- Email: [email protected]
Fabrication of letrozole formulation using chitosan nanoparticles through ionic gelation method
Accepted Manuscript Title: Fabrication of letrozole formulation using chitosan nanoparticles through ionic gelation method Authors: Thandapani Gomathi, P.N. Sudha, J. Annie Kamala Florence, Jayachandran Venkatesan, Anil Sukumaran PII: DOI: Reference:
S0141-8130(16)32076-1 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.01.147 BIOMAC 7078
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
19-10-2016 1-1-2017 18-1-2017
Please cite this article as: Thandapani Gomathi, P.N.Sudha, J.Annie Kamala Florence, Jayachandran Venkatesan, Anil Sukumaran, Fabrication of letrozole formulation using chitosan nanoparticles through ionic gelation method, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2017.01.147 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
Fabrication of letrozole formulation using chitosan nanoparticles through ionic gelation method 1
Thandapani Gomathi, 1*P.N. Sudha, 2J. Annie Kamala Florence, 4Jayachandran Venkatesan and 5Anil Sukumaran
1,1*
Department of Chemistry, D.K.M. College for Women, Vellore, Tamil Nadu, India 2 4
Department of Chemistry, Voorhees College, Vellore, Tamil Nadu, India
Marine Bioprocess Research Center and Department of Marine-bio Convergence Science, Pukyong National University, Busan, Republic of Korea
5
Division of Periodontics, Department of PDS, College of Dentistry, Prince Sattam Bin Abdulaziz University, Riyadh, Saudi Arabia
(*Corresponding author Email: [email protected]; [email protected]; Mobile: +91-9842910157) Graphical abstract
1
Highlights: Letrozole – sustained release from chitosan nanoparticles formulation. Average particle size of the formulation ranges from 58 to 91 nm. Freeze dried formulation have to size of 24 nm. Hemolysis analysis discloses the blood compatibility of the formulations. Abstract In this study, the anticancer drug letrozole (LTZ) was formulated using chitosan nanoparticles (CS-NPs) with the crosslinking agent sodium tripolyphosphate (TPP). The nanoformulation was optimized by varying the concentration of drug. The prepared particles were characterized using FTIR, TGA, XRD, SEM, TEM and DLS. From the FTIR results, the appearance of a new peak for =C-H, C=C and C=N confirms the formation of LTZ loaded chitosan nanoparticles. TEM images shows that the average particle size was in the range of 60 80 nm and 20 – 40 mm air dried and freeze dried samples respectively. Also the prepared formulation had been evaluated in vitro for determining its hemocompatability, biodegradability and serum stability. The preliminary studies supported that the chitosan nanoparticles formulation has biocompatibility and hemocompatible properties and it can act as an effective pharmaceutical excipient for letrozole. Keywords: chitosan nanoparticles, serum stability, optimization, hemocompatability, letrozole
Introduction Breast cancer is the most common type of cancer, especially in women, and, unfortunately, its incidence is increasing each year [1,2]. It is widely accepted that the majority of breast cancers are hormone-dependent and that estrogen is a key mediator in the progression and metastasis of breast tumors. Particularly, for postmenopausal women it has been reported that the concentration of 17b-estradiol (E2) in breast tumor can be tenfold higher than those in 2
plasma [3]. The high concentration of E2 in breast tumors could be attributed to increased uptake from plasma or in situ aromatization of androgens to estrogens [4]. Letrozole is considered to be one of the most effective non-steroidal, third generation aromatase inhibitors (AIs) which inhibit excess estrogen bio-synthesis within the body [5-8]. Its use as an estrogen receptor positive breast cancer drug is well recognized [9,10]. In practice letrozole was given to treat breast cancers and it was administered orally. Letrozole prevents the aromatase from producing estrogens by competitive, reversible binding to the heme of its cytochrome P450 unit [11] by >99%. Therefore it is a highly potent drug [12,13]. Polymeric nanoparticles have recently been considered as promising carriers for anticancer agents [14,15]. Especially chitosan plays a vital role in drug delivery applications. The amino group in chitosan has a pKa value of ~6.5, thus, chitosan is positively charged and soluble in acidic to neutral solution. Chitosan is bioadhesive and readily binds to negatively charged surfaces such as mucosal membranes and enhances the transport of polar drugs across epithelial surfaces, and is biocompatible and recyclable. Since in the conventional mode of administration, there is the need for the ideal drug delivery system owing to its adverse side effects caused by the non-specific targets. Therefore for designing ideal delivery systems, the parameters such as size and size distribution, drug loading capacity and stability are the important parameters to be taken into account [16,17]. The durg-polymer interactions can be proposed by increase drug loading capacity, non-covalent interactions including hydrogen-bonding and ionic interactions. Generally, the development of new materials always leads to advances in technology and creates innovative solutions to old problems. Thus the nanotechnology has gained wide acceptance in contemporary life [18]. In oncology, nanomaterials can be engineered to serve as anti-cancer agents or as ideal drug delivery vehicles [19-21]. Hence the present work was aimed
3
to synthesis the novel formulation using chitosan nanoparticles for the highly potent anticancer drug letrozole for sustained release and to overcome the problems of conventional method of administration of letrozole. In the present work through ionic cosslinking method a novel drug carrier of letrozole was synthesized which overcomes the drawbacks of low loading efficiency, higher particle size etc. The formulations were characterized for its formation and tested for effective in vitro drug release, biocompatibility, stability and blood compatibility. Chitosan nanoparticles were used as a carrier for letrozole and sodium tripolypohosphate was used as a crosslinker. Materials and methods Materials Chitosan (CS) with the degree of deacetylation 92% was procured from India Sea foods, Cochin, Kerala. Letrozole was synthesized as per the procedure given in US patent 7705159B2. Sodium tripolyphosphate (TPP) was purchased from finer chemicals and used without further purification. All other chemicals and reagents used are analytical grade. Preparation of durg unloaded and loaded chitosan nanoparticles 50mg of chitosan was dispersed in 5 ml of aqueous acetic acid (2% v/v) solution and continuously stirred for about 20 minutes at 600 rpm to obtain the homogeneous solution. 5 ml (0.8% w/v) of TPP solution was used as a crosslinker [22, 23]. The resulting chitosan nanoparticle suspension was subsequently centrifuged for about 45 minutes at 12,000 rpm and re-suspended in water for washing followed by drying. Similarly for preparing letrozole loaded chitosan nanoparticles, the drug letrozole (5, 10 and 15 mg in 0.5 ml acetic acid) solution was added dropwise to the chitosan solution, under continuous stirring up to 30 minutes. TPP solution (38.00 mg in 5 ml deionised water) was added dropwise to the chitosan/ Letrozole
4
solution over a period of 60 minutes at a stirring speed in the range of 550- 600 rpm (Scheme 1). After the complete addition, the suspension was centrifuged at 12,000 rpm. The supernatant solution was subjected to determine the loading efficiency. The filtered solid was slurried in water and centrifuged, the centrifuged material was kept for drying. Yield (% w/w) Yield (% w/w) was calculated as weight of the dried nanoparticles recovered from each batch divided by the sum of the initial dry weight of the starting materials multiplied by a hundred, i.e. %
50
38
100
Characterization of Nanoparticles FTIR spectroscopy was measured for the determination of the types of bonds present in the nanoparticulate. FT-IR spectra of CS – NPs, LTZ and LTZ – CS – NPs were carried out using KBr tablets (1% w/w of product in KBr) with a resolution of 4 cm-1 and 100 scans per sample on a Thermo Nicolet AVATAR 330 spectrophotometer. Thermogravimetric analysis was conducted to measure the thermal weight loss of the samples on a TGA Q500 V20.10 Build 36 instruments at a heating rate of 20°C per minute in nitrogen atmosphere. The weight losses at different stages were analysed. The scanning electron microscopy analysis (SEM, Leica, Cambridge, UK) was conducted to observe the size, shape and surface morphology of the nanoparticles. For the analysis, the samples are wiped with a thin gold – palladium layer by a sputter coater unit (VG – microtech, UCK field, UK), the shape and morphology were analysed with a Cambridge stereoscan 440 scanning electron microscope (SEM, Leica, Cambridge, UK). The transmission electron microscopy analysis (TEM) was conducted to observe the size, shape 5
and surface morphology of the loaded and unloaded chitosan nanoparticles. The shape and morphology were analysed with a HITACHI-H-7650 transmission electron microscope. The particle size and size distribution of the nanoparticles were measured by Dynamic light scattering method (DLS, Zetasizer Nano-S, Malvern, England). Suitable amount of the dried nanoparticles from each formulation was suspended in deionised water and was sonicated for a suitable time period before the measurement. The volume mean diameter, size distribution and polydispersity of the resulting homogeneous suspension were determined using DLS technique. X-ray diffraction (XRD) patterns were obtained using an X-ray diffractometer (XRD-SHIMADZU XD-D1). Letrozole loading measurements The quantity of letrozole (LTZ) entrapped in the nanoparticulate system was determined indirectly, by measuring the quantity of letrozole remaining in the supernatant based on the absorbance of the samples at 250 nm [24]. The standard curve was obtained and the sample absorbance was measured in 3 ml quartz cuvettes using Shimadzu UV-1700 Pharma spec UVVisible spectrophotometer (Suzhow, Jiangsu, China). The measurement for quantifying the amount of drug loaded in the nanoparticles is drug entrapment efficiency (EE). Determining the EE allows for the optimization of the amount added, reducing wastage, and is defined as follows:
∗ 100
Entrapment efficiency describes the quantity of the drug entrapped within the nanoparticle as it is related to the initial drug loading. 100% EE means that the entire drug quantity added has been incorporated into the nanoparticle. In vitro drug release studies
6
In vitro drug release profiles were done by direct dispersion method as explained in literature [25,26]. In vitro drug release studies were done for a period of one week at pH 7.4. 10 mg of drug loaded CS – NPs were taken in 50 mL of 10 mM phosphate buffer solution (PBS) in a beaker under magnetic stirring at 100 rpm. The receptor phase was stirred and thermally controlled at 37 ºC. The base absorbance of the release media was accounted by using the release medium as the solution in the UV-spectrophotometer reference cell, as well as the solution for zeroing the system. At fixed time intervals, 3 ml of the receptor phase was withdrawn, centrifuged to collect the supernatant and then substituted with fresh buffer. The drug release was assayed spectrophotometrically. The cumulative percentage amount of drug release was calculated and plotted against time. Kinetics studies To analyze the in vitro release data various kinetic models were used to describe the release kinetics. The zero order rate equation describes the systems where the drug release rate is independent of its concentration [27].
where Qo and Qt are the initial amount of drug and cumulative amount of drug released at time t. Ko is the zero order rate constant. The first order kinetics describes the release from system, where release rate is concentration dependent [28]. log
log
where k1 is the first order rate constant. Higuchi [29], described the release of drugs from insoluble matrix as a square root of time dependent process based on Fickian diffusion.
