranose linear chain and branches begins as α-1,3-linkages. It bears several functionalizable hydroxyl groups in its skeleton [17]. Owing to its excellent biocompatiblility, biodegradability, easy functionalization and low cost, it is extensively used in biomedical applications [18, 19]. Also several iron oxide-dextran formulations are currently available in biomedical field [20]. Glycidyl methacrylate (GMA) is a biocompatible monomer used in developing functionalized polymeric DDS. The presence of polymerizable vinyl end, reactive epoxy and ester groups make it a potential candidate for the synthesis of functionalized polymers [21]. As reported by van Dijk-Wolthuis et al, the grafting of GMA onto dextran resulted in the formation of a trans esterified product with vinyl end bearing dextran chain (Dex-MA) [22]. In the present work, Dex-MA is utilized for functionalizing iron oxide nanoparticles. Poly(N-vinylcaprolactam) (PNVCL) is a temperature sensitive biocompatible polymer having hydrophilic cyclic amide unit and hydrophobic carbon-carbon back bone used in drug
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delivery applications. Moreover its hydrolysis products are not toxic. It exhibits a LCST around 32 ºC which can be altered by changing chain length. The incorporation of PNVCL into a polymer backbone induces temperature sensitive behavior to the network [23-25]. Nvinylimidazole (NVI) offers ease for functionalization by free radical polymerization and exhibit high thermal stability. Imidazole ring is present in many biomacromolecules [26]. N, N'methylenebisacrylamide (MBA) is a hydrophilic crosslinker broadly used in DDS [27, 28]. 5Fluorouracil (5FU) is a hydrophilic anticancer drug commonly used for the treatment of solid tumors of breast, colorectal, liver, neck and brain. The fluoropyrimidine drug 5FU is an antimetabolite drug and considered as a structural analogue of uracil [29]. It can be used for combination therapy along with most of the other chemotherapeutic agents safely which makes it a widely accepted chemotherapeutic agent. But one of the disadvantage of 5FU is its fast metabolism, which necessitates frequent administration of the drug to maintain desired serum concentration level. A suitable DDS can overcome these limitations effectively [30]. In the present study, a magnetic nanosphere with temperature and pH sensitivity was fabricated by simple free radical polymerization process. Dex-MA coating of iron oxide was used as a new functionalization strategy so that free radical polymerization can be carried out at the vinyl end of Dex-MA. Dex-MA coating also helps in increasing drug loading efficiency. The use of NVI and N-vinyl caprolactam (NVCL) monomers for the preparation of DDS (Fe3O4Dex-MA-g-P(NVI/NVCL) helps in introducing pH and temperature sensitivity. Equilibrium swelling studies were carried out as a function of time and pH. In vitro drug release kinetic studies were conducted at different pH and temperatures using a model anticancer drug, 5FU. In vitro cell viabiity studies were conducted to find out the biocompatibility of the material to L929
5
normal cells and antiproliferative activity of the drug loaded material to MCF7 breast cancer cells.
2. Materials and methods 2.1. Materials Dextran ( Mn = 15000, Mw = 32500, as determined by GPC analysis) was purchased from Himedia, India. Dimethyl sulfoxide (DMSO, <0.01 % water), glycidylmethacrylate (GMA, 95 % by GC) were purchased from Tokyo Chemical Industries LTD, Japan. N-vinyl-ε-caprolactam (NVCL) was obtained from Alfa Aesar, UK. 1-vinylimidazole (NVI) was purchased from Aldrich, USA. Iron(III) chloride hexahydrate (FeCl3.6H2O), Iron(II) sulfate heptahydrate (FeSO4.7H2O), ammonia solution (25.0 -28.0 %), potassiumpersulphate (K2S2O8), N,N’methylenebisacrylamide (MBA), and methanol were procured from Merck, India. Double distilled water was used throughout the study. 2.2. Synthesis of DDS 2.2.1. Synthesis of Dex-MA Dex-MA was prepared using the method as reported elsewhere [22]. Typically 1.0 g of dextran was dissolved in DMSO (10.0 mL) in a round bottom flask under nitrogen atmosphere. 200 mg of DMAP was then added. After complete dissolution of DMAP, GMA (428 µL) was introduced into the solution. The solution was stirred at room temperature for 4 days. Methanol was used to precipitate the product, Dex-MA. The white precipitate was collected by centrifugation and freeze-dried. 2.2.2. Synthesis of Fe3O4-Dex-MA
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About 2.0 g of modified dextran (Dex-MA) was dissolved in 10 mL distilled water. Fe2+and Fe3+ salts in the ratio 1:2 were weighed and added to the dextran solution. After the complete dissolution of the iron salts, ammonia solution was quickly dropped into the mixture with vigorous stirring until the pH of the solution reached 11.0 [31]. The solution was stirred further for 1 h at 60 ºC. The black suspension was cooled. The precipitate was collected using a magnet and dried. 2.2.3. Synthesis of Fe3O4-Dex-MA-g-P(NVI/NVCL) Fe3O4- Dex-MA (0.5 g) was taken in 50 mL water and dispersed well by sonication. About 0.8 g of K2S2O8 initator was taken in a stoppered bottle and heated to 60 ºC and cooled. Fe3O4-Dex-MA dispersion was added to the above solution and stirred at 1000 rpm. To this 0.282 g (3.0 mmol) NVI, solution of 0.417 g (3.0 mmol) of NVCL dissolved in DMSO and 0.45 g of MBA dissolved in water were added. The mixture was stirred for 4 h at 70 ºC to complete the polymerization reaction. The brown precipitate obtained was collected and washed with hot water thrice to remove unreacted monomers. Then it was dried in an air oven at 50 ºC. 2.3. Instruments for Characterization The FTIR spectra of iron oxide containing samples were recorded with Schimadzu IR Prestige 21 FTIR spectrometer in the range 400 to 4000cm-1 by KBr pellet technique . XRD analysis was performed using Bruker AXS D8 advance X-ray diffractometer using Cu Kα radiation at a wavelength of 1.5406 Å. Morphology of the samples were investigated using Zeiss scanning electron microscope. FEI Tecnai F20 Transmission Electron Microscope (TEM) was used to find out the size and morphology of Fe3O4-Dex-MA-g-P(NVI/NVCL). UV visible spectroscopic analyses were done using Jasco UV- visible spectrophotometer (model 530, India). Concentration of 5FU was measured at a λmax of 266 nm. Room temperature magnetic hysteresis
7
curve of magnetic nanoparticles were recorded using Vibrating Sample Magnetometer [Quantum Design Physical Property Measurement System (PPMS, USA)]. Systronic Microprocessor pH meter (Model µ-362, India) was used to get all the pH measurements.
