In the present study, the nanocomposite of alginate - modi fied bentonite has been synthesised for the controlled release applica- tions of anti-cancerous drug 5-FU.. The precursors used [r]
(1)Original Article
Synthesis and characterization of a clay-alginate nanocomposite for the controlled release of 5-Flurouracil
R Surya, Manohar D Mullassery*, Noeline B Fernandez, Diana Thomas
Department of Chemistry, Fatima Mata National College, Kollam, 691001, India
a r t i c l e i n f o Article history:
Received January 2019 Received in revised form 29 July 2019
Accepted August 2019 Available online xxx Keywords: Bentonite Controlled release Nanocomposite Kinetics Alginate
a b s t r a c t
The scope of the present study is the synthesis and characterization of a nanocomposite based on natural bentonite clay and sodium alginate as a drug delivery system The nanocomposite was prepared by the grafted copolymerization of alginate, acrylamide and modified bentonite The characterization of the nanocomposite was carried out using FTIR, XRD, SEM, TG/DTA, Zeta potential, DLS and TEM analysis A hydrophilic anticancer drug 5-Flurouracil was chosen as the model drug to investigate the loading and release of the nanocomposite Swelling profile study revealed that maximum swelling was occurred at pH 6.8 Thefitting of Peppas's kinetic model was analysed at pH 6.8 and the release kinetics was found to be morefitted to Korsemeyer-Peppas kinetic model having R2¼ 0.9840 Human Colorectal
Adenocar-cinoma cells-HT 29 was used for analysing cell viability The percentage of cell viability decreases from 46.65% to 20.12% when the concentration increases from 2.5mg/ml to 10mg/ml As an alternative to in-vivo models the chick embryo chorioallantoic membrane (CAM) study was conducted The study showed the better biocompatibility and non-toxicity of the nanocomposite
© 2019 Publishing services by Elsevier B.V on behalf of Vietnam National University, Hanoi This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
1 Introduction
In cancer treatment, in order to eradicate tumour, the thera-peutics must be delivered in high doses to ensure sufficient and sustained therapy However, the sustained therapy in high dose is causing damages to healthy tissues such as liver, kidney and bone marrow along with the targeted cells Therefore it is desirable to develop stimuli-responsive controlled drug delivery systems (CDDS) CDDS work on the principle that drug is delivered only on the exposure to external stimuli thereby reducing the pre-mature release of drugs The study on controlled drug release has been getting wide acceptance from the researchers due to its main advantages such as high drug efficiency, continuous release, and reduced side effects compared with conventional drugs in dosage[1,2]
Recently preparation, characterization and applications of controlled drug delivery materials prepared from biopolymer/ inorganic compounds have much sought after owing to their peculiar properties such as biodegradability, controlled release characteristics and high encapsulation efficiency [3,4] Most
hydrogels are prepared by the copolymerization of different vinyl monomers containing hydrophilic side groups with natural poly-saccharides as well as their derivatives Apart from various ad-vantages such as excellent biocompatibility, biodegradability and nontoxicity, they suffer from disadvantages of low strength This disadvantage can be overcome by using natural clays asfiller
Clay minerals that predominantly have properties governed by smectites are called bentonites Montmorillonite is a major con-stituent of most bentonites (typically 80e90 wt%), the remainder being a mixture of mineral impurities including quartz, cristobalite, feldspar and various other clay minerals depending on the geological origin This group of clay minerals has a dioctahedral or tricotcahedral 2:1 layer structure, with isomorphous substitution that leads to a negative layer charge of less than 1.2 per formula unit Interlayer spacing varies between 10 and 15 Å and are generally dependent on the nature of the exchangeable cation and relative humidity Montmorillonites are dioctahedral smectites with layer charges predominantly in octahedral and tetrahedral sites, respectively The general formula of the montmorillonite group can be represented as (Mxỵ)ex[(Si8)tet (M(III)4-xM(II)x)octO20(OH)4]x, where Mỵis the exchangeable cation pre-sent in the interlayer (e.g Naỵ) and M(III) and M(II) are non-exchangeable octahedrally trivalent and divalent cations (e.g Al3ỵ and Mg2ỵ) respectively, and the layer charge is 0.5< x<1.2[5,6] The surface property of bentonite can be enhanced by introducing * Corresponding author
E-mail address:mdmullassery@gmail.com(M.D Mullassery)
Peer review under responsibility of Vietnam National University, Hanoi
Contents lists available atScienceDirect
Journal of Science: Advanced Materials and Devices
j o u r n a l h o m e p a g e : w w w e l s e v i e r c o m / l o c a t e / j s a m d
https://doi.org/10.1016/j.jsamd.2019.08.001