7
where kH is the Higuchi constant. The Hixson-Crowell [30], cube root law describes the release from systems where there is a change in surface area and diameter of particles or tablets.
where kHC is the Hixson-Crowell constant. Korsmeyer et al. [31] derived a simple relationship which described drug release from a polymeric system. where kKP is the Korsmeyer-Peppas constant and n is the release exponent describing the drug release mechanism. Modeling was performed using the parameters that provide the closest fit between experimental observations and the nonlinear function. The model that best fits the release data was selected based on the correlation coefficient (R2) in models described above. The model, which gives highest R2 value, is considered as the best fit of release data.
In vitro biodegradation behavior The rate of degradation of the prepared samples was studied according to the previous method [32]. The CS-NPs and LTZ-CS-NPs with TPP were weighed equally, and the initial weight was recorded as Wo, followed by immersion in 1xPBS containing lysozyme (10,000 µg/L) and incubated at 37 °C at different intervals (24, 48 and 72 h). After completion of the incubation period, the samples were washed with deionized water to remove ions and blot dried 8
with filter paper. The dry weights of the samples were noted as (Wt). The degradation was calculated by using the following formula: %
100
Hemolysis assay Blood compatibility was evaluated with hemolysis assay. Fresh human blood was used in this study. 1.5 mL acid citrate dextrose (ACD) was added to 10 mL fresh blood. To 1 mL of the blood sample, 100 µL of sample of concentration ranging from 0.01 to 1 mg/mL was added. The whole samples were incubated for 2 h with shaking in an incubator chamber at 37 °C. The samples were spin down at 6000 rpm for 15 min to obtain the plasma (plasma would be red in color if hemolysis happened). The plasma portion was collected [100 µL plasma+1 mL Na2CO3 (0.01%)]. The OD values were read at 450, 380 and 415 nm. The obtained OD values of the samples were compared with that of controls. To obtain 100% hemolysis, cells were lysed by dispersing in distilled water (positive control) and the negative control was obtained by treating blood with DMSO. Each assay was evaluated in triplicate. Stability studies To assess the colloidal stability of unloaded and drug loaded CS-NPs, Human Serum (HS) was first incubated at 37°C in a water bath incubator to simulate physiological media. HS was then added in the nanoparticles dispersion with the volume ratio of 1:1 in which the final concentration of 50% v/v of serum was yielded. Subsequently, the resulting mixture was incubated in the water bath incubator at 37 °C for 24 h. The mean particle size was measured at predetermined time points (0, 1, 6, 12 and 24 h). The nanoparticles were suspended in PBS (pH, 7.4) prior to mixing with HS. Statistical analysis 9
Statistical analysis was performed using origin software (origin pro8) for descriptive statistics (mean, standard deviation, 95% confidence interval, linear fit and so on). Statistical analysis was performed for different formulae by applying one-way analysis of variance ANOVA test. 2-sample t-tests were used to test the differences in mean values. A ρ-value of <0.05 was considered statistically significant. Result and Discussion Preparation of CS-NPs and LTZ-CS-NPs Chitosan nanoparticles were fabricated instantaneously at room temperature through ionic gelation method while mixing the homogenuous solution of chitosan and the cross linker TPP [33-36]. The amino groups in chitosan which are responsible for many of its advanced functions, including biological activity and cationic polymer properties [37], interacts with polyanions to form nanoparticles through inter- and intramolecular crosslinkages [38,39] which involves the addition of alkaline phase and acidic phase. The alkaline phase is polyanions dissolved in water and the acidic phase is chitosan dissolved in dilute acidic acid. Upon mixing, these two phases forms inter- and intramolecular linkages between TPP and NH2 group of chitosan. The development of appropriate vehicles to deliver the drug molecules is a meaningful challenge for pharmaceutical scientist. Here the chitosan nanoparticles paved a way for these challenges for delivering the aromatase inhibitor – letrozole. Chitosan molecules may likely adopt a spread conformation in solution because of electrostatic repulsion force existing between amine groups along the molecular chain. To this solution, when letrozole was added under constant stirring, the hetero atoms present in letrozole, involved in hydrogen bonding with protonated chitosan chain with certain sites at the spread chitosan chain to achieve high
10
encapsulation. Chitosan is polycationic in acidic media (pKa 6.5) and when the crosslinking agents TPP is added to the chitosan-drug mixture it interacts with the negatively charged TPP, leading to the formation of chitosan nanoparticles. The crosslinking agents contain both –OH and phosphoric ions with compete with each other to interact with the –NH3+ sites of chitosan. The mechanism of interaction in the formulation is given in the Figure 1. The formation of nanoparticles depends dramatically on the concentration of free amino groups, which strengthens the electrostatic interactions between the nanoparticles and the drug, helping to reduce the particle sizes [40,41]. Table 1 showed that the particle size of the nano formulation has been to be dependent on the drug load. As the drug load increases the particle size increases [42]. The particle size is also depends upon the drying method (Table 2). The result confirms that the particles size is drastically decreased on freeze drying process than air drying process. This was due to the fact that the adsorbed and bound moisture will went off during freeze drying process. The freezing effect, could prevent the nano-particles from agglomerating efficiently, so the samples are fluffy, the average particle sizes are small and the particle size distributions are narrow when compared to air drying method. FTIR studies of the formulation In this section, the formation of chitosan nanoparticles and letrozole loaded chitosan nanoparticles crosslinked with TPP in air drying method and freeze drying method were analyzed (Figure 2a – 2d) through FTIR spectroscopy. The FTIR spectrum of chitosan (CS) (spectrum not given) shows the band at 3425.58 cm-1 indicates the O-H stretching and N-H 11
stretching vibration. Bands at 2918.30 and 2881.65 cm-1 corresponds to anti-symmetric C-H stretching and symmetric C-H stretching modes of vibration. The band at 1658.78 cm-1 is for the amide I band (C=O stretching), since chitosan used here was 92% deacetylated. The presence of amide groups were confirmed through amide I (1658.78 cm-1), amide II (1577.78 cm-1) and amide III bands (1323.17 cm-1) [43,44]. Figure 2a and 2b shows the FTIR spectrum of CS-NPs-TPP-Air dried and Freeze dried sample. The results were consistent with previously published data [45]. A broad band at 3425.28 cm-1 was assigned to a combination of stretching modes of O-H and N-H bonds in chitosan matrix. In the sample of chitosan nanoparticles this band becomes wider and shifts to higher wavenumber, 3448.1 cm-1 in case of air dried sample and to lower wavenumber 3421.88 cm-1 for freeze dried sample, indicating the presence of strong electrostatic interactions and an enhancement of the hydrogen bond interactions [38, 47, 48]. The band for C=O of CS-NPs air dried and freeze dried samples was shifted to lower wavenumber of 1653 and 1656 cm-1 from 1658 cm-1 during nanoparticles formation, indicating that C=O groups also participating in the nonbonding interaction. In addition, the 1577.78 cm−1 band of the NH2 bending vibration of chitosan samples shifted to 1545.6 and 1562.5 cm−1 in the NPs. A similar result has been observed in literature on chitosan-TPP NPs [38] and on chitosan films treated with phosphate (NaH2PO4); the shift was attributed to the interaction between the amino group and the phosphate anion [49]. The other peaks observed in the sample of air dried and freeze dried nanoparticles were those of the P=O stretching at 1280.5 and 1234.38 cm−1 [35] and P-O bending at 1036 and 1051.62 cm−1 of the TPP [50]. Thus, the shift in the peak of OH, NH2 and C=O functional groups and also the
12
appearance of a new peak for P=O and P-O when compared with the fingerprint pure chitosan, confirms the formation of chitosan nanoparticles through ionotropic gelation method. LTZ alone (spectrum not given) showed major peaks at 2232.6 cm-1 for C≡N stretching, 3054.27 cm-1 for sp2 hybridized CH stretching, 690-900 cm-1 for out-of plane CH deformation modes of vibration. The FTIR spectrum of letrozole loaded chitosan nanoparticles formulation (Figure 2c and 2d) showed that no significant differences on shape and position of the absorption bands for the formulation. Most of the absorption peaks from pure LTZ and bare chitosan nanoparticles overlapped with the absorption peaks from LTZ-CS-NPs. It can be concluded that no strong covalent interaction occurred between drug and polymer. For LTZ-CSNPs new bands for C≡N group at 2377 and 2230.27 cm-1 for LTZ-CS-NPs-TPP-Air dried and LTZ-CS-NPs-TPP-Freeze dried samples, this confirms the incorporation of LTZ in chitosan nanoformulation. Also the LTZ incorporated CS-NPs showed intense band at 1657 and 1636.30 cm-1 corresponding to the hetero aromatic C=N stretching and phenyl C=C stretching vibrations in the higher wavenumber region due to the overlap with C=O stretching modes and appears to be broad. The asymmetric C=O stretching (amide I band) was observed at the lower wavenumber for LTZ-CS-NPs-TPP on compared with its respective bare chitosan nanoparticles indicating the involvement of C=O group in the electrostactic interaction as well as hydrogen bond interaction [51]. The bands in the region 1395 – 1415 cm-1, 1380 cm-1, 1247 – 1306 cm-1, 1154 – 1166 cm-1, 1076 – 1100 cm-1 and 1018 – 1030 cm-1 508 – 523 cm-1 are assigned to C-H and O-H deformation, C-N stretching modes, P=O stretching modes, C-O stretching and C-N stretching [26, 52, 53], C-O-C skeletal linkage and P-O stretching modes of vibrations respectively. The actual positions of these bands were comparatively shifted to the lower region while loading
13
letrozole, confirming the participation of these groups in non-covalent interactions. Thus, the shift in the peak of OH, NH2 and C=O functional groups and also the appearance of a new peak for =C-H, C=C and C=N when compared with the fingerprint bare chitosan nanoparticles, confirms the formation of LTZ loaded chitosan nanoparticles confirming the drug is intact and has not reacted with the excipient used in the formulation and hence they are compatible. TGA studies The change on the thermal degradation of chitosan showed the participation of functional groups of chitosan in modification [54]. The thermal stability and thermal decomposition of CSNPs-TPP and LTZ-CS-NPs-TPP Air dried and Freeze dried samples were investigated by TG and are given in Figure 3. The results showed the similar trend of thermal decomposition. Figure 3a and 3b shows the thermal behavior of CS-NPs-TPP-Air dried and freeze dried samples. Here three stages of decomposition were seen. The first stage of degradation is due to the elimination of water. Within 100 °C, 7.81 % and 12.69 % of CS-NPs-TPP-Air dried and freeze dried samples were dehydrated [55,56], confirming that substantially more water compared to chitosan which can be attributed to the higher hydrophilicity of chitosan nanoparticles. The second stage of degradation occurs in the range of 200 to 390 °C and 190 °C to 450 °C in case of CS-NPs-TPP-Air dried and freeze dried samples, corresponds to the cleavage of glycosidic linkages via dehydration and deamination [43,57-59]. The third stage was due to the degradation of polysaccharides backbone. The residue remained for air dried sample was more than freeze dried sample. For air dried 48 % and 29.14% of freeze dried sample remained as the residue at the end of the experiment 880 °C. This confirms the change in thermal