2.4. Determination of optimum encapsulation pH In order to find out the optimum encapsulation pH, 50 mg of Fe3O4-Dex-MA-gP(NVI/NVCL) was added to 100 mL of
buffer solutions of different pH containing 5FU
concentrations of 250 and 300 mgL-1 respectively and shaken in a water bath for 24 h at room temperature. The 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) was collected using an external magnet and washed with deionized water to remove the loosely attached 5FU. The supernatant was collected and analysed with UV –visible spectrophotometer at 266 nm. Triplicate experiments were conducted for each concentration. The drug loading efficiency (DLE) and encapsulation efficiency (EE) were calculated using equations, (1) and (2) respectively. DLE = EE =
(
)
×100
(1)
× 100
(2)
2.5. Determination of equilibrium swelling percentage Swelling of polymer is an important parameter for drug delivery applications. The swelling behaviour of Fe3O4-Dex-MA-g-P(NVI/NVCL) was studied as functions of pH and temperature. Exactly, 50 mg of dry Fe3O4-Dex-MA-g-P(NVI/NVCL) was transferred to a previously weighed tea bag and placed in 100.0 mL aqueous medium of desired pH (1.0-10.0) at 37 ºC for 24 h in order to attain equilibrium. At regular intervals tea bag was wiped to remove surface water and its weight was taken as Ws [32]. The swelling percentage was given by the formula, 8
Swelling Percentage =
+, ++-
× 100
(3)
Where Ws is the weight of swollen sample at time, t and Wd the weight of dry sample. The experiments were conducted in triplicate and mean of the swelling percentage was calculated. 2.6. In vitro drug release studies To measure the release performance of 5FU from 5FU loaded Fe3O4-Dex-MA-gP(NVI/NVCL), 100 mg of the 5FU loaded DDS was added to 50 mL of phosphate and acetate buffer solutions (pH 7.4 and 5.0 respectively) and were shaken in a waterbath at 37 ºC. At specified time intervals, 1 mL of solution was removed. Fresh buffer solutions were used to replenish the withdrawn volume. The accumulated amount of drug released into the solution was measured using a UV-visible spectrophotometer at 266 nm (λmax of 5FU). The tests were run in triplicate and the mean percentage cumulative drug release was calculated. The 5FU release studies were also conducted at 27 ºC. 2.7. In vitro cell viability study MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide]
assay
was
conducted to find out the effect of Fe3O4-Dex-MA-g-P(NVI/NVCL) on normal cells and 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) on cancer cells. L929 (mouse fibroblast) normal cell line and MCF-7 (human breast cancer) cell lines were obtained from National Centre for Cell Science, Pune, India. The cells were maintained in Dulbecco’s Modified Eagles Medium (DMEM). The cells were cultured in 25 cm2 flask and cells were trypsinized and transferred to 96-well plate and were grown to confluency in DMEM and incubated for 24 h at 37 ºC in a CO2 incubator. The supernatant was then removed and the cells were treated with different concentrations of sample followed by incubation for 24 h. After incubation, the supernatant was 9
removed and cells were treated with MTT solution. After 4 h of incubation, MTT containing medium was replaced with DMSO so as to solubilize formazan crystals. Using a microplate reader, the absorbance was measured at 570 nm. The cell viability was calculated using the formula Cell viability % =
3
4 3
5 4
5
× 100
(4)
2.8. Statistical analysis Triplicate experiments were conducted for each of the drug encapsulation, equilibrium swelling, in vitro drug release and in vitro cell viability studies. Results were presented as mean ± standard deviation (SD). Analysis of variance (ANOVA) was employed to compare data. A pvalue < 0.05 was considered statistically significant in all cases. 3. Results and discussion 3.1. Preparation of the drug carrier In the present study, a pH and temperature sensitive magnetic nano drug carrier was prepared as depicted in Scheme 1. Dextran is a hydrophilic and biocompatible natural polymer. In the first step, polymerizable vinyl ends were introduced into dextran using GMA. GMA reacts with dextran by transesterification reaction. Methacryloyl group is directly attached to the 2and 3- hydroxyl group of the glucopyranose ring in 1:1 ratio to form Dex-MA [22]. Then DexMA coated Fe3O4 nano particles were prepared by coprecipitation of Fe2+ and Fe3+in 1:2 ratio in Dex-MA solution. Thus Fe3O4-Dex-MA was formed by in situ coating of iron oxide with DexMA. Then temperature and pH responsive polymer layer was introduced to Fe3O4-Dex-MA through free radical polymerization with NVCL and NVI at the vinyl end to form Fe3O4-DexMA-g-P(NVI/NVCL). During free radical polymerization, K2S2O8 and MBA were used as initiator and crosslinker respectively. 10
3.2. Characterization studies All the steps in synthesis were closely examined with FT-IR spectroscopy and the spectra obtained for different samples are presented in Fig. 1 and Fig. 2. In the FT-IR spectrum of dextran, a broad band around 3600-3000 cm-1 region was due to O-H stretching vibrations. The peaks at 2928 and 1420 cm-1 were attributed to C-H vibrational modes. The peak at 1003 cm-1 was due to C-O stretching vibrations of alkoxy bonds. The band at 1150 cm-1 is assigned to C– O–C bond vibrations [33]. In the IR spectrum of GMA, peaks at 1702 and 811cm
-1
indicated
C=O and C=C stretching present in methacrylate group GMA. Epoxy group in GMA was represented by the peaks at 848 and 906 cm-1 [34]. IR spectrum of Dex-MA showed absence of such peaks. This implies that methacrylate group of GMA is sufficiently incorporated in dextran while epoxy group is eliminated as glycidol. In the IR spectra of Fe3O4-Dex-MA, Fe3O4-DexMA-g-P(NVI/NVCL) and 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL), a band at 580 cm-1 was present which is the characteristic stretching frequency of Fe-O in Fe3O4 molecule [35].This confirmed the incorporation of Fe3O4 is in these materials. In the IR spectrum of Fe3O4-DexMA-g-P(NVI/NVCL), the peak at 1634 cm-1 showed the carbonyl stretching band in caprolactam ring. C-N stretching band was observed at around 1447 and 3264 cm-1 was assigned to NH stretching vibration. The bands at 2915 and 2843 cm-1 indicate the CH stretching vibration. The peak at 1500 cm-1 indicate the C=N stretching present in polymerized NVI [36, 37]. A broad band between the 3000 and 3500 cm–1, is attributed to NH stretching vibrations in the spectrum of 5FU. The peak at 1275 cm–1 was due to C-F stretching in the spectrum of 5FU [38]. This peak was observed at 1280 cm–1 in the spectrum of drug loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) .
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The XRD patterns of Dex-MA, Fe3O4-Dex-MA, Fe3O4-Dex-MA-g-P(NVI/NVCL) and 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) are shown in Fig.3. The XRD pattern of Dex-MA clearly indicated its amorphous nature. The XRD pattern of Fe3O4-Dex-MA has 6 characteristic peaks at 2θ = 30.1, 35.5, 43.1, 53.4, 56.9 and 62.6 º which can be indexed to (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1), and (4 4 0) planes of cubic inverse spinel structure of Fe3O4 [29]. The broadening of bands indicated amorphous coating of Dex-MA onto them. The XRD patterns of Fe3O4-Dex-MA,
Fe3O4-Dex-MA-g-P(NVI/NVCL)
and
5FU
P(NVI/NVCL) contain an intense peak between 2θ values 30
loaded _
Fe3O4-Dex-MA-g-
40º due to the presence of
crystalline Fe3O4. However the peak intensities of the diffraction peaks were weakened and width was broadened due to polymer coating [39]. The XRD pattern of 5FU loaded Fe3O4-DexMA-g-P(NVI/NVCL) contains a sharp peak at 2θ = 28º which is the characteristic peak of 5FU.This suggested the proper incorporation of drug to Fe3O4-Dex-MA-g-P(NVI/NVCL). The surface morphology of the samples was examined using SEM and TEM techniques. In the SEM image of Fe3O4-Dex-MA (Fig.4A), particle aggregation was observed which might be due to small size. The SEM image of Fe3O4-Dex-MA-g-P(NVI/NVCL) (Fig. 4B) shows a collection of spherical particles with particle size around 450 nm. After 5FU loading, the spherical morphology was retained, but surface was smoothened (Fig. 4C). Fig. 4D shows the TEM image of Fe3O4-Dex-MA-g-P(NVI/NVCL) having uniform spheres with average particle size around 450 nm and it can be considered as a magnetic nanosphere [9]. Thus surface morphology results confirm that the prepared DDS could passively accumulate in tumour tissue through the fenestrations of endothelium [7]. Room temperature magnetization curves of Fe3O4-Dex-MA and Fe3O4-Dex-MA-gP(NVI/NVCL) are shown in Fig. 5. Significant hysteresis was not observed in the room
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temperature magnetization curve of both samples which indicated their superparamagnetic nature. Surface coating of Fe3O4 with Dex-MA or further polymerization did not alter the superparamagnetic behaviour of the materials. But magnetization was lowered by the presence of non-magnetic layer in its surface. The magnetization values of Fe3O4-Dex-MA and Fe3O4-DexMA-g-P(NVI/NVCL) at an applied field of 90 kOe were found to be 24.2 emu/g and 10.4 emu/g respectively. The magnetization value of Fe3O4-Dex-MA-g-P(NVI/NVCL) was lower than Fe3O4-Dex-MA due to the increased amount of polymer incorporated onto the surface. This confirmed the effective polymer coating on the surface of magnetic nanoparticles. The higher magnetization values even after coating indicate that they can be manipulated by using an external magnetic field [40, 41]. 3.3. Encapsulation pH In order to find out the effect of pH on drug loading, drug loading studies were carried out at different pH values (Fig. 6.). It was found that alkaline pH favoured higher encapsulation due to hydrogen bonding interaction between drug and the carrier. At lower pH values, the repulsion between protonated imidazole groups (pKa 7.3) [42] of polymer chain and positively charged drug (pKa 8.2) [43] decreases its encapsulation efficiency. The encapsulation efficiency was found to be 93.5 % and drug loading efficiency was found to be 46.8 % respectively for a drug concentration of 250 mg/L. The higher encapsulation efficiency might be due to the complexing ability of caprolactam units in polymer chain, and hydrophilic nature of imidazole moieties present in polymer chains at alkaline pH. 3.4. Equilibrium swelling studies Swelling studies were conducted at different pH values ranging from 4 to 8 so as to find out the effect of pH on swelling. A plot of swelling percentage against pH is shown in Fig.7.