2468-2179/© 2019 Publishing services by Elsevier B.V on behalf of Vietnam National University, Hanoi This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/)
(2)silylated amino functional groups[7] Tuneable pore size is a pre-requisite for the successful loading and the release of the drug, the release kinetics may be slower than expected if the pore size of the carrier is very small[8]
Clays exhibit many interesting features with active sites such as hydroxyl groups, Lewis and Brønsted acidity, and exchange-able interlayer cations In addition to the high aspect ratio of clay minerals and the small dimensions of the individual layer render them particularly attractive in several areas of material science[9]
Clay minerals were proposed as fundamental constituents of several modified therapeutic carriers and had different purposes and acted through various mechanisms Although clay minerals and polymers were often used in the pristine form as a single drug carrier, they did not meet all the requirements The preparation of polymer-layered silicate composite offered the possibility of improving the properties of individual components Different biodegradable and biocompatible polymers are suitable drug car-riers which can release therapeutics at a constant, predetermined rate[10] Alginate is a widely used mucoadhesive and biodegrad-able natural polymer for the controlled release of drugs But the low efficiency in trapping water-soluble drugs in alginate is one of the problems for developing a drug delivery system[11] To improve the drug entrapment efficiency and thereby modulating the drug release it is desirable to incorporate water-insoluble materials like bentonite Thus alginate-clay composite formed would decrease the drug release by increasing the drug absorption capacity in the composite matrix
Present study explores the utilisation of a nanocomposite prepared by the copolymer reaction of alginate and natural so-dium bentonite for the controlled release application of 5-Flurouracil
2 Materials and methods 2.1 Materials
Sodium alginate (SA), 5-Flurouracil (5-FU) and ethyl-eneglycoldimethacrylate (EGDMA), fetal bovine serum (FBS), 3-(4,5-dimethythiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT), Dulbecco's Modified Eagle's medium (DMEM), phosphate buffered saline (PBS) were purchased from Sigma Aldrich Bentonite clay was procured from Ashapura Clay Mines (India), which was further converted to sodium bentonite (NaeB) by immersing it in 1M NaOH Acrylamide (AM) was received from Merck Life Solence Pvt Ltd Ceric ammonium nitrate (CAN), DMSO and methanol were purchased from Merck Specialities Pvt Ltd 3-Aminopropyltriethoxysilane (APTES) was procured from Spectrochem Pvt Ltd NCCS, Pune, India supplied HT 29 cell line Distilled water with specific conductivity of less than 1mScm1 was used throughout the study
2.2 Synthesis of the drug delivery system involves the following steps
2.2.1 Preparation of 3-Aminopropyltriethoxysilane bentonite (APSB)
About 5.0 g of NaeB was dried at 60C for 24 h It was dispersed in 100 ml ethanol Separately a solution with 5.0 g of 3-Aminopropyltriethoxysilane (pur) was dissolved in 100 ml sol-vent This solution was added to the dispersion containing sodium bentonite The dispersion was stirred at 50 C under magnetic stirring for 72 h After the complete stirring, the product was centrifuged and was washed with distilled water, dried at 80C and grained tofine powder
2.2.2 Preparation of alginate-modified bentonite nanocomposite (ABNC)
About 1.0 g of sodium alginate was dissolved in 100 ml water taken in a three-neck RBflask, which was equipped with a reflux condenser, magnetic stirrer, and a nitrogen line Nitrogen was purged into the solution for 30 min, after that the solution was heated to 60C To this, an appropriate amount of ceric ammo-nium nitrate was added After 10 min, a mixture of a suitable amount of acrylamide and modified bentonite (APSB) and the cross-linking agent EGDMA were added The above mixture was magnetically stirred for h at 450 rpm and at 60C The pH was adjusted to 7.0 by using M NaOH solution and was precipitated by using a methanol-water mixture (5:1) The sample was dried at 60 C to a constant weight The product was milled and had a uniform particle size
2.3 Characterization
The FTIR spectral analysis was performed to characterize the presence of functional groups present in NaeB, APSB, ABNC nanocomposite and the spectrum was recorded in the scanning range between 400 and 4000 cm1 using a Bruker-spectrophotometer (Germany) X-ray diffraction study was used tofind out the crystallinity of a substance XRD measurement was carried out on a Rigaku Geigerflex X-ray diffractometer with Ni filtered Cu Karadiation at 40 kV, 20 mA and a diffraction angle of 2qscanning from 1to 100 The thermal analyses were made on a Metler Toledo Star system under nitrogen atmosphere with a heating rate of 20C min1 A Philips model XL 30 CP scanning electron microscope (SEM) was used to take micrographs In this instrument, cryofreezing method was used for taking SEM pho-tographs at 15 kV and 20 kV with a working distance of mm, in which frozen samples were coated with a thin layer of gold to make the surface conductive towards electron beam The samples were ultrasonicated (PCi Electronics, Mumbai, 230 V, 50 Hz) for a definite period before the Dynamic Light Scattering (DLS) analysis and were performed by BI-200SM multiangle dynamic/static laser scattering instrument (Brookhaven, USA) The Zeta potential (z) of the samples was measured using Horiba SZ-100 equipped with a 532 nm Diode Pumped Solid State (DPSS) laser, operated at a temperature of 25C TEM images of the sample were recorded using JEOL-1200 TEM instrument