14
behavior [60] and this anomaly could be related with crystalline and/or morphological variations in nanoparticles with respect to those of the pure chitosan [61]. The degradation profile of letrozole loaded air dried CS-NPs-TPP and freeze dried CSNPs-TPP (Figure 3c and 3d), showed a similar decomposition stages as seen in the bare chitosan nanoparticles. Around 30 – 180 °C the first stage, 200 – 350 °C the second stage and the third stage starts from 350 °C. Comparatively the high residue is present for air dried samples than freeze dried samples. The 10 % degradation is higher for LTZ-CS-NPs-TPP Air dried sample than the Freeze dried sample. 50% decomposition temperature of air dried CS-NPs-TPP samples is >870 °C and freeze dried CS-NPs-TPP is 520.15 °C. Therefore the decreasing trend of percentage decomposition is clearly depicted in the TGA of drug loaded chitosan nanoparticles compared with bare chitosan nanoparticles. This change is thermal behavior is owing to the formation of new interaction which would have formed between the drug, polymer and crosslinker. XRD The X-ray diffraction pattern of letrozole, chitosan nanoparticles and letrozole loaded chitosan nanoparticles nanoparticles prepared with TPP crosslinker in air drying and freeze drying method is presented in Table 3 and Figure 4a – 4e. The pure LTZ exhibits a strong characteristic peak at 2θ = 11.2°, 13.12°, 14.16°, 16.24°, 17.16°, 19.72°, 21°, 21.44°, 22°, 23.16°, 25.08°, 25.52°, 26.8°, indicating its crystalline structure. The number of peaks confirms the different crystalline forms present in letrozole. For LTZ loaded chitosan nanoparticles, only a broad peak with a shoulder is observed, corresponding to the amorphous nature. This is due the interaction present between LTZ with CS and TPP, which acts as the main driving force for LTZ loading can be ascribed to intermolecular
15
hydrogen bonding [62,63]. Hydrogen bonding is formed based on the complexes between the Nitrogen atoms in LTZ and H atoms of NH2, NH3+, OH and P=O groups in CS-NPs. For all the LTZ-loaded CS-NPs, the X-ray diffraction pattern shows the presence of the typical peaks for both LTZ and CS-NPs, which indicates that the crystal state of LTZ in CS-NPs remains the same. While the position of the peaks shifts, and the intensity of these peaks is evidently reduced, revealing a certain degree of interaction between LTZ and CS-NPs. This indicates that the conjugated and entrapped forms of the drugs are unable to form their own crystal lattice inside the polymer and they are in amorphous state. These evidences lead to the conclusion that the drugs are distributed over the nanostructure which satisfies the essential requirement of a good drug delivery system (DDS). SEM The SEM microphotography of the letrozole loaded chitosan nanoparticles prepared with TPP crosslinker under air drying and freeze drying method of ratio 1:0.8:0.1 are shown in Figure 5a – 5d. SEM images shows that the loaded nanoparticles have a rough morphology after loading. The image shows the aggregation of particles during drying process. Also the image reveals that the drug gets encapsulation and some of the drug molecules are adhered on the surface. Compared with the air dried formulation the freeze dried formulations have well established porosity, which is depicted in the micrography. This porosity is due to the amorphous nature of the prepared formulation, which proved that the nanoparticles are good candidates for sustained drug delivery systems, with their sizes in nano range required for its parentral chemotherapy. TEM 16
The surface morphology and particle size of the prepared letrozole formulations were studied using TEM analysis. The images (Figure 6a – 6d) depicted that the prepared formulations are in the nano size range. For LTZ-CS-TPP air dried and freeze dried samples the particles are found in the range of 60 - 80 nm and 20 – 40 nm respectively. These measurements are supported by DLS studies which will give the exact size distribution of the nanoparticles. TEM images of letrozole loaded chitosan nano formulations also confirms the particles aggregation which was probably due to drying process. Entrapment efficiency of chitosan nanoparticles The entrapment efficiency of chitosan nanoparticles during loading of letrozole was determined through indirect measurements using UV-spectrophotomer in the absorbance at 240 nm. The letrozole formulations which were prepared by varying drug concentration and drying method are studied for the effective loading. While increasing the concentration of the drug from 5 mg to 15 mg (0.1 to 0.3 ratio) the entrapment efficiency measurement showed a slight decrease with respect to the concentration. The encapsulation efficiency depends upon the number of available sites present in the formulating material. About 98.4 % of entrapment efficiency was found for LTZ-CS-NPs-TPP (1:0.8:0.1) ratio of air dried and freeze dried samples. Whereas 97.8% and 97.1% of drug is encapsulated in (1:0.8:0.2) and (1:0.8:0.3) ratio of air dried samples respectively. Biodegradation studies Biodegradation study was performed on LTZ loaded air dried formulations of ratio 1:0.8:0.1, 1:0.8:0.2 and 1:0.8:0.3 with TPP crosslinker. The incorporation of LTZ into the chitosan nanoparticles had improved the biodegradability. It was seen that the nano formulation
17
improved the biodegradability to a great extent in the presence of lysozyme when compared with bare chitosan nanoparticles. The results were shown in Figure 7. In vitro drug release studies A drug release mechanism of nanoparticles has been well described in the literature as (i) desorption, (ii) diffusion, and (iii) matrix degradation [64]. The release of the drug was found to be dependent on the amount of the drug and its formulation. Drug release was found to be inversely related to the amount of release retardant and directly proportional to amount of swellable nano polymer matrix. The release was studied at pH 7.4 to observe the parentral mode of administration, which is a physiological pH of the body. The cumulative release profile of the letrozole from the matrix is shown in the Figure 8. The release profile shows the brust release initially, which is due to the release of adhered drug molecules on the surface of the excipient, followed by the slow release from the interior of the excipient. The drug release profile showed an initial burst release of 28.28 %, 18.04 %, 29.29 % and 33.63 % for polymer-crosslinker-drug ratio of (1:0.8:0.1)-TPP-Air dried, (1:0.8:0.1)-TPP-Freeze dried, (1:0.8:0.2)-TPP-Air dried and (1:0.8:0.3)-TPP-Air dried samples respectively followed by a slow and increased release of letrozole. The slow release of letrozole is due to the diffusion process (Figure 8). In this process, the water molecules first penetrated into the matrix and convert the matrix into the swollen one. Here the polymer will be in the rubbery state. After this the matrix tends to degraded. The release profile is controlled by the process called erosion. Through all the three process the sustained drug release were taken place from the nanoparticulate. From the release data, it was seen that
18
the maximum of 58.81 %, 42.01 %, 63.65 %,and 74.27 % of the drug was released within in 70 h in the respective formulations. On comparing the air dried and freeze dried formulations of ratio 1:0.8:0.1, the slow release was noticed in case of freeze dried samples. Also on increasing the concentration of letrozole from 5 mg to 15 mg in the nano formulation, the initial burst release increases accordingly, confirming that more amount of the drug was adhered on the surface of the formulation. The mathematical models were used in this study to determine the release mechanism of letrozole loaded chitosan nano formulations. The results of the mathematical models are very important in the optimization of the suitable formulation for letrozole. Table 4 enlists the regression parameters obtained after fitting various release kinetic models to the in vitro drug release data. All the kinetic models are fitted with the release data with respect to earlier time periods. Since the extent of drug release at given time points depends on the interaction and varied largely between inter- and intra-drug-carrier dispersions. The goodness of fit for various models investigated for binary systems ranked in the order of HixsonCrowell > first-order > zero-order > Higuchi > Korsemeyer-Peppas. The most fitted model is Hixson-Crowell followed by first order kinetics model. On analysing the values of diffusional exponent n of Korsemeyer–Peppas model, the values are lesser than 0.45 and ranged between 0.09 and 0.15, which depicts that the drug release exhibit Fickian diffusion release. Blood compatibility studies
19
Blood compatibility is a significant parameter for the biological safety of the formulation. This is of great significance when the material is intended for injections. Thus in this present work the blood compatibility study was done for the prepared formulations and the results are depicted in the Figure 9. Hemolysis assay indicates that the prepared formulations are heme compatibility. As per the results, the hemolytic ratio of the sample was 2.4 %, much below 5% which is the safe haemolytic ratio for bio-materials in accordance with ISO/TR 7406. It was evident from the results that the nanoparticles are free from the risk of hemolysis. Serum stability The stability of the letrozole formulation was carried out in Human Serum with the volume ratio of 1:1 in the time intervals of 0, 1, 6, 12 and 24 h. After intravenous administration, the size and charge of nanoparticles are mainly determined by the adsorbed serum components [65]. The obtained data revealed that an increment in the particle size was occurred during incubation in HS (Figure 10). All the formulations were to be found stable in HS up to 24 h. Comparatively the particle size increases for air dried samples than the freeze dried one. Despite the fact that there is no drastic increase in the particle sizes of the formulation, therefore more biocompatibility. Conclusion The aromatase inhibitor Letrozole was synthesized and formulated using chitosan nanoparticles with TPP crosslinker through simple ionic gelation method and characterized by FTIR, TGA, SEM, TEM and DLS. The drug encapsulation was confirmed by FTIR. The change in thermal behavior was well exhibited in the TGA results. TEM results confirmed that the prepared LTZ-CS-NPs were nano size range. The size distribution of LTZ-CS-NPs was
20
supported by DLS study. The particles are in the size range of 20 – 80 nm. The in vitro drug release study showed that letrozole is released in a controlled manner from the nano formulation in the physiological pH 7.4. From the release data it was confirmed that it can also be administered parenterally. The biodegradability assay and serum stability studies prove that the prepared formulations were biodegradable and stable. The hemolysis analysis discloses the blood compatibility of the formulations. The in vitro evaluations of the letrozole proved the successful loading and sustained release profile of chitosan nanoparticles, which satisfies the need of cancer chemotherapy. References [1]
I.E. Smith, Letrozole versus tamoxifen in the treatment of advanced breast cancer and as neoadjuvant therapy. J. Steroid Biochem. Mol. Biol. 86 (2003) 289–295.