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Maximum swelling of 380.0 % was observed at acidic pH. The pKa of imidazole group in NVI is approximately 7.3, consequently it gets protonated at acidic pH. The protonation of imidazole nitrogen at acidic pH induces repulsion between polymer chains. This causes the relaxation of polymer chains and hence swelling of the polymer. But at alkaline pH only 120.0 % swelling was observed. At this pH, the imidazole moieties remain almost neutral. Swelling of Fe3O4-Dex-MA-g-P(NVI/NVCL) was studied at pH 5.0 and pH 7.4 as a function of time (Fig. 8). It was observed that swelling increased with time for both pH values. But the swelling percentage was higher (380.0 %) for pH 5.0 due to protonation of imidazole nitrogen and subsequent chain relaxation at this pH. The water permeability of PNVI also helps in swelling. The slight swelling at pH 7.4 might be due to the hydrophilicity of imidazole ring which helps in water permeation. 3.5. In vitro drug release studies The drug release behaviour of 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) was studied at pH 7.4 phosphate buffer and 5.0 acetate buffer solutions (Fig.9). It was observed that drug release percentage was higher at pH 5.0 than at pH 7.4. About 88.2 % of drug was released at acidic pH within 24 h while only 24.1% drug was released at pH 7.4. This substantiated the pH sensitivity of the prepared nano carrier. Also the drug release followed a sustained drug release pattern. At pH 5.0, the nitrogen of imidazole ring get protonated and there is a repulsion between the polymer chains. This leads to relaxation of polymer chains. The polymer chain containing caprolactam ring may shrink at 37 ºC and there may be a hydrophilic to hydrophobic transition which also triggers the drug release. Also at acidic pH, 5FU is positively charged. So there is a repulsion between protonated imidazole and 5FU. This synergistically affects the drug release. The lower drug release at pH 7.4 can be explained as follows. Imidazole groups remains neutral
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at this pH. So there is hydrogen bonding interaction between 5FU and imidazole and caprolactam ring of the polymer. So the drug is almost encapsulated by this hydrogen bonding interaction and only a small amount of drug is released from the matrix. In order to study the effect of temperature on drug release, drug release studies were conducted at two temperatures, at 27 and 37 ºC respectively and the results are shown in Fig 10. It was found that cumulative drug release was higher at high temperature. 88.2 % drug release was observed at 37 ºC while only 80.0 % release was observed at 27 ºC. This is due to the presence of thermoresponsive PNVCL blocks in the drug carrier. PNVCL has a LCST of 32 ºC. So above that temperature it becomes hydrophobic. This reduces the interaction between the drug and the carrier. Therefore the drug is released at a faster rate. Shrinking of PNVCL chain above its LCST may also facilitate drug release [44, 45]. 3.6. In vitro drug release kinetics To get an insight into the mechanism of drug release, drug release kinetics at pH 5.0 and 7.4 were analysed using Korsmeyer-Peppas kinetic model [46]. It is described by the equation 67 68
= kt
(5)
Where M is the amount of 5FU released at time t and M; is the amount of 5FU released at infinite time. Release constant and release exponent are denoted by k and n respectively. In the present study, the obtained drug release kinetic data were examined using this kinetic model with the help of origin pro 8 software by nonlinear regression analysis. Korsmeyer–Peppas kinetic model describes the mechanism of drug release based on the value of release exponent, n. The drug release follows fickian diffusion when n<0.43 and drug is released from polymer matrix due to diffusion. If the value of n is in between 0.43 and 0.85, a combined action of swelling and diffusion of the polymer results in drug release. This is termed as anomalous transport. The drug 15
release is non-fickian, if the value of n>0.85. In this case, the drug release is controlled by the swelling of polymer matrix [47].
Korsmeyer-Peppas fit of drug release data at pH 5.0 (Fig. 11) gave the value of n as 0.585 with a R2 value of 0.997. This reflects anomalous transport of the drug. The drug is released from the matrix as a result of both swelling of the polymer chain and diffusion of drug from the matrix. Due to the protonation of imidazole moiety in the polymer coating, it swells in acidic pH. The initial release was due to the swelling of the polymer. The presence of hydrophilic imidazole group enhances the water permeability. 5FU is a water soluble drug, it diffuses out from the matrix due to a chemical potential gradient. Korsmeyer-Peppas fit for the drug release kinetic data at pH 7.4 (Fig. 12) presented a R2 value of 0.971. The value of n obtained was 0.302 which indicates that drug is released as a result of diffusion of drug from the drug carrier. The initial burst release was due to the diffusion of drug from the surface in the immediate vicinity of the solid-liquid interface. The value of n also rules out the possibility of swelling controlled release at alkaline pH. From the drug release kinetic data, it was concluded that the prepared magnetic nano drug carrier could be used for the controlled and sustained release of 5FU at acidic pH. Also during intravenous administration, only slight amount of drug will be released during blood circulation. But in the acidic tumor environment the drug will be released in controlled way due to both swelling and diffusion. 3.7. In vitro cell viability studies The viability profiles of Fe3O4-Dex-MA-g-P(NVI/NVCL) and 5FU loaded Fe3O4-Dex-MAg-P(NVI/NVCL) were analysed using MTT assay. A DDS should be biocompatible while considering its application in real systems. To ensure this, cell viability of the prepared material was assessed on L929 normal cells (Fig. 13.). It was found that the prepared material cause less 16
harm to the normal cells. This pointed out the biocompatibility of the material. The viability of cells decreased only to a small extent when concentration increased from 0.5 to 10.0 µg/mL . About 98.5 % cells were viable at 0.5 µg/mL concentration of the carrier. At a carrier concentration of 10.0 µg/mL, 85.3 % cells were viable. This result indicates that the prepared DDS, Fe3O4-Dex-MA-g-P(NVI/NVCL) might be safe for administration.” In vitro cell viability study of 5FU loaded material on MCF-7 breast cancer cell line (Fig. 14) indicates that the 5FU loaded material shows higher toxicity to MCF-7 cells. Only 60.2 % cells were viable at a concentration of 25.0 µg/mL. From cell viability studies, it was concluded that the drug loaded magnetic DDS could be employed to selectively kill neoplastic cells. 4. Conclusions Herein we reported the synthesis of a pH/temperature dual sensitive functionalized iron oxide based drug delivery system for the delivery of a model anticancer drug, 5FU . VSM analysis revealed the superparamagnetic nature and the applicability of material in the magnetic field guided therapy for localized cancer treatment. The developed DDS was able to encapsulate a large amount of drug with an EE of 93.5 %. Temperature and pH sensitivity of the carrier were well demonstrated by conducting in vitro drug release studies. The drug was retained in the DDS at alkaline pH and controlled release was achieved at acidic tumor pH. The biocompatibility of the material was evident from in vitro cell viability studies. From the In vitro cell viability study of 5FU loaded material on MCF-7 breast cancer cell line, 60.2 % cells were viable at a concentration of 25.0 µg/mL. The cytotoxicity towards cancer cells can further be improved by attaching suitable targeting ligands to the drug carrier. All these results imply that the prepared DDS could be used for the magnetic field guided, pH and temperature controlled release of 5FU to neoplastic cells.
17
Conflict of interest The authors declare no conflicts of interest.
Acknowledgements The authors are grateful to the Professor and Head, Department of Chemistry, School of Physical and Mathematical Sciences, University of Kerala, Trivandrum, India for providing the laboratory facilities. One of the author, J. Christa is grateful to University Grants Commission, New Delhi, India for providing financial assistance in the form of research fellowship. References [1]
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List of Figures Scheme 1: Schematic representation of the synthesis of Fe3O4-Dex-MA-g-P(NVI/NVCL). Fig. 1. FTIR spectra of Dextran (A), GMA (B), Dex-MA (c), and 5FU (D). Fig. 2. FTIR spectra of Fe3O4-Dex-MA (A), Fe3O4-Dex-MA-g-P(NVI/NVCL) (B) and 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) (C). Fig. 3. XRD patterns of Dex-MA (A), Fe3O4-Dex-MA (B), Fe3O4-Dex-MA-g-P(NVI/NVCL) (C) and 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) (D). Fig. 4. SEM images of Fe3O4-Dex-MA (A) and Fe3O4-Dex-MA-g-P(NVI/NVCL) (B), 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) and TEM image of Fe3O4-Dex-MA-gP(NVI/NVCL) (D). Fig. 5. Room temperature hysteresis curves of Fe3O4-Dex-MA and Fe3O4-Dex-MA-gP(NVI/NVCL). Fig. 6. Effect of pH on encapsulation efficiency of 5FU by Fe3O4-Dex-MA-g-P(NVI/NVCL). Data are presented as mean ± SD (n=3). Fig. 7. Swelling (%) of Fe3O4-Dex-MA-g-P(NVI/NVCL) as a function of pH. Data are presented as mean ± SD (n=3). Fig. 8. Swelling (%) of Fe3O4-Dex-MA-g-P(NVI/NVCL) as a function of time at pH 5.0 and 7.4. Data are presented as mean ± SD (n=3). Fig. 9. pH responsive 5FU release profile of 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) at 37 o
C at pH of 5.0 and 7.4. Data are presented as mean ± SD (n=3).