The cell viability of the prepared sample in HT 29 cell line was measured using MTT assay at different concentrations of 2.5, 5.0, 10.0, 20.0 and 40.0mg/ml A temperature-controlled water bath shaker (Lab line, India) with a temperature variation of±1.0C was used for the controlled shaking experiments
All pH measurements of the solution were carried out using a pH meter (Systronics modelm362, India) The absorbance mea-surements of the 5-FU solution were performed on a UV-Visible spectrophotometer (Systronics, India) at 266 nm Accurate weights of samples were taken using electronic balance (Shimadzu, Japan)
2.4 Swelling study
(3)Swelling%ị ẳWwetWdry
Wdry 100
(1)
2.5 Drug encapsulation studies
About 50 ml of 5-FU solution at a concentration of 5.0 mg/ml was added to 0.1 g of ABNC in a stoppered bottle; the pH was adjusted to 5.0 using phosphate buffer and stirred for h at 1000 rpm The composite was further washed with distilled water to remove the loosely bound drug molecules on the surface and dried The drug encapsulation efficiency (DEE) was calculated using the equation given below
DEE¼ðTotalamount of FlurouracilÞ ðFree FlurouracilÞ Total amount 5 Flurouracil
100%
(2) The total amount of 5-Flurouracil indicates the initial concen-tration of the drug and free 5-Flurouracil indicates the concentra-tion of the drug in the supernatant after encapsulaconcentra-tion onto ABNC The concentration (mg/mL) of 5-FU was measured by using the UV-Visible spectrophotometer at 266 nm by comparing the value with a standard calibration curve
2.6 In vitro drug release study
Release study of 5-FU from the ABNC was studied under two different physiological pH conditions of 1.2 and 7.4 It is known that pH values differ in different tissues and cellular compartments within the human body such as that in the gastrointestinal tract Most tumour tissues, as well as inflamed or wound tissues, exhibit a pH that differs from the pH value of 7.4 found in normal tissues The change in pH along the gastrointestinal tract from acidic to basic in the intestine has been utilized to explore the pH-responsive com-posite for oral drug delivery Therefore the swelling profile of the composite in aqueous solution was carried out at two different pH conditions of 1.2 and 7.4 by varying time intervals About 0.1 g of 5-FU loaded ABNC was placed in 100 ml of buffer solution at physi-ological temperature At specific intervals, about ml of the su-pernatant solution was taken and the concentration of released drug was measured using UV-Visible spectrophotometer at 266 nm
% of drug release¼ amount of drug released
Total amount of drug loaded 100 (3) The in vitro release kinetics was analysed using the Korsmeyer-Peppas kinetic relation[12]equation(4)
Mt M∞¼ K t
n (4)
where Mtis the amount of drug released at time t and M is the amount of the drug released completely K is the rate constant and n is the diffusion exponential
2.7 Cell line and cell culture conditions
HT 29e Human Colorectal Adenocarcinoma cells were taken as the cell line The HT cells were cultured in DMEM, supplemented with 10% FBS and kept at 37C in a humidified 5% CO2incubator (NBS Eppendorf, Germany) The cells were trypsinized with buff-ered saline solution contains 0.25% trypsin and 0.03% EDTA The cells were plated to the culture plate for 24 h
2.8 Cytotoxicity analysis/MTT assay
MTT assay is a colorimetric assay used for the determination of cell proliferation and cytotoxicity which is based on the reduction of the yellow coloured water-soluble tetrazolium dye MTT to formazan crystals Mitochondrial lactate dehydrogenase produced by live cells reduces MTT to insoluble formazan crystals, which upon dissolution into an appropriate solvent exhibits purple colour, the intensity of which is proportional to the number of viable cells and can be measured spectrophotometrically at 570 nm Briefly, seeded using a 200ml cell suspension in a 96-well plate at a cell density of 20,000 cells per well without the test agent Allow the cells to grow for about 12 h Add appropriate concentrations of the test agent (2.5mg/ml, 5mg/ml, 10mg/ml, 20mg/ml and 40mg/ml) Incubate the plate for 24 h at 37C in a 5% CO2atmosphere After the incubation period, the plates are taken out from the incubator, the spent media is removed and MTT reagent is added to afinal concentration of 0.5 mg/ml of total volume Wrap the plate with aluminium foil to avoid exposure to light Return the plates to the incubator and incubate for h Remove the MTT reagent and then add 100ml of solubilisation so-lution (DMSO) Gentle stirring in a gyratory shaker will enhance the dissolution Occasionally, pipetting up and down may be required to completely dissolve the MTT formazan crystals especially in dense cultures Read the optical density on a spectrophotometer at 570 nm and 630 nm used as the reference wavelength The cell viability % can be calculated by (Equation (5)) From the % of cell viability, it is possible to calculate the % of cytotoxicity (Equation(6))