[2]
C. De Santis, R. Siegel, P. Bandi, A. Jemal, Breast cancer statistics, 2011. CA: A Cancer Journal for Clinicians, 61 (2011) 408–418.
[3]
A.A.Van Landeghem, J. Poortman, M. Nabuurs, J.H. Thijssen, Endogenous concentration and subcellular distribution of androgens in normal and malignant human breast tissue. Cancer Res., 45 (1985) 2907-2912.
[4]
W. Yue, J.P. Wang, C.J. Hamilton, L.M. Demers, R.J. Santen, In situ aromatization enhances breast tumor estradiol levels and cellular proliferation, Cancer Res. 58 (1998) 927-932.
[5]
H.M. Lamb, J.C. Adkins, Letrozole. A review of its use in postmenopausal women with advanced breast cancer, Drugs. 56 (1998) 1125-1140.
21
[6]
G.F. Meresman, M. Bilotas, V. Abello, Effects of aromatase inhibitors on proliferation and apoptosis in eutopic endometrial cell cultures from patients with endometriosis. Fertil. Steril., 84 (2005) 459-463.
[7]
L.L. Amsterdam, W. Gentry, S. Jobanputra, Anastrazole and oral contraceptives: a novel treatment for endometriosis, Fertil. Steril. 84 (2005) 300-304.
[8]
H.M. Fatemi, H.A. Turki, E.G. Papanikolaou, Successful treatment of an aggressive recurrent post-menopausal endometriosis with an aromatase inhibitor, Reprod. Biomed. 11 (2005) 455-457.
[9]
M.R. Saboktakin, R.M. Tabatabaie, A. Maharramov, M.A. Ramazanov, Synthesis and in vitro studies of biodegradable thiolated chitosan hydrogels for breast cancer therapy, Int. J. Biol. Macromol. 48 (2011) 747-752.
[10] N. Mondal, T.K. Pal, S.K. Ghosal, Development, physical characterization, micromeritics and in vitro release kinetics of letrozole loaded biodegradable nanoparticles, Pharmazie 63 (2008) 361-365. [11] J. L. Scott, J.S. Keam, Letrozole : in postmenopausal hormone-responsive early-stage breast cancer, Drugs 66 (2006) 353. [12] P. Furet, C. Batzl, A. Bhatnagar, E. Francotte, G. Rihs, M. Lang, Aromatase inhibitors: synthesis, biological activity, and binding mode of azole-type compounds, J. Med. Chem., 36 (1993) 1393–1400. [13] M. Lang, C. Batzl, P. Furet, R. Bowman, A. Hausler, A.S. Bhatnagar, Structure-activity relationships and binding model of novel aromatase inhibitors, J. Steroid Biochem. Mol. Biol. 44 (1993) 421–428.
22
[14] I. Brigger, C. Dubernet, P. Couvreur, Nanoparticles in cancer therapy and diagnosis. Adv Drug Del Rev. 54 (2002) 631–51. [15] C. Vauthier, C. Dubernet, C. Chauvierre, I. Brigger, P. Couvreur, Drug delivery to resistant tumors: the potential of poly(alkyl cyanoacrylate) nanoparticles, J Controlled Release. 93 (2003) 151–160. [16] K. Kataoka, A. Harada, Y. Nagasaki, Block copolymer micelles for drug delivery Design, characterization and biological significance, Advance Drug Delivery Review 47 (2001) 113-131. [17] S.H. Kim, J.P. Tan, F. Nederberg, K. Fukushima, J. Colson, C. Yang, A. Nelson, Y.Y. Yang, J.L. Hedrick, Hydrogen bonding-enhanced micelle assemblies for drug delivery, Biomaterials, 31 (2010) 8063-71. [18]
A.G. Cuenca, H. Jiang, S.N. Hochwald, M. Delano, W.G. Cance, S.R. Grobmyer, Emerging implications of nanotechnology on cancer diagnostics and therapeutics. Cancer, 107 (2006) 459-66.
[19] R.L. Manthe, S.P. Foy, N. Krishnamurthy, B. Sharma, V. Labhasetwar, Tumor ablation and nanotechnology. Mol Pharm, 7 (2010) 1880-98. [20] A.Z. Wang, R.S. Langer, O.C. Farokhzad, Nanoparticle delivery of cancer drugs. Annu Rev Med 2011. [21] P. Sharma, A. Singh, S.C. Brown, N. Bengtsson, G.A. Walter, S.R. Grobmyer, N. Iwakuma, S. Santra, E.W. Scott, B.M. Moudgil, Multimodal nanoparticulate bioimaging contrast agents. Methods Mol Biol. 624(2010) 67-81. [22] P. Calvo, C. Remunan, J.J.L. Vila, M.J. Alonso, Novel hydrophilic chitosan-polyethylene oxide nanoparticles as protein carriers. Journal ofApplied Polymer Science, 63(1997) 125–132.
23
[23] M.
Prabaharan,
R.L.
Reis,
J.F.
Mano,
Carboxymethyl
chitosan-graft-
phosphotidylethanolamine: amphiphilic matrices for controlled drug delivery.”- Reactive and Functional Polymers, 67 (2007) 43 – 52. [24] F.Albertoni, B. Pettersson, V. Reichelova, G. Juliusson, J. Liliemark, Analysis of 2Chloro-2'-Deoxyadenosine in Human Blood Plasma and Urine by High-Performance Liquid Chromatography Using Solid-Phase Extraction. Therapeutic Drug Monitoring, 16 (1994) 413-418. [25] S. Bisht, G. Feldman, S. Soni, R. Ravi, C. Karikar, A. Maitra, A. Maitra, Polymeric nanoparticle-encapsulated curcumin (“nanocurcumin") a novel strategy for human cancer therapy. Journal of Nanobiotechnology, 5 (2007) 1–18. [26] A. Anitha, S. Maya, N. Deepa, K.P. Chennazhi, S.V. Nair, R. Jayakumar, Curcuminloaded N,O-carboxymethyl chitosan nanoparticles for cancer drug delivery. Journal of Biomaterials Science, 23(11) (2012) 1381-1400. [27] T.P. Hadjiioannou, G.D. Christian, M.A. Koupparis, P.E.
Macheras, Quantitative
Calculations in Pharmaceutical Practice and Research, VCH Publishers Inc., New York, (1993) 345-348. [28] D.W.A. Bourne, Pharmacokinetics. In: Banker GS, Rhodes CT, Modern Pharmaceutics, 4th ed., Marcel Dekker Inc., New York, (2002) 67-92. [29] T.J. Higuchi, Mechanism of sustained medication. Theoretical analysis of rate of release of solid drugs dispersed in solid matrices., Pharm Sci.,84 (1963) 1464-1477. [30] A.W. Hixson, J.H. Crowell, Dependence of reaction velocity upon surface and agitation (I) theoretical consideration. Ind. Eng. Chem., 23 (1931) 923-931.
24
[31] R.W. Korsmeyer, R. Gurny, E. Doelker, P. Buri, N.A. Peppas, Mechanisms of solute release from porous hydrophilic polymers. Int. J. Pharm., 15 (1983) 25-35. [32] J. Venkatesan, I. Bhatnagar, S.K. Kim, Chitosan-Alginate Biocomposite Containing Fucoidan for Bone Tissue Engineering Chitosan-Alginate Biocomposite Containing Fucoidan for Bone Tissue Engineering. Mar. Drugs, 12 (2014) 300-316. [33] Q. Gan, T. Wang, Chitosan nanoparticle as protein delivery carrier-Systematic examination of fabrication conditions for efficient loading and release. Colloids and Surfaces B:Biointerfaces, 59 (2007) 24-34. [34] Q. Gan, T. Wang, C. Cochrane, P. McCarron, Modulation of surface charge, particle size and morphological properties of chitosan-TPP nanoparticles intended for gene delivery, Colloids and Surfaces B, 44 (2005) 65–73. [35] L. Qi, Z. Xu, X. Jiang, C. Hu, X. Zou, Preparation and antibacterial activity of chitosan nanoparticles. Carbohydrate Research, 339(16) (2004) 2693–2700. [36] M. Prabaharan, and J.F. Mano, Hydroxypropyl chitosan bearing β-cyclodextrin cavities: synthesis and slow release of its inclusion complex with a model hydrophobic drug”. – Macromolecular Bioscience, 5 (2005) 965 – 973. [37] Y.J. Liu, Y. Jiang, Y.F. Feng, D.L. Han, Study on the chitosan hydrolysis catalyzed by special cellulase and preparation of chitooligosaccharide. J. Func. Polym., 18 (2005) 325– 329. [38] Y. Xu, Y. Du, Effect of molecular structure of chitosan on protein delivery properties of chitosan nanoparticles. International Journal of Pharmaceutics, 250(1) (2003) 215–226.