Fig. 10. In vitro 5FU release of profile of 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) at pH 5.0, temperature 27 and 37 ºC. Data are presented as mean ± SD (n=3).
24
Fig.11. Korsmeyer-Peppas fit for 5FU release from 5FU loaded Fe3O4-Dex-MA-gP(NVI/NVCL) at pH 5.0. Fig.12. Korsmeyer-Peppas fit for 5FU release from 5FU loaded Fe3O4-Dex-MA-gP(NVI/NVCL) at pH 5.0. Fig.13. In vitro cell viability studies of Fe3O4-Dex-MA-g-P(NVI/NVCL) on L929 cells. Data are presented as mean ± SD (n=3). Fig.14. In vitro cell viability studies of 5 FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) on MCF7 breast cancer cells. Data are presented as mean ± SD (n=3).
25
Scheme 1: Schematic representation of the synthesis of Fe3O4-Dex-MA-g-P(NVI/NVCL).
1
D
Transmittance (%)
C
B
A
4000
3500
3000
2500
2000
1500
1000
-1 Wavenumber (cm )
Fig.1. FTIR spectra of Dextran (A), GMA (B), Dex-MA (C), and 5FU (D).
Transmittance (%)
C
B
A
4000 3500 3000 2500 2000 1500 1000
500
-1
Wavenumber (cm )
Fig. 2. FTIR spectra of Fe3O4-Dex-MA (A), Fe3O4-Dex-MA-g-P(NVI/NVCL) (B) and 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) (C). 2
D
Intensity (a.u.)
C
B
A
10
20
30
40
50
60
70
80
2θ (Degree)
Fig. 3. XRD patterns of Dex-MA (A), Fe3O4-Dex-MA (B), Fe3O4-Dex-MA-g-P(NVI/NVCL) (C) and 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) (D).
Fig. 4. SEM images of Fe3O4-Dex-MA (A), Fe3O4-Dex-MA-g-P(NVI/NVCL) (B), 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) (C) and TEM image of Fe3O4-Dex-MA-g-P(NVI/NVCL) (D). 3
B A
Magnetization (emu/g)
20
0
-20
-100000
0
100000
Applied magnetic field (Oe)
Fig. 5. Room temperature hysteresis curves of Fe3O4-Dex-MA (A) and Fe3O4-Dex-MA-gP(NVI/NVCL) (B).
100
250 mg 5FU/L 80
Encapsulation Efficiency (%)
300 mg 5FU/L
60
40
20
0 3
4
5
6
7
8
9
10
pH
Fig. 6. Effect of pH on encapsulation efficiency of 5FU by Fe3O4-Dex-MA-g-P(NVI/NVCL). Data are presented as mean ± SD (n=3).
4
Fe3O4-Dex-MA-g-P(NVI/NVCL) 400
Swelling (%)
300
200
100
4
6
8
pH
Fig. 7. Swelling (%) of Fe3O4-Dex-MA-g-P(NVI/NVCL) as a function of pH. Data are presented as mean ± SD (n=3).
500
pH 5.0 pH 7.4
Swelling (%)
400
300
200
100
0 0
500
1000
1500
Time (Minutes)
Fig. 8. Swelling (%) of Fe3O4-Dex-MA-g-P(NVI/NVCL) as a function of time at pH 5.0 and 7.4. Data are presented as mean ± SD (n=3). 5
100
pH 5.0 pH 7.4
Cumulative drug release(%)
80
60
40
20
0 0
500
1000
1500
Time (Minutes)
Fig.9. pH responsive 5FU release profile of 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) at 37 ºC at pH of 5.0 and 7.4. Data are presented as mean ± SD (n=3).
6
100 ο
37 C o 27 C
Cumulative drug release (%)
80
60
40
20
0 0
500
1000
1500
Time (Minutes)
Fig. 10. In vitro 5FU release of profile of 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) at pH 5.0, temperatures 27 and 37 ºC. Data are presented as mean ± SD (n=3).
1.0
Drug release data at pH 5.0 Korsmeyer Peppas equation 0.8
Mt/M∞
0.6
0.4
0.2
0.0 0
500
1000
1500
Time (minutes)
Fig.11. Korsmeyer-Peppas fit for 5FU release from 5FU loaded Fe3O4-Dex-MA-gP(NVI/NVCL) at pH 5.0.
7
Drug release data pH7.4 Korsmeyer-Peppas equation 0.3
Mt/M∞
0.2
0.1
0.0 0
400
800
1200
Time (Minutes)
Fig.12. Korsmeyer-Peppas fit for 5FU release from 5FU loaded Fe3O4-Dex-MA-gP(NVI/NVCL) at pH 7.4.
120
L929
Cell Viability(%)
100
80
60
40
20
0
Control
0.5
1.5
3.0
6.25
Concentration (µg/ mL)
8
10.0
Fig.13. In vitro cell viability studies of Fe3O4-Dex-MA-g-P(NVI/NVCL) on L929 cells. Data are presented as mean ± SD (n=3).
100
MCF7
Cell viability (%)
80
60
40
20
0
control
1.5
3.0
6.25
12.5
25.0
Concentration (µg/ mL)
Fig.14. In vitro cell viability studies of 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) on MCF7 breast cancer cells. Data are presented as mean ± SD (n=3).
9
Conflict of interest The authors declare no conflicts of interest.
Author statement The authors, T. S. Anirudhan and J. Christa contributed equally to this work.
S1773-2247(19)30651-3
DOI:
https://doi.org/10.1016/j.jddst.2019.101476
Reference:
JDDST 101476
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 8 May 2019 Revised Date:
14 December 2019
Accepted Date: 22 December 2019
Please cite this article as: T.S. Anirudhan, J. Christa, Temperature and pH sensitive multi-functional magnetic nanocomposite for the controlled delivery of 5-fluorouracil, an anticancer drug, Journal of Drug Delivery Science and Technology (2020), doi: https://doi.org/10.1016/j.jddst.2019.101476. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Graphical Abstract
Diagrammatic representation of release of 5-FU from the 5-FU loaded Fe3O4-Dex-MA-gP(NVI/NVCL).
Temperature and pH Sensitive Multi-Functional Magnetic Nanocomposite for the Controlled Delivery of 5-Fluorouracil, an Anticancer Drug T.S. Anirudhan*, J. Christa Department of Chemistry, School of Physical and Mathematical Sciences, University of Kerala, Kariavattom, Trivandrum -695 581, India
*Corresponding author: Tel: 0471-2308682 Email Address: [email protected]
1
Abstract With the purpose of avoiding leakage of drug during blood circulation and burst release of the drug at the tumor site, a functionalized magnetic Fe3O4 drug delivery system (DDS) with pH and temperature sensitivity was developed for the controlled delivery of 5-fluorouracil (5FU). Fe3O4 nanoparticles were coated with glycidyl methacrylate grafted dextran and then N-vinyl caprolactam and N-vinylimidazole monomers were used for further modification so as to introduce temperature and pH sensitivity respectively. The drug release profiles were assessed at different pH values which mimic blood circulation pH and tumor pH. The pH sensitivity of the carrier helps in modulating the drug release by decreasing the drug release percentage at the blood circulation pH and facilitating the drug release at acidic pH. Drug release profiles at different temperatures indicated the temperature sensitivity of the material. Analysis of drug release kinetic data at pH 5.0, with Korsmeyer-Peppas model pointed out both swelling and diffusion controlled drug release. In vitro cell viability studies of 5FU loaded DDS on MCF-7 breast cancer cells substantiated the cytotoxicity of the drug loaded carrier towards cancer cells. Keywords: Magnetic nanocomposite; Controlled Delivery; 5-Fluorouracil; N-vinylcaprolactam; N-vinylimidazole
2
1. Introduction Cancer is one of the life threatening diseases that affect humanity worldwide. Chemotherapy is largely adopted for treating various types of cancers. But direct administration of chemotherapeutic drug leads to several drawbacks such as poor biodistribution, lack of selectivity, accumulation of drug at the unwanted site etc [1]. Different modalities have been reported in literature to overcome these shortcomings. Encapsulation of drug in stimuli responsive materials is one of such strategy which could enhance the therapeutic efficacy of the drug by delivering the drug to the tumor cells only. The stimuli responsive polymers are able to change their conformation, solubility, transition between hydrophilic and hydrophobic behavior as per the external stimuli such as pH and temperature [2]. Neoplastic cells are characterized by their acidic microenvironment and leaky vasculature. The tumor extracellular pH is ~6.5, pH of endosome and lysosome is further low, ~5.0-5.5 while normal cells having a pH ~7.4. Temperature sensitive polymers show either a lower critical solution temperature (LCST) or an upper critical solution temperature, which causes change in their properties like dissolution/precipitation, hydrophilic/ hydrophobic surface change etc [3, 4]. Hence pH and temperature sensitive polymers play an important role in anticancer drug delivery systems (DDS) [5, 6]. Due to rapid growth and multiplication, tumour tissues demand more blood supply. Therefore new blood vessels are formed which are typified by discontinous endothelium with large fenestrations in the size range 200-780 nm [7]. Particles less than 500 nm are usually accumulated in tumour tissue due to enhanced permeability and retention effect offered by the leaky vasculature [8].