Cell viability %¼Optical density of controlOptical density of test 100 (5)
Cytotoxicity %¼ 100 e cell viability % (6)
2.9 Chorioallantoic membrane (CAM) assay protocol
The anti-angiogenic activity of the drug 5-FU loaded in the nanocomposite was analysed by CAM assay Fertilized chick embryos were collected from Kerala State Poultry Farm, Kudappanakunnu, Trivandrum (India) and were incubated in a humidified incubator at 37C After four days of incubation, a small window on the eggshell of about cm width was introduced Afixed concentration of the sample was prepared in PBS and introduced through the hole by a sterilized needle on the eighth day The window was tightly sealed by wax and the incubation was continued After eleven days, the eggs were taken out and the window was opened to see the formation of blood vessels and photographs were taken using a digital camera All experiments were conducted under the sterilized condition in order to avoid the contamination[13]
2.10 Statistical analysis
All the results were expressed as mean ± standard deviation (SD) Statistical analysis was performed with origin 8.0 (Origin eLab Corporation- USA)
3 Result and discussion
3.1 Synthesis and characterisation of the drug delivery system 3.1.1 Synthesis of the drug delivery system (ABNC)
(4)synthesis of the drug delivery system are sodium alginate, acryl-amide and silylated bentonite
During the synthesis of the DDS, one of the precursors, NaeB was silylated using 3-Aminopropyltriethoxysilane in order to in-crease the interlayer spacing NaeB was silylated by exchanging intercalated water molecules with 3-aminopropyltriethoxysilane The water molecules present in the ethanol medium catalysed the interlayer surface silylation on NaeB[6]
During the first step of the synthesis, ceric ions induced the sodium alginate (SA) through a redox initiation method Ceric ammonium nitrate can act as redox initiator both in aqueous and acidic conditions (~0.1 N HNO3)[14] Due to the redox reaction, alginate is converted into corresponding alginate radicals These macromolecular radicals of alginate initiate the acrylamide monomer molecules followed by grafted copolymerization on the alginate back bone In the presence of the cross linking agent EGDMA, the crosslinked grafted copolymer was formed
In the next step, the layer structured inorganic filler of the modified bentonite was introduced into the polymeric matrices followed by the strong intermolecular hydrogen bonds between theeOH group present in the clay and the electronegative groups present in the cross-linked polymer By the alkaline hydrolysis using NaOH, the amide groups present in the cross-linked polymer was converted into carboxylate anions The scheme of preparation of the nanocomposite was presented as supplementary information S1
3.1.2 Fourier transform infrared spectroscopy (FTIR)
Fourier transfer infrared spectroscopy was one of the simplest methods for the characterization and identification of functional groups present in a compound Additional information regarding the structure and the chemical bonds between chemical species was obtained by carrying out by the infrared spectroscopy in 4000-400 cm1wavenumber range By comparing the IR spectra of the starting materials with the final modified composite, it was possible to ascertain whether the required modification was incorporated
The FTIR spectra of NaeB, APSB, ABNC and 5-FU-loaded ABNC are shown inFig In NaeB, the band at 3630 cm1corresponds to the stretching vibrations of the hydroxyl group in MgeOHeAl and AleOHeAl The bands at 3420 cm1and 1628 cm1are due to the stretching vibrations and bending vibrations of the HeOeH bonds of water molecules intercalated in the clay minerals[15] There is an intense band at 980 cm1present in both NaeB and APSB due to the SieO stretching in SieOeSi in clay materials In APSB there are two bands observed at 2930 cm1 and 2860 cm1, due to the stretching asymmetric and symmetric vibrations ofeCH2groups And also the two bands observed at 1555 cm1and 1490 cm1are corresponding to the bending vibrations of eNH2 and eCH2 respectively[16]
Sodium alginate showed asymmetric and symmetric stretching vibrations at 1627 and 1415 cm1, due to the carboxyl anion and the peak at 1034 cm1 was due to oxygen stretching in cyclic ether bridge The presence of a broadband at around 3450 cm1 corre-sponds toeOH stretching vibrations[11]