25
[39] A. Vila, A. Sánchez, K. Janes, I. Behrens, T. Kissel, J.L. Vila Jato, M.J. Alonso, Low molecular weight chitosan nanoparticles as new carriers for nasal vaccine delivery in mice. Eur J Pharm Biopharm, 57(1) (2004) 123-131. [40] H.C. Yang, M.H. Hon, The effect of the molecular weight of chitosan nanoparticles and its application on drug delivery. Microchemical Journal, 92(1) (2009) 87–91. [41] K.A. Janes, M.P. Fresneau, A. Marazuela, A. Fabra, M.J. Alonso, Chitosan nanoparticles as delivery systems for doxorubicin. Journal of Controlled Release, 73(2-3) (2001) 255– 267. [42] D.D. Ankola, B. Viswanad, V. Bhardwaj, P.R. Rao, M.N.V. Kumar, Development of potent oral nanoparticulate formulation of coenzyme Q10 for treatment of hypertension: can the simple nutritional supplements be used as first line therapeutic agents for prophylaxis/therapy? Eur. J. Pharm. Biopharm., 67 (2007) 361– 369. [43] A. Pawlak, M. Mucha, Thermogravimetric and FTIR studies of chitosan blends. Thermochimica Acta, 396 (2003) 153-166. [44] Y. Dong, C. Xu, J. Wang, Y. Wu, M. Wang, Y. Ruan, Influence of degree of deacetylation on critical concentration of chitosan/dichlorocatic acid liquid crystalline solution. Journal of Applied Polymer Science, 83(6) (2002) 1204–1208. [45] C. Qin, H. Li, Q. Xiao, Y. Liu, J. Zhu, Y. Du, Water solubility of chitosan and its antimicrobial activity. Carbohydrate Polymers, 63(3) (2006) 367–374. [46] A.R. Dudhani, S.L. Kosaraju, Bioadhesive chitosan nanoparticles: preparationand characterization. Carbohydr. Polym., 81 (2010) 243–251.
26
[47] Y. Wu, W. Yang, C. Wang, J. Hu, S. Fu, Chitosan nanoparticles as a novel delivery system for ammonium glycyrrhizinate. International Journal of Pharmaceutics, 295(1–2) (2005) 235–245. [48] Y. Jia-hui, D. Yu-min, Z. Hua,
Blend films of chitosan–gelatin. Journal of Wuhan
University, 45 (1999) 440–444. [49] J.Z. Knaul, S.M. Hudson, K.A.M. Creber, Improved mechanical properties of chitosan fibers. J. Appl. Polym. Sci., 72 (1999) 1721–1731. [50] S. Vimal, S. Abdul Majeed, G. Taju, Nambi, K.S.N. Sundar Raj, N. Madan, M.A. Farook, T. Rajkumar, D. Gopinath, A.S. Sahul Hameed, Chitosan tripolyphosphate (CS/TPP) nanoparticles: Preparation,characterization and application for gene delivery in shrimp. Acta Tropica, 128 (2013) 486– 493. [51] N.S. Rejinold, K.P. Chennazhi, S.V. Nair, H. Tamura, R. Jayakumar, Biodegradable and thermo-sensitive chitosan-g-poly (N-vinylcaprolactam) nanoparticles as a 5-fluorouracil carrier. Carbohydr. Polym., 83 (2011a) 76–786. [52] N.S. Rejinold, M. Muthunarayanan, K.P. Chennazhi, S.V. Nair, R. Jayakumar,5Fluorouracil loaded fibrinogen nanoparticles for cancer drug delivery applications. International Journal of Biological Macromolecules, 48(1) (2011b) 98–105. [53] E.B. Yassin, M.K. Anwer, H.A. Mowafy, I.M. Elbagory, M.A. Bayomi, I.A. Alsarra, Optimization of 5-fluorouracil solid-lipid nanoparticles: a preliminary study to treat colon cancer. Int. J. Med. Sci., 7 (2010) 398–408 [54] H.K. Holme, H. Foros, H. Pettersen, M. Dornish, O. Smidsrod, Thermal depolymerization of chitosan chloride. Carbohydrate polym., 46(3) (2001) 287-294
27
[55] A.P. Martínez-Camacho, M.O. Cortez-Rocha, J.M. Ezquerra-Brauer, A.Z. GracianoVerdugo, F. Rodriguez-Félix, M.M. Castillo-Ortega, M.S. Yépiz-Gómez, M. PlascenciaJatomea, Chitosan composite films: Thermal, structural, mechanical and antifungal properties. Carbohydr. Polym., 82 (2010) 305–315. [56] A. Casariego, B.W.S. Souza, M.A. Cerqueira, J.A. Teixeira, L. Cruz, R. Díaz, A.A. Vicente, Chitosan/clay films' properties as affected by biopolymer and clay micro/nanoparticles' concentrations. Food Hydrocolloids, 23(2009) 1895–1902. [57] E. Mansfield, A. Kar, S.A. Hooker, Applications of TGA in quality control of SWCNTs. Analytical and Bioanalytical Chemistry, 396(3) (2010) 1071–1077. [58] C.Y. Ou, C.H. Zhang, S.D. Li, L. Yang, J.J. Dong, X.L. Mo, Mu-Ting Zeng, Thermal degradation kinetics of chitosan–cobalt complex as studied by thermogravimetric analysis. Carbohydrate Polymers, 82(4) (2010) 1284–1289. [59] J. Zawadzki, H. Kaczmarek, Thermal treatment of chitosan in various conditions. Carbohydrate Polymers, 80(2) (2010) 394–400. [60] P.A. Sandford, In G. Skjak-Braek, T. Anthonsen, & P. A. Sandford (Eds.), Chitin/chitosan: Sources, chemistry, biochemistry, physical properties, and applications. Amsterdam: Elsevier, (1990). [61] M.T. Viciosa, M. Dionisio, R.M. Silva, R.L. Reis, J.E. Mano, Molecular motions of chitosan studied by dielectric relaxation spectroscopy. Biomacromolecules, 5 (2004) 2073–2078. [62] M.E. Mathew, J.C. Mohan, K. Manzoor, S.V. Nair, H. Tamura, R. Jayakumar, Folate conjugated carboxymethyl chitosan–manganese doped zinc sulphide nanoparticles for
28
targeted drug delivery and imaging of cancer cells. Carbohyd. Polym., 80 (2010) 442– 448. [63] Y.M. Hao, F.L. Zhao, N. Li, Y.H. Yang, K.A. Li, Studies on a high encapsulation of colchicine by a noisome system. Int. J. Pharm., 244 (2002) 73–80. [64] S.T.R. Aydm, M. Pulat, 5-Fluorouracil Encapsulated Chitosan Nanoparticles for pHStimulated Drug Delivery: Evaluation of Controlled Release Kinetics. Journal of Nanomaterials, (2012) 1-10. [65] C. Lourenco, M. Teixeira, S. Simoes, R. Gaspar, Steric stabilization of nanoparticles: size and surface properties. Int. J. Pharm., 138 (1996) 1–12.