Moreover, magnetic nanoparticles embedded in polymeric
nanosphere drug carrier can be easily controlled by applying an external magnetic field [9].
3
Magnetic nanoparticles can be passively targeted to the tumour site via applying an external magnetic field. They can be coated with stimuli responsive polymers, which enhance their colloidal stability and blood circulation time. The easy synthesis, stability towards oxidation and biocompatibility make Fe3O4 nanoparticle an accepted material to introduce magnetism [10,11]. These properties make iron oxide based hybrid systems suitable for magnetic field guided localized cancer therapy and controlled anticancer drug delivery [12, 13]. However it is difficult to target tumors present at deep tissues using an applied magnetic field. In such cases, magnetic implants are another alternative which can be used to locate the DDS to the tumor site [14-16]. Dextran is a hydrophilic, biocompatible natural polymer produced by microorganism. Structurally dextran is a soluble branched homo polysaccharide of glucose with α -1,6-linked Dglucopyranose linear chain and branches begins as α-1,3-linkages. It bears several functionalizable hydroxyl groups in its skeleton [17]. Owing to its excellent biocompatiblility, biodegradability, easy functionalization and low cost, it is extensively used in biomedical applications [18, 19]. Also several iron oxide-dextran formulations are currently available in biomedical field [20]. Glycidyl methacrylate (GMA) is a biocompatible monomer used in developing functionalized polymeric DDS. The presence of polymerizable vinyl end, reactive epoxy and ester groups make it a potential candidate for the synthesis of functionalized polymers [21]. As reported by van Dijk-Wolthuis et al, the grafting of GMA onto dextran resulted in the formation of a trans esterified product with vinyl end bearing dextran chain (Dex-MA) [22]. In the present work, Dex-MA is utilized for functionalizing iron oxide nanoparticles. Poly(N-vinylcaprolactam) (PNVCL) is a temperature sensitive biocompatible polymer having hydrophilic cyclic amide unit and hydrophobic carbon-carbon back bone used in drug
4
delivery applications. Moreover its hydrolysis products are not toxic. It exhibits a LCST around 32 ºC which can be altered by changing chain length. The incorporation of PNVCL into a polymer backbone induces temperature sensitive behavior to the network [23-25]. Nvinylimidazole (NVI) offers ease for functionalization by free radical polymerization and exhibit high thermal stability. Imidazole ring is present in many biomacromolecules [26]. N, N'methylenebisacrylamide (MBA) is a hydrophilic crosslinker broadly used in DDS [27, 28]. 5Fluorouracil (5FU) is a hydrophilic anticancer drug commonly used for the treatment of solid tumors of breast, colorectal, liver, neck and brain. The fluoropyrimidine drug 5FU is an antimetabolite drug and considered as a structural analogue of uracil [29]. It can be used for combination therapy along with most of the other chemotherapeutic agents safely which makes it a widely accepted chemotherapeutic agent. But one of the disadvantage of 5FU is its fast metabolism, which necessitates frequent administration of the drug to maintain desired serum concentration level. A suitable DDS can overcome these limitations effectively [30]. In the present study, a magnetic nanosphere with temperature and pH sensitivity was fabricated by simple free radical polymerization process. Dex-MA coating of iron oxide was used as a new functionalization strategy so that free radical polymerization can be carried out at the vinyl end of Dex-MA. Dex-MA coating also helps in increasing drug loading efficiency. The use of NVI and N-vinyl caprolactam (NVCL) monomers for the preparation of DDS (Fe3O4Dex-MA-g-P(NVI/NVCL) helps in introducing pH and temperature sensitivity. Equilibrium swelling studies were carried out as a function of time and pH. In vitro drug release kinetic studies were conducted at different pH and temperatures using a model anticancer drug, 5FU. In vitro cell viabiity studies were conducted to find out the biocompatibility of the material to L929
5
normal cells and antiproliferative activity of the drug loaded material to MCF7 breast cancer cells.
2. Materials and methods 2.1. Materials Dextran ( Mn = 15000, Mw = 32500, as determined by GPC analysis) was purchased from Himedia, India. Dimethyl sulfoxide (DMSO, <0.01 % water), glycidylmethacrylate (GMA, 95 % by GC) were purchased from Tokyo Chemical Industries LTD, Japan. N-vinyl-ε-caprolactam (NVCL) was obtained from Alfa Aesar, UK. 1-vinylimidazole (NVI) was purchased from Aldrich, USA. Iron(III) chloride hexahydrate (FeCl3.6H2O), Iron(II) sulfate heptahydrate (FeSO4.7H2O), ammonia solution (25.0 -28.0 %), potassiumpersulphate (K2S2O8), N,N’methylenebisacrylamide (MBA), and methanol were procured from Merck, India. Double distilled water was used throughout the study. 2.2. Synthesis of DDS 2.2.1. Synthesis of Dex-MA Dex-MA was prepared using the method as reported elsewhere [22]. Typically 1.0 g of dextran was dissolved in DMSO (10.0 mL) in a round bottom flask under nitrogen atmosphere. 200 mg of DMAP was then added. After complete dissolution of DMAP, GMA (428 µL) was introduced into the solution. The solution was stirred at room temperature for 4 days. Methanol was used to precipitate the product, Dex-MA. The white precipitate was collected by centrifugation and freeze-dried. 2.2.2. Synthesis of Fe3O4-Dex-MA
6
About 2.0 g of modified dextran (Dex-MA) was dissolved in 10 mL distilled water. Fe2+and Fe3+ salts in the ratio 1:2 were weighed and added to the dextran solution. After the complete dissolution of the iron salts, ammonia solution was quickly dropped into the mixture with vigorous stirring until the pH of the solution reached 11.0 [31]. The solution was stirred further for 1 h at 60 ºC. The black suspension was cooled. The precipitate was collected using a magnet and dried. 2.2.3. Synthesis of Fe3O4-Dex-MA-g-P(NVI/NVCL) Fe3O4- Dex-MA (0.5 g) was taken in 50 mL water and dispersed well by sonication. About 0.8 g of K2S2O8 initator was taken in a stoppered bottle and heated to 60 ºC and cooled. Fe3O4-Dex-MA dispersion was added to the above solution and stirred at 1000 rpm. To this 0.282 g (3.0 mmol) NVI, solution of 0.417 g (3.0 mmol) of NVCL dissolved in DMSO and 0.45 g of MBA dissolved in water were added. The mixture was stirred for 4 h at 70 ºC to complete the polymerization reaction. The brown precipitate obtained was collected and washed with hot water thrice to remove unreacted monomers. Then it was dried in an air oven at 50 ºC. 2.3. Instruments for Characterization The FTIR spectra of iron oxide containing samples were recorded with Schimadzu IR Prestige 21 FTIR spectrometer in the range 400 to 4000cm-1 by KBr pellet technique . XRD analysis was performed using Bruker AXS D8 advance X-ray diffractometer using Cu Kα radiation at a wavelength of 1.5406 Å. Morphology of the samples were investigated using Zeiss scanning electron microscope. FEI Tecnai F20 Transmission Electron Microscope (TEM) was used to find out the size and morphology of Fe3O4-Dex-MA-g-P(NVI/NVCL). UV visible spectroscopic analyses were done using Jasco UV- visible spectrophotometer (model 530, India). Concentration of 5FU was measured at a λmax of 266 nm. Room temperature magnetic hysteresis
7
curve of magnetic nanoparticles were recorded using Vibrating Sample Magnetometer [Quantum Design Physical Property Measurement System (PPMS, USA)]. Systronic Microprocessor pH meter (Model µ-362, India) was used to get all the pH measurements.