From the spectrum of ABNC, it can be observed that the chem-ical structure of ABNC is similar to alginate, which is the major fraction in its composition The eOH stretching vibration gets decreased in intensity than that of pure alginate and also it gets shifted to 3462.2 cm1, indicating the participation ofeOH groups in the composite formation Band corresponds to 1059 cm1 in-dicates the formation ofeCH-O-CH2during the grafting The strong band at 1047 cm1is due to SieOeSi stretching which is found to be more sharp with a decrease in intensity may be due to the better participation ofeOH groups on the clay Presence of bands at 1459
and 1552 cm1 are due to the symmetric stretching and asym-metric stretching modes ofeCOO-group The FTIR spectrum of the drug-loaded composite material resembles the superimposition of the spectra corresponding to the drug and to the ABNC This simi-larity between the spectra indicates the interaction between the drug and the composite is rather weak
3.1.3 X-ray diffraction (XRD)
Fig 2shows the XRD pattern of NaeB, APSB, ABNC and 5-FU-L-ABNC The XRD pattern of NaeB showed a characteristic (001) reflection peak at 2q¼ 6.89corresponding to the d-spacing value of 13.02 A
̊
(Fig 2) Silylation of NaeB had resulted in the shift of d001 of Na-bentonite to lower 2q value from 6.89 to 4.17, which had resulted in an increase in the basal spacing d001from 13.02 Ato 21.15 A[16] The peak at 2q ¼ 20.63 Awas attributed to (002) reflection[17] Interaction of alginate with silylated bentonite had not resulted in any change in the XRD pattern of silylated bentonite, but the spectra was broadened due to the dispersion of clay into the polymeric matrix The spectra of the drug-loaded system was again
Wavenumber (cm-1)
T
ran
sm
ittan
ce
(a.u
.)
400 900 1400 1900 2400 2900 3400 3900
Na-alginate APSB ABNC
5-Flurouracil 5-FU-L-ABNC
Fig FT-IR of Na-alginate, APSB, alginate-clay nanocomposite, 5-Flurouracil and 5-Fu-L-nanocomposite
5-FU-Loaded-nanocomposite Alg-clay nanocomposite
APSB
Na-B
0 20 40 60 80
2 Theta (degree)
Int
ensi
ty
(a
.u
.)
(5)broadened, indicated the loading of drug molecules into the poly-meric system[18]
3.1.4 Thermo analytical techniques (TG/DTA)
Fig 3shows the thermal analysis of ABNC and 5-FU-L-ABNC Alginate powder DTA curve showed that three weight loss events at 252 C, 374 C and 450 C which are associated with alginate decomposition The weight loss above 600 C may due to the destruction of the carbon skeleton
The drug-loaded composite showed four weight-loss events at 50C, 260C, 342C and 464C The initial weight loss at 50C was due to the loss of physisorbed drug weakly bonded on the surface and absorbed water The second weight loss at 269C was due to the drug decomposition The third weight loss at 342C may be due to the strongly bonded drug molecules The weight loss at 464C may due to the alginate decomposition The decrease in % weight loss at a higher temperature for the composite was 1.613%, whereas for alginate the decrease in percentage was 2.93% This fact attri-butes to the extra thermal stability of the composite compared to the alginate
3.1.5 Zeta potential analysis
The stability of the colloidal particles depends on the magnitude of zeta potential[19] Dispersion of particles with zeta potential higher thanỵ30 mV or lower than 30 mV is considered as stable Zeta potential of modified clay, APSB is slightly positive in an aqueous medium The slightly positive charge means the overall instability of the nanoparticles Due to the lack of stabilization, the particles may aggregate to form a precipitate and later they dispersed in water[20].Supplementary information S2shows the zeta potential of ABNC and 5-FU-L-ABNC ABNC shows the zeta potential value of39.8 mV indicates the better stability of nano-composite The high negative zeta potential of the composite is due to the repulsion betweeneCOO-groups on the surface After the loading of 5-FU, the zeta potential is changed to45.4 mV, implied the successful loading of the drug molecule with appropriate sta-bility It was reported that 5-FU was negatively charged[21] The encapsulation of negatively charged drug molecule may cause electrostatic repulsion with the anionic part of the composite and which may lead to change in the zeta potential to45.4 mV. 3.1.6 Particle size determination of the nanocomposite
Particle size determination can be done by dynamic light scat-tering (DLS), transmission electron microscopy (TEM) and field emission scanning electron microscopy (FE-SEM) analysis It was observed that the particle size by DLS analysis was larger than that obtained from TEM because DLS gave the hydrodynamic size of the
particles The nanostructure of the ABNC composite was confirmed by TEM analysis (Fig 4) The particle size of the DDS was calculated with the help of IMAGE J software Using the software scale the image with the magnification mentioned (200 nm) and then picking the particles using the selection tool to get the area of a single particle Then after selecting a minimum of 10 points the software will calculate the average, maximum and minimum area of the entire particle From the area obtained the average radius and diameter of the particles can be calculated For the minimum area of 4910 nm2, the diameter obtained was 80 nm and for the maximum area of 14,577 nm2, it showed a diameter of 136 nm For the average value of 8988 nm2, the particle diameter was found to be 106 nm Thus from the TEM data using the IMAGE J software the approximate diameter lies in the range of 100 nm for the DDS particles