Figure 1: Structure of LTZ loaded chitosan nanoparticles using sodium tripolyphosphate Figure 2: FTIR spectrum of (a) CS-NPs-TPP-Air dried; (b) CS-NPs-TPP-Freeze dried; (c) LTZ-CS-NPs-TPP-Air dried; (d) LTZ-CS-NPs-TPP-Freeze dried samples Figure 3: TGA thermogram of (a) CS-NPs-TPP-Air dried; (b) CS-NPs-TPP-Freeze dried; (c) LTZ-CS-NPs-TPP-Air dried; (d) LTZ-CS-NPs-TPP-Freeze dried samples Figure 4: XRD of (a) Letrozole; (b) CS-NPs-TPP-Air dried; (c) CS-NPs-TPP-Freeze dried; (d) LTZ-CS-NPs-TPP-Air dried; (e) LTZ-CS-NPs-TPP-Freeze dried samples Figure 5: SEM images of (a) CS-NPs-TPP-Air dried;(b) CS-NPs-TPP-Freeze dried; (c) LTZ-CS-NPs-TPP-Air dried; (d) LTZ-CS-NPs-TPP-Freeze dried samples Figure 6: TEM images of (a) CS-NPs-TPP-Air dried;(b) CS-NPs-TPP-Freeze dried; (c) LTZ-CS-NPs-TPP-Air dried; (d) LTZ-CS-NPs-TPP-Freeze dried samples Figure 7: Biodegradation assay of unloaded and LTZ loaded chitosan nanoparticles Figure 8: Percentage cumulative drug release of LTZ from chitosan nanoparticles Figure 9: Hemo compatibility studies of LTZ loaded chitosan nanoparticles Figure 10: Serum Stability studies of LTZ loaded chitosan nanoparticles Scheme 1: Preparation of Letrozole loaded chitosan nanoparticles
29
Figure 1
30
(a)
1280.5
3448.1
1653
1183
% T
(b)
1656
3421.88 2377
1234
1223 1171.88
(c) 1657
3448 2230.27
(d)
1220.72 3411.46 1636.30
4000
3500
3000
2500
cm-1
2000
Figure 2
31
1500
1000
500
110
1.6
100
(b)
1.4
90 1.2
(c)
(a)
70
(d)
60
0.8 50
(c) 0.6
40 30
0.4
(b) (d) (a)
20
0.2
10 0 0
200
400
600
Temperature (°C) Figure 3
32
1.0
800
0.0 1000
Deriv. weight (%/°C)
Weight (%)
80
Figure 4
33
(a)
(c)
(b)
(d)
Figure 5
34
(a)
(b)
(c)
(d)
Figure 6
35
Figure 7
36
Figure 8
37
Figure 9
38
Figure 10
Scheme 1
39
Table 1: Effect of drug concentration on the particle size of LTZ loaded CS-NPs Ratio LTZ (mg) CS (mg) TPP (mg) Avg. Particle size (nm) (CS:TPP:LTZ) 5 50 40 1:0.8:0.1 58.77 10 50 40 1:0.8:0.2 78.82 15 50 40 1:0.8:0.3 91.28
Table 2: Effect of drying method on the particle size of LTZ-CS-NPs formulation Ratio Drying method Avg. Particle size (nm) CS TPP LTZ 1 0.8 0.1 58.77 Air 1 0.8 0.1 24.36 Freeze
Table 3: XRD details of letrozole, chitosan nanoparticles and letrozole loaded chitosan nanoparticles Samples 2θ LTZ 11.2°, 13.12°, 14.16°, 16.24°, 17.16°, 19.72°, 21°, 21.44°, 22°, 23.16°, 25.08°, 25.52°, 26.8° CS-NPs-TPP-Air dried 11.36, 20.64 and 22 CS-NPs-TPP-Freeze dried 13.68, 18.4 and 23.38 LTZ-CS-NPs-TPP-Air dried 11.44° and 18.04° LTZ-CS-NPs-TPP-Freeze dried 11.38°, 18.36° and 23.04° Table 4: Statistical parameters of various formulations obtained after fitting the drug release data to various release kinetic models LTZ-CS-NPs Zero First Higuchi HixsonKorsmeyerorder order Crowell Peppas (1:0.8:0.1)-TPP-Air dried (1:0.8:0.1)-TPP-Freeze dried (1:0.8:0.2)- TPP-Air dried (1:0.8:0.3)- TPP-Air dried
R2 0.9833 0.9944 0.9888 0.9967
R2 0.9766 0.9969 0.9829 0.9935
40
R2 0.9159 0.9868 0.9366 0.9598
R2 0.9884 0.9845 0.9918 0.9950
n 0.0941 0.1546 0.1203 0.1063
R2 0.7187 0.9618 0.8304 0.8356
S0141-8130(16)32076-1 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.01.147 BIOMAC 7078
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
19-10-2016 1-1-2017 18-1-2017
Please cite this article as: Thandapani Gomathi, P.N.Sudha, J.Annie Kamala Florence, Jayachandran Venkatesan, Anil Sukumaran, Fabrication of letrozole formulation using chitosan nanoparticles through ionic gelation method, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2017.01.147 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
Fabrication of letrozole formulation using chitosan nanoparticles through ionic gelation method 1
Thandapani Gomathi, 1*P.N. Sudha, 2J. Annie Kamala Florence, 4Jayachandran Venkatesan and 5Anil Sukumaran
1,1*
Department of Chemistry, D.K.M. College for Women, Vellore, Tamil Nadu, India 2 4
Department of Chemistry, Voorhees College, Vellore, Tamil Nadu, India
Marine Bioprocess Research Center and Department of Marine-bio Convergence Science, Pukyong National University, Busan, Republic of Korea
5
Division of Periodontics, Department of PDS, College of Dentistry, Prince Sattam Bin Abdulaziz University, Riyadh, Saudi Arabia
(*Corresponding author Email: [email protected]; [email protected]; Mobile: +91-9842910157) Graphical abstract
1
Highlights: Letrozole – sustained release from chitosan nanoparticles formulation. Average particle size of the formulation ranges from 58 to 91 nm. Freeze dried formulation have to size of 24 nm. Hemolysis analysis discloses the blood compatibility of the formulations. Abstract In this study, the anticancer drug letrozole (LTZ) was formulated using chitosan nanoparticles (CS-NPs) with the crosslinking agent sodium tripolyphosphate (TPP). The nanoformulation was optimized by varying the concentration of drug. The prepared particles were characterized using FTIR, TGA, XRD, SEM, TEM and DLS. From the FTIR results, the appearance of a new peak for =C-H, C=C and C=N confirms the formation of LTZ loaded chitosan nanoparticles. TEM images shows that the average particle size was in the range of 60 80 nm and 20 – 40 mm air dried and freeze dried samples respectively. Also the prepared formulation had been evaluated in vitro for determining its hemocompatability, biodegradability and serum stability. The preliminary studies supported that the chitosan nanoparticles formulation has biocompatibility and hemocompatible properties and it can act as an effective pharmaceutical excipient for letrozole. Keywords: chitosan nanoparticles, serum stability, optimization, hemocompatability, letrozole
Introduction Breast cancer is the most common type of cancer, especially in women, and, unfortunately, its incidence is increasing each year [1,2]. It is widely accepted that the majority of breast cancers are hormone-dependent and that estrogen is a key mediator in the progression and metastasis of breast tumors. Particularly, for postmenopausal women it has been reported that the concentration of 17b-estradiol (E2) in breast tumor can be tenfold higher than those in 2
plasma [3]. The high concentration of E2 in breast tumors could be attributed to increased uptake from plasma or in situ aromatization of androgens to estrogens [4]. Letrozole is considered to be one of the most effective non-steroidal, third generation aromatase inhibitors (AIs) which inhibit excess estrogen bio-synthesis within the body [5-8]. Its use as an estrogen receptor positive breast cancer drug is well recognized [9,10]. In practice letrozole was given to treat breast cancers and it was administered orally. Letrozole prevents the aromatase from producing estrogens by competitive, reversible binding to the heme of its cytochrome P450 unit [11] by >99%. Therefore it is a highly potent drug [12,13]. Polymeric nanoparticles have recently been considered as promising carriers for anticancer agents [14,15]. Especially chitosan plays a vital role in drug delivery applications. The amino group in chitosan has a pKa value of ~6.5, thus, chitosan is positively charged and soluble in acidic to neutral solution. Chitosan is bioadhesive and readily binds to negatively charged surfaces such as mucosal membranes and enhances the transport of polar drugs across epithelial surfaces, and is biocompatible and recyclable. Since in the conventional mode of administration, there is the need for the ideal drug delivery system owing to its adverse side effects caused by the non-specific targets. Therefore for designing ideal delivery systems, the parameters such as size and size distribution, drug loading capacity and stability are the important parameters to be taken into account [16,17]. The durg-polymer interactions can be proposed by increase drug loading capacity, non-covalent interactions including hydrogen-bonding and ionic interactions. Generally, the development of new materials always leads to advances in technology and creates innovative solutions to old problems. Thus the nanotechnology has gained wide acceptance in contemporary life [18]. In oncology, nanomaterials can be engineered to serve as anti-cancer agents or as ideal drug delivery vehicles [19-21]. Hence the present work was aimed
3
to synthesis the novel formulation using chitosan nanoparticles for the highly potent anticancer drug letrozole for sustained release and to overcome the problems of conventional method of administration of letrozole. In the present work through ionic cosslinking method a novel drug carrier of letrozole was synthesized which overcomes the drawbacks of low loading efficiency, higher particle size etc. The formulations were characterized for its formation and tested for effective in vitro drug release, biocompatibility, stability and blood compatibility. Chitosan nanoparticles were used as a carrier for letrozole and sodium tripolypohosphate was used as a crosslinker. Materials and methods Materials Chitosan (CS) with the degree of deacetylation 92% was procured from India Sea foods, Cochin, Kerala. Letrozole was synthesized as per the procedure given in US patent 7705159B2. Sodium tripolyphosphate (TPP) was purchased from finer chemicals and used without further purification. All other chemicals and reagents used are analytical grade. Preparation of durg unloaded and loaded chitosan nanoparticles 50mg of chitosan was dispersed in 5 ml of aqueous acetic acid (2% v/v) solution and continuously stirred for about 20 minutes at 600 rpm to obtain the homogeneous solution. 5 ml (0.8% w/v) of TPP solution was used as a crosslinker [22, 23]. The resulting chitosan nanoparticle suspension was subsequently centrifuged for about 45 minutes at 12,000 rpm and re-suspended in water for washing followed by drying. Similarly for preparing letrozole loaded chitosan nanoparticles, the drug letrozole (5, 10 and 15 mg in 0.5 ml acetic acid) solution was added dropwise to the chitosan solution, under continuous stirring up to 30 minutes. TPP solution (38.00 mg in 5 ml deionised water) was added dropwise to the chitosan/ Letrozole
4
solution over a period of 60 minutes at a stirring speed in the range of 550- 600 rpm (Scheme 1). After the complete addition, the suspension was centrifuged at 12,000 rpm. The supernatant solution was subjected to determine the loading efficiency. The filtered solid was slurried in water and centrifuged, the centrifuged material was kept for drying.