2.4. Determination of optimum encapsulation pH In order to find out the optimum encapsulation pH, 50 mg of Fe3O4-Dex-MA-gP(NVI/NVCL) was added to 100 mL of
buffer solutions of different pH containing 5FU
concentrations of 250 and 300 mgL-1 respectively and shaken in a water bath for 24 h at room temperature. The 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) was collected using an external magnet and washed with deionized water to remove the loosely attached 5FU. The supernatant was collected and analysed with UV –visible spectrophotometer at 266 nm. Triplicate experiments were conducted for each concentration. The drug loading efficiency (DLE) and encapsulation efficiency (EE) were calculated using equations, (1) and (2) respectively. DLE = EE =
(
)
×100
(1)
× 100
(2)
2.5. Determination of equilibrium swelling percentage Swelling of polymer is an important parameter for drug delivery applications. The swelling behaviour of Fe3O4-Dex-MA-g-P(NVI/NVCL) was studied as functions of pH and temperature. Exactly, 50 mg of dry Fe3O4-Dex-MA-g-P(NVI/NVCL) was transferred to a previously weighed tea bag and placed in 100.0 mL aqueous medium of desired pH (1.0-10.0) at 37 ºC for 24 h in order to attain equilibrium. At regular intervals tea bag was wiped to remove surface water and its weight was taken as Ws [32]. The swelling percentage was given by the formula, 8
Swelling Percentage =
+, ++-
× 100
(3)
Where Ws is the weight of swollen sample at time, t and Wd the weight of dry sample. The experiments were conducted in triplicate and mean of the swelling percentage was calculated. 2.6. In vitro drug release studies To measure the release performance of 5FU from 5FU loaded Fe3O4-Dex-MA-gP(NVI/NVCL), 100 mg of the 5FU loaded DDS was added to 50 mL of phosphate and acetate buffer solutions (pH 7.4 and 5.0 respectively) and were shaken in a waterbath at 37 ºC. At specified time intervals, 1 mL of solution was removed. Fresh buffer solutions were used to replenish the withdrawn volume. The accumulated amount of drug released into the solution was measured using a UV-visible spectrophotometer at 266 nm (λmax of 5FU). The tests were run in triplicate and the mean percentage cumulative drug release was calculated. The 5FU release studies were also conducted at 27 ºC. 2.7. In vitro cell viability study MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide]
assay
was
conducted to find out the effect of Fe3O4-Dex-MA-g-P(NVI/NVCL) on normal cells and 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) on cancer cells. L929 (mouse fibroblast) normal cell line and MCF-7 (human breast cancer) cell lines were obtained from National Centre for Cell Science, Pune, India. The cells were maintained in Dulbecco’s Modified Eagles Medium (DMEM). The cells were cultured in 25 cm2 flask and cells were trypsinized and transferred to 96-well plate and were grown to confluency in DMEM and incubated for 24 h at 37 ºC in a CO2 incubator. The supernatant was then removed and the cells were treated with different concentrations of sample followed by incubation for 24 h. After incubation, the supernatant was 9
removed and cells were treated with MTT solution. After 4 h of incubation, MTT containing medium was replaced with DMSO so as to solubilize formazan crystals. Using a microplate reader, the absorbance was measured at 570 nm. The cell viability was calculated using the formula Cell viability % =
3
4 3
5 4
5
× 100
(4)
2.8. Statistical analysis Triplicate experiments were conducted for each of the drug encapsulation, equilibrium swelling, in vitro drug release and in vitro cell viability studies. Results were presented as mean ± standard deviation (SD). Analysis of variance (ANOVA) was employed to compare data. A pvalue < 0.05 was considered statistically significant in all cases. 3. Results and discussion 3.1. Preparation of the drug carrier In the present study, a pH and temperature sensitive magnetic nano drug carrier was prepared as depicted in Scheme 1. Dextran is a hydrophilic and biocompatible natural polymer. In the first step, polymerizable vinyl ends were introduced into dextran using GMA. GMA reacts with dextran by transesterification reaction. Methacryloyl group is directly attached to the 2and 3- hydroxyl group of the glucopyranose ring in 1:1 ratio to form Dex-MA [22]. Then DexMA coated Fe3O4 nano particles were prepared by coprecipitation of Fe2+ and Fe3+in 1:2 ratio in Dex-MA solution. Thus Fe3O4-Dex-MA was formed by in situ coating of iron oxide with DexMA. Then temperature and pH responsive polymer layer was introduced to Fe3O4-Dex-MA through free radical polymerization with NVCL and NVI at the vinyl end to form Fe3O4-DexMA-g-P(NVI/NVCL). During free radical polymerization, K2S2O8 and MBA were used as initiator and crosslinker respectively. 10
3.2. Characterization studies All the steps in synthesis were closely examined with FT-IR spectroscopy and the spectra obtained for different samples are presented in Fig. 1 and Fig. 2. In the FT-IR spectrum of dextran, a broad band around 3600-3000 cm-1 region was due to O-H stretching vibrations. The peaks at 2928 and 1420 cm-1 were attributed to C-H vibrational modes. The peak at 1003 cm-1 was due to C-O stretching vibrations of alkoxy bonds. The band at 1150 cm-1 is assigned to C– O–C bond vibrations [33]. In the IR spectrum of GMA, peaks at 1702 and 811cm
-1
indicated
C=O and C=C stretching present in methacrylate group GMA. Epoxy group in GMA was represented by the peaks at 848 and 906 cm-1 [34]. IR spectrum of Dex-MA showed absence of such peaks. This implies that methacrylate group of GMA is sufficiently incorporated in dextran while epoxy group is eliminated as glycidol. In the IR spectra of Fe3O4-Dex-MA, Fe3O4-DexMA-g-P(NVI/NVCL) and 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL), a band at 580 cm-1 was present which is the characteristic stretching frequency of Fe-O in Fe3O4 molecule [35].This confirmed the incorporation of Fe3O4 is in these materials. In the IR spectrum of Fe3O4-DexMA-g-P(NVI/NVCL), the peak at 1634 cm-1 showed the carbonyl stretching band in caprolactam ring. C-N stretching band was observed at around 1447 and 3264 cm-1 was assigned to NH stretching vibration. The bands at 2915 and 2843 cm-1 indicate the CH stretching vibration. The peak at 1500 cm-1 indicate the C=N stretching present in polymerized NVI [36, 37]. A broad band between the 3000 and 3500 cm–1, is attributed to NH stretching vibrations in the spectrum of 5FU. The peak at 1275 cm–1 was due to C-F stretching in the spectrum of 5FU [38]. This peak was observed at 1280 cm–1 in the spectrum of drug loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) .