From the TEM analysis of the drug delivery system, the average diameter of DDS was around 100 nm The DLS of nanocomposite and 5-FU-nano composites were 115.1 nm and 228.5 nm indicating encapsulation of the drug (S3) From SEM analysis, the increase in particle size from the precursor to thefinal product gives a positive evidence of successful synthesis of nanocomposite and loading of 5-FU (Fig 5) In SEM, each and every sample can be distinguished based on their surface morphology
3.2 Drug encapsulation study
Fig 6shows the encapsulation profile at variable pH conditions The maximum drug encapsulation efficiency was found to be at pH 5.0 (89.8%) This may due to the fact that at pH 5.0 the nano-composite can be appropriately swollen So the drug molecules can enter the inner gallery where it can form H-bond or electrostatic attraction with the modified clay Better encapsulation was observed within the pH of range 4.5e5.5 At very low pH conditions the swelling of the composite was extremely low therefore the entry of drug molecules into the DDS was limited Also at pH from to 10, the encapsulation efficiency was found to be too low Since at alkaline pH condition 5-FU attains mono or di-anionic tautomeric form[22,23] This may lead to anioneanion repulsive interaction and preventing the entry of drug molecules into the drug delivery system
3.3 Swelling kinetics and swelling capacity of the nanocomposite It was reported that the swelling kinetics of a nanocomposite gel depends on the variable factors such as swelling capacity, the particle size of composite and the composition of the composite
[24].Fig 7depicts the swelling capacity of the composite under
-1.991 -1.491 -0.991 -0.491 0.009 0.509 1.009 60 70 80 90 100
40 240 440 640
Weight % (%) Derivative Weight % (%/m
Temperature (°C) We ig h t (% ) D eriv a tiv e W eig h t % (% /m in ) 5-Fu -L-Nanocomposite -3.243 -2.743 -2.243 -1.743 -1.243 -0.743 -0.243 0.257 50 60 70 80 90 100
40 240 440 640
Weight % (%) Derivative Weight % (%/m
Temperature (°C) We ig h t (% ) D er iva tive We ig h t % (% /m in ) ABNC
Fig TG/DTA of alginate-clay nanocomposite and 5-Fu-L- nanocomposite
(6)variable pH conditions by taking stock solutions of NaOH and HCl During the study, it was found that the swelling ratio of the com-posite was found to increase with the increase in pH After pH 3.0 the swelling profile was changed This may due to the ionization of
eCOOH group leading to anioneanion repulsion [25] The maximum swelling was found to be at pH 6.8, after that there was a gradual decrease in the swelling ratio due to screening effect of excess sodium ions, which may reduce the anioneanion repulsion Fig TEM of alginate-clay nanocomposite
(7)[26] It was reported that the swelling behaviour of the polybasic composite was influenced by the composition as well as the pKa of the carboxylic group present[26,27] In order to attain the better swelling of the composite, the pH of the medium should be greater than the pKa of the carboxylate groups in the composite Moreover, alginate polymer composite has the tendency to attain the maximum swelling nearly at neutral pH conditions than at acidic/ alkaline pH The pKa of alginic acid lies between 3.4 and 4.4 and at above pH 5.0 most of the carboxylic groups get protonated Above the pH of 7.4, the swelling of the composite was found to be retarded due to the screening effect Therefore, the maximum swelling was occurred within the pH range of 6.5e7.0 The size of the composite also influences the swelling index Nanocomposite can absorb more and more water molecules due to the large surface area, so the nanogels can swell easily
In order to study the dependence of the composition of the nanocomposite gel on the swelling kinetics, various proportions of precursors were taken From the studies, it was observed that the swelling property of the composite was increased with increase in the concentration of SA It was observed that, by varying the con-centration of SA from 0.1 to 0.9 g, the swelling capacity of the com-posite was getting increased by ~10 times Further, an increase in the concentration of SA would decrease the swelling capacity This was because SA dispersion in water resulted in the increase in viscosity hence it was very difficult for the monomer units of acrylamide to