50
38
100
Characterization of Nanoparticles FTIR spectroscopy was measured for the determination of the types of bonds present in the nanoparticulate. FT-IR spectra of CS – NPs, LTZ and LTZ – CS – NPs were carried out using KBr tablets (1% w/w of product in KBr) with a resolution of 4 cm-1 and 100 scans per sample on a Thermo Nicolet AVATAR 330 spectrophotometer. Thermogravimetric analysis was conducted to measure the thermal weight loss of the samples on a TGA Q500 V20.10 Build 36 instruments at a heating rate of 20°C per minute in nitrogen atmosphere. The weight losses at different stages were analysed. The scanning electron microscopy analysis (SEM, Leica, Cambridge, UK) was conducted to observe the size, shape and surface morphology of the nanoparticles. For the analysis, the samples are wiped with a thin gold – palladium layer by a sputter coater unit (VG – microtech, UCK field, UK), the shape and morphology were analysed with a Cambridge stereoscan 440 scanning electron microscope (SEM, Leica, Cambridge, UK). The transmission electron microscopy analysis (TEM) was conducted to observe the size, shape 5
and surface morphology of the loaded and unloaded chitosan nanoparticles. The shape and morphology were analysed with a HITACHI-H-7650 transmission electron microscope. The particle size and size distribution of the nanoparticles were measured by Dynamic light scattering method (DLS, Zetasizer Nano-S, Malvern, England). Suitable amount of the dried nanoparticles from each formulation was suspended in deionised water and was sonicated for a suitable time period before the measurement. The volume mean diameter, size distribution and polydispersity of the resulting homogeneous suspension were determined using DLS technique. X-ray diffraction (XRD) patterns were obtained using an X-ray diffractometer (XRD-SHIMADZU XD-D1). Letrozole loading measurements The quantity of letrozole (LTZ) entrapped in the nanoparticulate system was determined indirectly, by measuring the quantity of letrozole remaining in the supernatant based on the absorbance of the samples at 250 nm [24]. The standard curve was obtained and the sample absorbance was measured in 3 ml quartz cuvettes using Shimadzu UV-1700 Pharma spec UVVisible spectrophotometer (Suzhow, Jiangsu, China). The measurement for quantifying the amount of drug loaded in the nanoparticles is drug entrapment efficiency (EE). Determining the EE allows for the optimization of the amount added, reducing wastage, and is defined as follows:
∗ 100
Entrapment efficiency describes the quantity of the drug entrapped within the nanoparticle as it is related to the initial drug loading. 100% EE means that the entire drug quantity added has been incorporated into the nanoparticle. In vitro drug release studies
6
In vitro drug release profiles were done by direct dispersion method as explained in literature [25,26]. In vitro drug release studies were done for a period of one week at pH 7.4. 10 mg of drug loaded CS – NPs were taken in 50 mL of 10 mM phosphate buffer solution (PBS) in a beaker under magnetic stirring at 100 rpm. The receptor phase was stirred and thermally controlled at 37 ºC. The base absorbance of the release media was accounted by using the release medium as the solution in the UV-spectrophotometer reference cell, as well as the solution for zeroing the system. At fixed time intervals, 3 ml of the receptor phase was withdrawn, centrifuged to collect the supernatant and then substituted with fresh buffer. The drug release was assayed spectrophotometrically. The cumulative percentage amount of drug release was calculated and plotted against time. Kinetics studies To analyze the in vitro release data various kinetic models were used to describe the release kinetics. The zero order rate equation describes the systems where the drug release rate is independent of its concentration [27].
where Qo and Qt are the initial amount of drug and cumulative amount of drug released at time t. Ko is the zero order rate constant. The first order kinetics describes the release from system, where release rate is concentration dependent [28]. log
log
where k1 is the first order rate constant. Higuchi [29], described the release of drugs from insoluble matrix as a square root of time dependent process based on Fickian diffusion.
7
where kH is the Higuchi constant. The Hixson-Crowell [30], cube root law describes the release from systems where there is a change in surface area and diameter of particles or tablets.
where kHC is the Hixson-Crowell constant. Korsmeyer et al. [31] derived a simple relationship which described drug release from a polymeric system. where kKP is the Korsmeyer-Peppas constant and n is the release exponent describing the drug release mechanism. Modeling was performed using the parameters that provide the closest fit between experimental observations and the nonlinear function. The model that best fits the release data was selected based on the correlation coefficient (R2) in models described above. The model, which gives highest R2 value, is considered as the best fit of release data.
In vitro biodegradation behavior The rate of degradation of the prepared samples was studied according to the previous method [32]. The CS-NPs and LTZ-CS-NPs with TPP were weighed equally, and the initial weight was recorded as Wo, followed by immersion in 1xPBS containing lysozyme (10,000 µg/L) and incubated at 37 °C at different intervals (24, 48 and 72 h). After completion of the incubation period, the samples were washed with deionized water to remove ions and blot dried 8
with filter paper. The dry weights of the samples were noted as (Wt). The degradation was calculated by using the following formula: %
100
Hemolysis assay Blood compatibility was evaluated with hemolysis assay. Fresh human blood was used in this study. 1.5 mL acid citrate dextrose (ACD) was added to 10 mL fresh blood. To 1 mL of the blood sample, 100 µL of sample of concentration ranging from 0.01 to 1 mg/mL was added. The whole samples were incubated for 2 h with shaking in an incubator chamber at 37 °C. The samples were spin down at 6000 rpm for 15 min to obtain the plasma (plasma would be red in color if hemolysis happened). The plasma portion was collected [100 µL plasma+1 mL Na2CO3 (0.01%)]. The OD values were read at 450, 380 and 415 nm. The obtained OD values of the samples were compared with that of controls. To obtain 100% hemolysis, cells were lysed by dispersing in distilled water (positive control) and the negative control was obtained by treating blood with DMSO. Each assay was evaluated in triplicate. Stability studies To assess the colloidal stability of unloaded and drug loaded CS-NPs, Human Serum (HS) was first incubated at 37°C in a water bath incubator to simulate physiological media. HS was then added in the nanoparticles dispersion with the volume ratio of 1:1 in which the final concentration of 50% v/v of serum was yielded. Subsequently, the resulting mixture was incubated in the water bath incubator at 37 °C for 24 h. The mean particle size was measured at predetermined time points (0, 1, 6, 12 and 24 h). The nanoparticles were suspended in PBS (pH, 7.4) prior to mixing with HS. Statistical analysis 9
Statistical analysis was performed using origin software (origin pro8) for descriptive statistics (mean, standard deviation, 95% confidence interval, linear fit and so on). Statistical analysis was performed for different formulae by applying one-way analysis of variance ANOVA test. 2-sample t-tests were used to test the differences in mean values. A ρ-value of <0.05 was considered statistically significant. Result and Discussion Preparation of CS-NPs and LTZ-CS-NPs Chitosan nanoparticles were fabricated instantaneously at room temperature through ionic gelation method while mixing the homogenuous solution of chitosan and the cross linker TPP [33-36]. The amino groups in chitosan which are responsible for many of its advanced functions, including biological activity and cationic polymer properties [37], interacts with polyanions to form nanoparticles through inter- and intramolecular crosslinkages [38,39] which involves the addition of alkaline phase and acidic phase. The alkaline phase is polyanions dissolved in water and the acidic phase is chitosan dissolved in dilute acidic acid. Upon mixing, these two phases forms inter- and intramolecular linkages between TPP and NH2 group of chitosan. The development of appropriate vehicles to deliver the drug molecules is a meaningful challenge for pharmaceutical scientist. Here the chitosan nanoparticles paved a way for these challenges for delivering the aromatase inhibitor – letrozole. Chitosan molecules may likely adopt a spread conformation in solution because of electrostatic repulsion force existing between amine groups along the molecular chain. To this solution, when letrozole was added under constant stirring, the hetero atoms present in letrozole, involved in hydrogen bonding with protonated chitosan chain with certain sites at the spread chitosan chain to achieve high
10
encapsulation. Chitosan is polycationic in acidic media (pKa 6.5) and when the crosslinking agents TPP is added to the chitosan-drug mixture it interacts with the negatively charged TPP, leading to the formation of chitosan nanoparticles. The crosslinking agents contain both –OH and phosphoric ions with compete with each other to interact with the –NH3+ sites of chitosan. The mechanism of interaction in the formulation is given in the Figure 1. The formation of nanoparticles depends dramatically on the concentration of free amino groups, which strengthens the electrostatic interactions between the nanoparticles and the drug, helping to reduce the particle sizes [40,41].
stretching vibration. Bands at 2918.30 and 2881.65 cm-1 corresponds to anti-symmetric C-H stretching and symmetric C-H stretching modes of vibration. The band at 1658.78 cm-1 is for the amide I band (C=O stretching), since chitosan used here was 92% deacetylated. The presence of amide groups were confirmed through amide I (1658.78 cm-1), amide II (1577.78 cm-1) and amide III bands (1323.17 cm-1) [43,44]. Figure 2a and 2b shows the FTIR spectrum of CS-NPs-TPP-Air dried and Freeze dried sample. The results were consistent with previously published data [45]. A broad band at 3425.28 cm-1 was assigned to a combination of stretching modes of O-H and N-H bonds in chitosan matrix. In the sample of chitosan nanoparticles this band becomes wider and shifts to higher wavenumber, 3448.1 cm-1 in case of air dried sample and to lower wavenumber 3421.88 cm-1 for freeze dried sample, indicating the presence of strong electrostatic interactions and an enhancement of the hydrogen bond interactions [38, 47, 48]. The band for C=O of CS-NPs air dried and freeze dried samples was shifted to lower wavenumber of 1653 and 1656 cm-1 from 1658 cm-1 during nanoparticles formation, indicating that C=O groups also participating in the nonbonding interaction.