11
The XRD patterns of Dex-MA, Fe3O4-Dex-MA, Fe3O4-Dex-MA-g-P(NVI/NVCL) and 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) are shown in Fig.3. The XRD pattern of Dex-MA clearly indicated its amorphous nature. The XRD pattern of Fe3O4-Dex-MA has 6 characteristic peaks at 2θ = 30.1, 35.5, 43.1, 53.4, 56.9 and 62.6 º which can be indexed to (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1), and (4 4 0) planes of cubic inverse spinel structure of Fe3O4 [29]. The broadening of bands indicated amorphous coating of Dex-MA onto them. The XRD patterns of Fe3O4-Dex-MA,
Fe3O4-Dex-MA-g-P(NVI/NVCL)
and
5FU
P(NVI/NVCL) contain an intense peak between 2θ values 30
loaded _
Fe3O4-Dex-MA-g-
40º due to the presence of
crystalline Fe3O4. However the peak intensities of the diffraction peaks were weakened and width was broadened due to polymer coating [39]. The XRD pattern of 5FU loaded Fe3O4-DexMA-g-P(NVI/NVCL) contains a sharp peak at 2θ = 28º which is the characteristic peak of 5FU.This suggested the proper incorporation of drug to Fe3O4-Dex-MA-g-P(NVI/NVCL). The surface morphology of the samples was examined using SEM and TEM techniques. In the SEM image of Fe3O4-Dex-MA (Fig.4A), particle aggregation was observed which might be due to small size. The SEM image of Fe3O4-Dex-MA-g-P(NVI/NVCL) (Fig. 4B) shows a collection of spherical particles with particle size around 450 nm. After 5FU loading, the spherical morphology was retained, but surface was smoothened (Fig. 4C). Fig. 4D shows the TEM image of Fe3O4-Dex-MA-g-P(NVI/NVCL) having uniform spheres with average particle size around 450 nm and it can be considered as a magnetic nanosphere [9]. Thus surface morphology results confirm that the prepared DDS could passively accumulate in tumour tissue through the fenestrations of endothelium [7]. Room temperature magnetization curves of Fe3O4-Dex-MA and Fe3O4-Dex-MA-gP(NVI/NVCL) are shown in Fig. 5. Significant hysteresis was not observed in the room
12
temperature magnetization curve of both samples which indicated their superparamagnetic nature. Surface coating of Fe3O4 with Dex-MA or further polymerization did not alter the superparamagnetic behaviour of the materials. But magnetization was lowered by the presence of non-magnetic layer in its surface. The magnetization values of Fe3O4-Dex-MA and Fe3O4-DexMA-g-P(NVI/NVCL) at an applied field of 90 kOe were found to be 24.2 emu/g and 10.4 emu/g respectively. The magnetization value of Fe3O4-Dex-MA-g-P(NVI/NVCL) was lower than Fe3O4-Dex-MA due to the increased amount of polymer incorporated onto the surface. This confirmed the effective polymer coating on the surface of magnetic nanoparticles. The higher magnetization values even after coating indicate that they can be manipulated by using an external magnetic field [40, 41]. 3.3. Encapsulation pH In order to find out the effect of pH on drug loading, drug loading studies were carried out at different pH values (Fig. 6.). It was found that alkaline pH favoured higher encapsulation due to hydrogen bonding interaction between drug and the carrier. At lower pH values, the repulsion between protonated imidazole groups (pKa 7.3) [42] of polymer chain and positively charged drug (pKa 8.2) [43] decreases its encapsulation efficiency. The encapsulation efficiency was found to be 93.5 % and drug loading efficiency was found to be 46.8 % respectively for a drug concentration of 250 mg/L. The higher encapsulation efficiency might be due to the complexing ability of caprolactam units in polymer chain, and hydrophilic nature of imidazole moieties present in polymer chains at alkaline pH. 3.4. Equilibrium swelling studies Swelling studies were conducted at different pH values ranging from 4 to 8 so as to find out the effect of pH on swelling. A plot of swelling percentage against pH is shown in Fig.7.
13
Maximum swelling of 380.0 % was observed at acidic pH. The pKa of imidazole group in NVI is approximately 7.3, consequently it gets protonated at acidic pH. The protonation of imidazole nitrogen at acidic pH induces repulsion between polymer chains. This causes the relaxation of polymer chains and hence swelling of the polymer. But at alkaline pH only 120.0 % swelling was observed. At this pH, the imidazole moieties remain almost neutral. Swelling of Fe3O4-Dex-MA-g-P(NVI/NVCL) was studied at pH 5.0 and pH 7.4 as a function of time (Fig. 8). It was observed that swelling increased with time for both pH values. But the swelling percentage was higher (380.0 %) for pH 5.0 due to protonation of imidazole nitrogen and subsequent chain relaxation at this pH. The water permeability of PNVI also helps in swelling. The slight swelling at pH 7.4 might be due to the hydrophilicity of imidazole ring which helps in water permeation. 3.5. In vitro drug release studies The drug release behaviour of 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) was studied at pH 7.4 phosphate buffer and 5.0 acetate buffer solutions (Fig.9). It was observed that drug release percentage was higher at pH 5.0 than at pH 7.4. About 88.2 % of drug was released at acidic pH within 24 h while only 24.1% drug was released at pH 7.4. This substantiated the pH sensitivity of the prepared nano carrier. Also the drug release followed a sustained drug release pattern. At pH 5.0, the nitrogen of imidazole ring get protonated and there is a repulsion between the polymer chains. This leads to relaxation of polymer chains. The polymer chain containing caprolactam ring may shrink at 37 ºC and there may be a hydrophilic to hydrophobic transition which also triggers the drug release. Also at acidic pH, 5FU is positively charged. So there is a repulsion between protonated imidazole and 5FU. This synergistically affects the drug release. The lower drug release at pH 7.4 can be explained as follows. Imidazole groups remains neutral
14
at this pH. So there is hydrogen bonding interaction between 5FU and imidazole and caprolactam ring of the polymer. So the drug is almost encapsulated by this hydrogen bonding interaction and only a small amount of drug is released from the matrix. In order to study the effect of temperature on drug release, drug release studies were conducted at two temperatures, at 27 and 37 ºC respectively and the results are shown in Fig 10. It was found that cumulative drug release was higher at high temperature. 88.2 % drug release was observed at 37 ºC while only 80.0 % release was observed at 27 ºC. This is due to the presence of thermoresponsive PNVCL blocks in the drug carrier. PNVCL has a LCST of 32 ºC. So above that temperature it becomes hydrophobic. This reduces the interaction between the drug and the carrier. Therefore the drug is released at a faster rate. Shrinking of PNVCL chain above its LCST may also facilitate drug release [44, 45]. 3.6. In vitro drug release kinetics To get an insight into the mechanism of drug release, drug release kinetics at pH 5.0 and 7.4 were analysed using Korsmeyer-Peppas kinetic model [46]. It is described by the equation 67 68
= kt
(5)
Where M is the amount of 5FU released at time t and M; is the amount of 5FU released at infinite time. Release constant and release exponent are denoted by k and n respectively. In the present study, the obtained drug release kinetic data were examined using this kinetic model with the help of origin pro 8 software by nonlinear regression analysis. Korsmeyer–Peppas kinetic model describes the mechanism of drug release based on the value of release exponent, n. The drug release follows fickian diffusion when n<0.43 and drug is released from polymer matrix due to diffusion. If the value of n is in between 0.43 and 0.85, a combined action of swelling and diffusion of the polymer results in drug release. This is termed as anomalous transport. The drug 15
release is non-fickian, if the value of n>0.85. In this case, the drug release is controlled by the swelling of polymer matrix [47].
Korsmeyer-Peppas fit of drug release data at pH 5.0 (Fig. 11) gave the value of n as 0.585 with a R2 value of 0.997. This reflects anomalous transport of the drug. The drug is released from the matrix as a result of both swelling of the polymer chain and diffusion of drug from the matrix. Due to the protonation of imidazole moiety in the polymer coating, it swells in acidic pH. The initial release was due to the swelling of the polymer. The presence of hydrophilic imidazole group enhances the water permeability. 5FU is a water soluble drug, it diffuses out from the matrix due to a chemical potential gradient. Korsmeyer-Peppas fit for the drug release kinetic data at pH 7.4 (Fig. 12) presented a R2 value of 0.971. The value of n obtained was 0.302 which indicates that drug is released as a result of diffusion of drug from the drug carrier. The initial burst release was due to the diffusion of drug from the surface in the immediate vicinity of the solid-liquid interface. The value of n also rules out the possibility of swelling controlled release at alkaline pH. From the drug release kinetic data, it was concluded that the prepared magnetic nano drug carrier could be used for the controlled and sustained release of 5FU at acidic pH. Also during intravenous administration, only slight amount of drug will be released during blood circulation. But in the acidic tumor environment the drug will be released in controlled way due to both swelling and diffusion. 3.7. In vitro cell viability studies The viability profiles of Fe3O4-Dex-MA-g-P(NVI/NVCL) and 5FU loaded Fe3O4-Dex-MAg-P(NVI/NVCL) were analysed using MTT assay. A DDS should be biocompatible while considering its application in real systems. To ensure this, cell viability of the prepared material was assessed on L929 normal cells (Fig. 13.). It was found that the prepared material cause less 16
harm to the normal cells. This pointed out the biocompatibility of the material. The viability of cells decreased only to a small extent when concentration increased from 0.5 to 10.0 µg/mL . About 98.5 % cells were viable at 0.5 µg/mL concentration of the carrier. At a carrier concentration of 10.0 µg/mL, 85.3 % cells were viable. This result indicates that the prepared DDS, Fe3O4-Dex-MA-g-P(NVI/NVCL) might be safe for administration.” In vitro cell viability study of 5FU loaded material on MCF-7 breast cancer cell line (Fig. 14) indicates that the 5FU loaded material shows higher toxicity to MCF-7 cells. Only 60.2 % cells were viable at a concentration of 25.0 µg/mL. From cell viability studies, it was concluded that the drug loaded magnetic DDS could be employed to selectively kill neoplastic cells. 4. Conclusions Herein we reported the synthesis of a pH/temperature dual sensitive functionalized iron oxide based drug delivery system for the delivery of a model anticancer drug, 5FU . VSM analysis revealed the superparamagnetic nature and the applicability of material in the magnetic field guided therapy for localized cancer treatment. The developed DDS was able to encapsulate a large amount of drug with an EE of 93.5 %. Temperature and pH sensitivity of the carrier were well demonstrated by conducting in vitro drug release studies. The drug was retained in the DDS at alkaline pH and controlled release was achieved at acidic tumor pH. The biocompatibility of the material was evident from in vitro cell viability studies. From the In vitro cell viability study of 5FU loaded material on MCF-7 breast cancer cell line, 60.2 % cells were viable at a concentration of 25.0 µg/mL. The cytotoxicity towards cancer cells can further be improved by attaching suitable targeting ligands to the drug carrier. All these results imply that the prepared DDS could be used for the magnetic field guided, pH and temperature controlled release of 5FU to neoplastic cells.