reach the active sites But on increasing the amount of the clay the swelling of the composite was found to decrease due to the more cross-linkage with the polymer The optimum amount of SA: NB was approximately 1: And also the concentration of the crosslinking agent had a profound effect on the swelling % It was seen that the cross-linking of the nanocomposite increased with increase in the concentration of EGDMA, but there was a possibility for lowering the absorption of water by the polymer due to the high extent of cross-linking This tendency was not at all favouring the better swelling property of nanocomposite gel The optimisation of the concentra-tion of crosslinking agent was determined by taking variable con-centrations of EGDMA and studied its influence in the swelling and encapsulation properties of the composite and the results were presented astable in S4 From the table, it could be shown that the optimised concentration of EGDMA for better swelling behaviour was found to be 2.5 mol/l The equilibrium swelling of the nano-composite was attained within 30 The maximum swelling un-der optimum conditions (AAme 4.83 mol/l, SA- 0.0462 mol/l and EGDMA- 2.5 mol/l) was found to be 280 g/g at pH¼ 6.8
3.4 In vitro release study
Fig 8shows the in vitro release study carried out at two different physiological pH conditions of simulated gastric pH of 1.2 and simulated intestinal pH of 7.4 The pH-responsive nature of alginate leads the scientist to develop new drug delivery systems using alginate as the starting material The in vitro release studies of 5-FU from alginate-chitosan-MMT has been reported by Azhar et al
[28] In that study, 5-FU was loaded in MMT and further it was coated with alginate followed by chitosan; with the maximum release of drug was occurring at pH 7.4, and the time taken for 50% (I50) release was h Iliescu et al.[11]reported a DDS of alginate-MMT composite beads for the release of the drug irinotecan According to them, the MMT-drug hybrid was synthesized and was again coated with alginate Maximum release of drug was occurring at pH 7.4
In the present study, the maximum release of drug was occur-ring at pH 7.4 About 81.5% of drug release was occuroccur-ring within 12 h, with the I50of h In our study, we synthesized a grafted hybrid of alginate and modified bentonite and further it was loaded with 5-FU From the swelling and in vitro release study of the nanocomposite, it was clear that the swelling profile of the com-posite had a direct influence on the in-vitro release The maximum swelling of the composite occurred at pH 6.8 (~7.0) and the in-vitro release occurred at a pH of 7.4, this may due to the peculiarity of alginate component in the nanocomposite The equilibrium swelling was attained within 60 and about 35% of drug release
20 40 60 80 100
0 10
Temperature = 37 oC
pH
E
nca
ps
ul
at
io
n ef
fi
cen
cy
(%
)
Fig Encapsulation of 5-Flurouracil at different pH (triplicates for each sample were analyzed and each datum point represents the mean value±standard devia-tion; n¼ 3)
150 175 200 225 250 275
6 6.2 6.4 6.6 6.8
pH
S
w
ellin
g (
%
)
0 50 100 150 200 250
1 10
pH
S
w
ell
in
g
(%
)
Temperature = 37 oC Temperature = 37 oC
Fig Swelling study of the alginatee clay nanocomposite at variable pH conditions (triplicates for each sample were analyzed and each datum point represents the mean value ±standard deviation; n ¼ 3)
(8)was occurring within h This is a good evidence for the influence of swelling property on in-vitro release kinetics
The in-vitro drug release study was analysed using Korsmeyer-Peppas kinetic equation (Fig 8) In Peppas equation, the value of k gives the idea about the interaction of drug on to the drug de-livery system Smaller the value of K, weaker is the interaction between the drug and drug delivery system[12] The value of n determines the release mechanism If the value of n 0.43, the drug release is mainly due to the diffusion of drug known as Fickian diffusion If the value of n¼ 0.85, the drug release is mainly due to the swelling of the polymer and if the value between is 0.43 and 0.85, it accounts for the non-Fickian diffusion In the non-Fickian diffusion, the release kinetics is controlled by both the diffusion and swelling process The fitting of Peppas's kinetic model was analysed at pH 7.4 and the release kinetics was found to be more fitted to Korsemeyer-Peppas kinetic model having R2¼ 0.9840 The value of n¼ 0.6223 means, the release of the drug follows non-Fickian mechanism The release of the drug depends both on the diffusion of drug and the swelling of the composite The value of K¼ 0.0250, indicates the weaker interaction between the drug and the drug delivery system The release of the drug may due to both the swelling of alginate covering and also due to the diffusion of drug from the modified clay
3.5 Cell viability assay (MTT assay)