17
Conflict of interest The authors declare no conflicts of interest.
Acknowledgements The authors are grateful to the Professor and Head, Department of Chemistry, School of Physical and Mathematical Sciences, University of Kerala, Trivandrum, India for providing the laboratory facilities. One of the author, J. Christa is grateful to University Grants Commission, New Delhi, India for providing financial assistance in the form of research fellowship. References [1]
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List of Figures Scheme 1: Schematic representation of the synthesis of Fe3O4-Dex-MA-g-P(NVI/NVCL). Fig. 1. FTIR spectra of Dextran (A), GMA (B), Dex-MA (c), and 5FU (D). Fig. 2. FTIR spectra of Fe3O4-Dex-MA (A), Fe3O4-Dex-MA-g-P(NVI/NVCL) (B) and 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) (C). Fig. 3. XRD patterns of Dex-MA (A), Fe3O4-Dex-MA (B), Fe3O4-Dex-MA-g-P(NVI/NVCL) (C) and 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) (D). Fig. 4. SEM images of Fe3O4-Dex-MA (A) and Fe3O4-Dex-MA-g-P(NVI/NVCL) (B), 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) and TEM image of Fe3O4-Dex-MA-gP(NVI/NVCL) (D). Fig. 5. Room temperature hysteresis curves of Fe3O4-Dex-MA and Fe3O4-Dex-MA-gP(NVI/NVCL). Fig. 6. Effect of pH on encapsulation efficiency of 5FU by Fe3O4-Dex-MA-g-P(NVI/NVCL). Data are presented as mean ± SD (n=3). Fig. 7. Swelling (%) of Fe3O4-Dex-MA-g-P(NVI/NVCL) as a function of pH. Data are presented as mean ± SD (n=3). Fig. 8. Swelling (%) of Fe3O4-Dex-MA-g-P(NVI/NVCL) as a function of time at pH 5.0 and 7.4. Data are presented as mean ± SD (n=3). Fig. 9. pH responsive 5FU release profile of 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) at 37 o
C at pH of 5.0 and 7.4. Data are presented as mean ± SD (n=3).
Fig. 10. In vitro 5FU release of profile of 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) at pH 5.0, temperature 27 and 37 ºC. Data are presented as mean ± SD (n=3).
24
Fig.11. Korsmeyer-Peppas fit for 5FU release from 5FU loaded Fe3O4-Dex-MA-gP(NVI/NVCL) at pH 5.0. Fig.12. Korsmeyer-Peppas fit for 5FU release from 5FU loaded Fe3O4-Dex-MA-gP(NVI/NVCL) at pH 5.0. Fig.13. In vitro cell viability studies of Fe3O4-Dex-MA-g-P(NVI/NVCL) on L929 cells. Data are presented as mean ± SD (n=3). Fig.14. In vitro cell viability studies of 5 FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) on MCF7 breast cancer cells. Data are presented as mean ± SD (n=3).
25
Scheme 1: Schematic representation of the synthesis of Fe3O4-Dex-MA-g-P(NVI/NVCL).
1
D
Transmittance (%)
C
B
A
4000
3500
3000
2500
2000
1500
1000
-1 Wavenumber (cm )
Fig.1. FTIR spectra of Dextran (A), GMA (B), Dex-MA (C), and 5FU (D).
Transmittance (%)
C
B
A
4000 3500 3000 2500 2000 1500 1000
500
-1
Wavenumber (cm )
Fig. 2. FTIR spectra of Fe3O4-Dex-MA (A), Fe3O4-Dex-MA-g-P(NVI/NVCL) (B) and 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) (C). 2
D
Intensity (a.u.)
C
B
A
10
20
30
40
50
60
70
80
2θ (Degree)
Fig. 3. XRD patterns of Dex-MA (A), Fe3O4-Dex-MA (B), Fe3O4-Dex-MA-g-P(NVI/NVCL) (C) and 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) (D).
Fig. 4. SEM images of Fe3O4-Dex-MA (A), Fe3O4-Dex-MA-g-P(NVI/NVCL) (B), 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) (C) and TEM image of Fe3O4-Dex-MA-g-P(NVI/NVCL) (D). 3
B A
Magnetization (emu/g)
20
0
-20
-100000
0
100000
Applied magnetic field (Oe)
Fig. 5. Room temperature hysteresis curves of Fe3O4-Dex-MA (A) and Fe3O4-Dex-MA-gP(NVI/NVCL) (B).
100
250 mg 5FU/L 80
Encapsulation Efficiency (%)
300 mg 5FU/L
60
40
20
0 3
4
5
6
7
8
9
10
pH
Fig. 6. Effect of pH on encapsulation efficiency of 5FU by Fe3O4-Dex-MA-g-P(NVI/NVCL). Data are presented as mean ± SD (n=3).
4
Fe3O4-Dex-MA-g-P(NVI/NVCL) 400
Swelling (%)
300
200
100
4
6
8
pH
Fig. 7. Swelling (%) of Fe3O4-Dex-MA-g-P(NVI/NVCL) as a function of pH. Data are presented as mean ± SD (n=3).
500
pH 5.0 pH 7.4
Swelling (%)
400
300
200
100
0 0
500
1000
1500
Time (Minutes)
Fig. 8. Swelling (%) of Fe3O4-Dex-MA-g-P(NVI/NVCL) as a function of time at pH 5.0 and 7.4. Data are presented as mean ± SD (n=3). 5
100
pH 5.0 pH 7.4
Cumulative drug release(%)
80
60
40
20
0 0
500
1000
1500
Time (Minutes)
Fig.9. pH responsive 5FU release profile of 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) at 37 ºC at pH of 5.0 and 7.4. Data are presented as mean ± SD (n=3).
6
100 ο
37 C o 27 C
Cumulative drug release (%)
80
60
40
20
0 0
500
1000
1500
Time (Minutes)
Fig. 10. In vitro 5FU release of profile of 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) at pH 5.0, temperatures 27 and 37 ºC. Data are presented as mean ± SD (n=3).
1.0
Drug release data at pH 5.0 Korsmeyer Peppas equation 0.8
Mt/M∞
0.6
0.4
0.2
0.0 0
500
1000
1500
Time (minutes)
Fig.11. Korsmeyer-Peppas fit for 5FU release from 5FU loaded Fe3O4-Dex-MA-gP(NVI/NVCL) at pH 5.0.
7
Drug release data pH7.4 Korsmeyer-Peppas equation 0.3
Mt/M∞
0.2
0.1
0.0 0
400
800
1200
Time (Minutes)
Fig.12. Korsmeyer-Peppas fit for 5FU release from 5FU loaded Fe3O4-Dex-MA-gP(NVI/NVCL) at pH 7.4.
120
L929
Cell Viability(%)
100
80
60
40
20
0
Control
0.5
1.5
3.0
6.25
Concentration (µg/ mL)
8
10.0
Fig.13. In vitro cell viability studies of Fe3O4-Dex-MA-g-P(NVI/NVCL) on L929 cells. Data are presented as mean ± SD (n=3).
100
MCF7
Cell viability (%)
80
60
40
20
0
control
1.5
3.0
6.25
12.5
25.0
Concentration (µg/ mL)
Fig.14. In vitro cell viability studies of 5FU loaded Fe3O4-Dex-MA-g-P(NVI/NVCL) on MCF7 breast cancer cells. Data are presented as mean ± SD (n=3).
9
Conflict of interest The authors declare no conflicts of interest.
Author statement The authors, T. S. Anirudhan and J. Christa contributed equally to this work.