The cell viability was analysed by MTT method using HT 29e Human Colorectal Adenocarcinoma cells, in which it was incu-bated to 48 h under variable concentration of 2.5 mg/ml to 40.0mg/ml The % cell viability of above 80 was considered as cytocompatible and non-toxic[29] The % of cell viability of the drug delivery system was not below 80% even at higher con-centration of 40.0mg/ml The % of cell viability was found to be decreased with increase in the concentration Fig represents the profile of cell viability assay and the % of cell activity (cell toxicity/cell viability) of the drug delivery system In the case of the drug-loaded composite, the % of cell viability was decreased from 46.65% to 20.12% when the concentration was changed from 2.5mg/ml to 10mg/ml This was due to the fact that the % of cell toxicity was increased by the factor of 26.53%, when the concentration was increased times The IC 50 value means the drug concentration required to achieve 50% inhibition time, which is found to be 10.21mg/ml for the 5-FU-loaded composite and 18.64% for Flurouracil Due to the larger IC 50 value of 5-FU-loaded composite than the free 5-Flurouracil, the cellular drug release may be better for 5-FU-loaded composite than pure drug
0 10 20 30 40 50 60 70 80 90
0 200 400 600 800
pH =7.4 pH = 1.8
Dr
ug
r
el
ea
se
(%
)
Time (min)
-1.4 -1.2 -1 -0.8 -0.6 -0.4 -0.2 0
0 1 2 3
y = 0.6223x - 1.6018 R2 = 0.9840
n = 0.6223 K = 0.0250 Log (time) in minute
Lo
g (
∞
)
Fig In-vitro release profile of 5-Flurouracil from alginate-clay nanocomposite and the kinetic profile fitted with Korsmeyer-Peppas kinetic model ((triplicates for each sample were analyzed and each datum point represents the mean value±standard deviation; n ¼ 3)
(9)3.6 In vivo study model- CAM assay
Angiogenesis is the development of new blood capillaries from pre-existing capillaries and post-capillary venules[30] Among the different kinds of angiogenic diseases, tumour growth and metathesis is the most drastic and dangerous one A tumor stim-ulates the blood capillaries to enhance its own growth and spreading A permanent remedial to prevent the tumour growth is the retardation of blood vessels Drugs like 5-FU generally called anti-angiogenic drugs can prevent the tumors from growing their blood vessels So the tumour can be starved by blocking oxygen and nutrients
The chick embryo chorioallantoic membrane (CAM) is an extraembryonic membrane with dense capillary blood vessels CAM is a better alternative of animals in vivo models to study both angiogenesis and antiangiogenesis Thefirst in vivo angiogenesis study using CAM was reported in 1913 There are so many advan-tages of CAM than the mammalian models such as low cost, easy to carry out, reproducibility and easy availability of materials, etc[31] During the analysis, three sets of eggs were taken for the study of 1) Controlled 2) ABNC and 3) 5-FU-loaded ABNC The image of the controlled study shows the angiogenic nature of CAM as shown inFig 10 The growth of blood capillaries are so dense and can be visualized clearly Similarly, there was no drastic differentiation between ABNC and the control It shows the better biocompatibility and non-toxicity of the nanocomposite By examining the images of 5-FU-loaded nanocomposite, the blood vessels are found to be much more retarded indicating the potent drug release by the nanocomposite without any premature leakage
4 Conclusion
Alginate-modified bentonite nanocomposite (ABNC) was syn-thesized by combining the copolymer of alginate-acrylamide using EGDMA as crosslinking agent followed by silylated bentonite (APSB) for the controlled release of 5-FU The composite was characterized by FTIR, XRD, SEM, and thermal analysis Swelling studies of the composite proposed that the maximum swelling of the composite occured at a pH of 6.8 The profile of the encapsu-lation study suggests that the encapsuencapsu-lation efficiency gets decreased with the increase in pH The release kinetics is controlled by both the diffusion and swelling process Thefitting of Peppas's kinetic model was analysed at pH 6.8 and the release kinetics was
found to be morefitted to Korsemeyer-Peppas kinetic model having R2¼ 0.9840 The cell viability was analysed by MTT method using HT 29e Human Colorectal Adenocarcinoma cells In the case of the drug-loaded composite, the % of cell viability was decreased from 46.65% to 20.12% when the concentration was changed from 2.5mg/ ml to 10 mg/ml This indicated that the % of cell toxicity was increased by the factor of 26.53%, when the concentration was increased times The chick embryo chorioallantoic membrane (CAM) study is a better alternative to animals in in-vivo models The study showed the better biocompatibility and non-toxicity of the nanocomposite By examining the images of the 5-FU-loaded nanocomposite, the blood vessels are found to be much more retarded indicating the potent drug release by the nanocomposite without any premature leakage
Acknowledgments
The authors are expressing sincere gratitude to The Head, Department of Chemistry, Fatima Mata National College, Kollam for providing laboratory facilities The corresponding author thanks UGC, New Delhi for financial assistance in the form of Minor Research Project (2324-MRP/15e16/KLKE015/UGC-SWRO) The authors sincerely acknowledging the services rendered by Indian Institute of Science, Bangalore for their assistance in the charac-terization of the samples
Appendix A Supplementary data
Supplementary data to this article can be found online at
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