12
appearance of a new peak for P=O and P-O when compared with the fingerprint pure chitosan, confirms the formation of chitosan nanoparticles through ionotropic gelation method. LTZ alone (spectrum not given) showed major peaks at 2232.6 cm-1 for C≡N stretching, 3054.27 cm-1 for sp2 hybridized CH stretching, 690-900 cm-1 for out-of plane CH deformation modes of vibration. The FTIR spectrum of letrozole loaded chitosan nanoparticles formulation (Figure 2c and 2d) showed that no significant differences on shape and position of the absorption bands for the formulation. Most of the absorption peaks from pure LTZ and bare chitosan nanoparticles overlapped with the absorption peaks from LTZ-CS-NPs. It can be concluded that no strong covalent interaction occurred between drug and polymer. For LTZ-CSNPs new bands for C≡N group at 2377 and 2230.27 cm-1 for LTZ-CS-NPs-TPP-Air dried and LTZ-CS-NPs-TPP-Freeze dried samples, this confirms the incorporation of LTZ in chitosan nanoformulation. Also the LTZ incorporated CS-NPs showed intense band at 1657 and 1636.30 cm-1 corresponding to the hetero aromatic C=N stretching and phenyl C=C stretching vibrations in the higher wavenumber region due to the overlap with C=O stretching modes and appears to be broad. The asymmetric C=O stretching (amide I band) was observed at the lower wavenumber for LTZ-CS-NPs-TPP on compared with its respective bare chitosan nanoparticles indicating the involvement of C=O group in the electrostactic interaction as well as hydrogen bond interaction [51]. The bands in the region 1395 – 1415 cm-1, 1380 cm-1, 1247 – 1306 cm-1, 1154 – 1166 cm-1, 1076 – 1100 cm-1 and 1018 – 1030 cm-1 508 – 523 cm-1 are assigned to C-H and O-H deformation, C-N stretching modes, P=O stretching modes, C-O stretching and C-N stretching [26, 52, 53], C-O-C skeletal linkage and P-O stretching modes of vibrations respectively. The actual positions of these bands were comparatively shifted to the lower region while loading
13
letrozole, confirming the participation of these groups in non-covalent interactions. Thus, the shift in the peak of OH, NH2 and C=O functional groups and also the appearance of a new peak for =C-H, C=C and C=N when compared with the fingerprint bare chitosan nanoparticles, confirms the formation of LTZ loaded chitosan nanoparticles confirming the drug is intact and has not reacted with the excipient used in the formulation and hence they are compatible. TGA studies The change on the thermal degradation of chitosan showed the participation of functional groups of chitosan in modification [54]. The thermal stability and thermal decomposition of CSNPs-TPP and LTZ-CS-NPs-TPP Air dried and Freeze dried samples were investigated by TG and are given in Figure 3. The results showed the similar trend of thermal decomposition.
14
behavior [60] and this anomaly could be related with crystalline and/or morphological variations in nanoparticles with respect to those of the pure chitosan [61]. The degradation profile of letrozole loaded air dried CS-NPs-TPP and freeze dried CSNPs-TPP (Figure 3c and 3d), showed a similar decomposition stages as seen in the bare chitosan nanoparticles. Around 30 – 180 °C the first stage, 200 – 350 °C the second stage and the third stage starts from 350 °C. Comparatively the high residue is present for air dried samples than freeze dried samples. The 10 % degradation is higher for LTZ-CS-NPs-TPP Air dried sample than the Freeze dried sample. 50% decomposition temperature of air dried CS-NPs-TPP samples is >870 °C and freeze dried CS-NPs-TPP is 520.15 °C. Therefore the decreasing trend of percentage decomposition is clearly depicted in the TGA of drug loaded chitosan nanoparticles compared with bare chitosan nanoparticles. This change is thermal behavior is owing to the formation of new interaction which would have formed between the drug, polymer and crosslinker. XRD The X-ray diffraction pattern of letrozole, chitosan nanoparticles and letrozole loaded chitosan nanoparticles nanoparticles prepared with TPP crosslinker in air drying and freeze drying method is presented in Table 3 and Figure 4a – 4e. The pure LTZ exhibits a strong characteristic peak at 2θ = 11.2°, 13.12°, 14.16°, 16.24°, 17.16°, 19.72°, 21°, 21.44°, 22°, 23.16°, 25.08°, 25.52°, 26.8°, indicating its crystalline structure. The number of peaks confirms the different crystalline forms present in letrozole. For LTZ loaded chitosan nanoparticles, only a broad peak with a shoulder is observed, corresponding to the amorphous nature. This is due the interaction present between LTZ with CS and TPP, which acts as the main driving force for LTZ loading can be ascribed to intermolecular
15
hydrogen bonding [62,63]. Hydrogen bonding is formed based on the complexes between the Nitrogen atoms in LTZ and H atoms of NH2, NH3+, OH and P=O groups in CS-NPs. For all the LTZ-loaded CS-NPs, the X-ray diffraction pattern shows the presence of the typical peaks for both LTZ and CS-NPs, which indicates that the crystal state of LTZ in CS-NPs remains the same. While the position of the peaks shifts, and the intensity of these peaks is evidently reduced, revealing a certain degree of interaction between LTZ and CS-NPs. This indicates that the conjugated and entrapped forms of the drugs are unable to form their own crystal lattice inside the polymer and they are in amorphous state. These evidences lead to the conclusion that the drugs are distributed over the nanostructure which satisfies the essential requirement of a good drug delivery system (DDS). SEM The SEM microphotography of the letrozole loaded chitosan nanoparticles prepared with TPP crosslinker under air drying and freeze drying method of ratio 1:0.8:0.1 are shown in Figure 5a – 5d. SEM images shows that the loaded nanoparticles have a rough morphology after loading. The image shows the aggregation of particles during drying process. Also the image reveals that the drug gets encapsulation and some of the drug molecules are adhered on the surface. Compared with the air dried formulation the freeze dried formulations have well established porosity, which is depicted in the micrography. This porosity is due to the amorphous nature of the prepared formulation, which proved that the nanoparticles are good candidates for sustained drug delivery systems, with their sizes in nano range required for its parentral chemotherapy.
The surface morphology and particle size of the prepared letrozole formulations were studied using TEM analysis. The images (Figure 6a – 6d) depicted that the prepared formulations are in the nano size range. For LTZ-CS-TPP air dried and freeze dried samples the particles are found in the range of 60 - 80 nm and 20 – 40 nm respectively. These measurements are supported by DLS studies which will give the exact size distribution of the nanoparticles. TEM images of letrozole loaded chitosan nano formulations also confirms the particles aggregation which was probably due to drying process. Entrapment efficiency of chitosan nanoparticles The entrapment efficiency of chitosan nanoparticles during loading of letrozole was determined through indirect measurements using UV-spectrophotomer in the absorbance at 240 nm. The letrozole formulations which were prepared by varying drug concentration and drying method are studied for the effective loading. While increasing the concentration of the drug from 5 mg to 15 mg (0.1 to 0.3 ratio) the entrapment efficiency measurement showed a slight decrease with respect to the concentration. The encapsulation efficiency depends upon the number of available sites present in the formulating material. About 98.4 % of entrapment efficiency was found for LTZ-CS-NPs-TPP (1:0.8:0.1) ratio of air dried and freeze dried samples. Whereas 97.8% and 97.1% of drug is encapsulated in (1:0.8:0.2) and (1:0.8:0.3) ratio of air dried samples respectively. Biodegradation studies Biodegradation study was performed on LTZ loaded air dried formulations of ratio 1:0.8:0.1, 1:0.8:0.2 and 1:0.8:0.3 with TPP crosslinker. The incorporation of LTZ into the chitosan nanoparticles had improved the biodegradability. It was seen that the nano formulation
17
improved the biodegradability to a great extent in the presence of lysozyme when compared with bare chitosan nanoparticles. The results were shown in Figure 7.
18
the maximum of 58.81 %, 42.01 %, 63.65 %,and 74.27 % of the drug was released within in 70 h in the respective formulations. On comparing the air dried and freeze dried formulations of ratio 1:0.8:0.1, the slow release was noticed in case of freeze dried samples. Also on increasing the concentration of letrozole from 5 mg to 15 mg in the nano formulation, the initial burst release increases accordingly, confirming that more amount of the drug was adhered on the surface of the formulation. The mathematical models were used in this study to determine the release mechanism of letrozole loaded chitosan nano formulations. The results of the mathematical models are very important in the optimization of the suitable formulation for letrozole.
19
Blood compatibility is a significant parameter for the biological safety of the formulation. This is of great significance when the material is intended for injections. Thus in this present work the blood compatibility study was done for the prepared formulations and the results are depicted in the Figure 9. Hemolysis assay indicates that the prepared formulations are heme compatibility. As per the results, the hemolytic ratio of the sample was 2.4 %, much below 5% which is the safe haemolytic ratio for bio-materials in accordance with ISO/TR 7406. It was evident from the results that the nanoparticles are free from the risk of hemolysis. Serum stability The stability of the letrozole formulation was carried out in Human Serum with the volume ratio of 1:1 in the time intervals of 0, 1, 6, 12 and 24 h. After intravenous administration, the size and charge of nanoparticles are mainly determined by the adsorbed serum components [65]. The obtained data revealed that an increment in the particle size was occurred during incubation in HS (Figure 10). All the formulations were to be found stable in HS up to 24 h. Comparatively the particle size increases for air dried samples than the freeze dried one. Despite the fact that there is no drastic increase in the particle sizes of the formulation, therefore more biocompatibility.
20
supported by DLS study. The particles are in the size range of 20 – 80 nm. The in vitro drug release study showed that letrozole is released in a controlled manner from the nano formulation in the physiological pH 7.4. From the release data it was confirmed that it can also be administered parenterally. The biodegradability assay and serum stability studies prove that the prepared formulations were biodegradable and stable. The hemolysis analysis discloses the blood compatibility of the formulations. The in vitro evaluations of the letrozole proved the successful loading and sustained release profile of chitosan nanoparticles, which satisfies the need of cancer chemotherapy. References [1]
I.E. Smith, Letrozole versus tamoxifen in the treatment of advanced breast cancer and as neoadjuvant therapy. J. Steroid Biochem. Mol. Biol. 86 (2003) 289–295.
[2]
C. De Santis, R. Siegel, P. Bandi, A. Jemal, Breast cancer statistics, 2011. CA: A Cancer Journal for Clinicians, 61 (2011) 408–418.
[3]
A.A.Van Landeghem, J. Poortman, M. Nabuurs, J.H. Thijssen, Endogenous concentration and subcellular distribution of androgens in normal and malignant human breast tissue. Cancer Res., 45 (1985) 2907-2912.
[4]
W. Yue, J.P. Wang, C.J. Hamilton, L.M. Demers, R.J. Santen, In situ aromatization enhances breast tumor estradiol levels and cellular proliferation, Cancer Res. 58 (1998) 927-932.
[5]
H.M. Lamb, J.C. Adkins, Letrozole. A review of its use in postmenopausal women with advanced breast cancer, Drugs. 56 (1998) 1125-1140.
21
[6]
G.F. Meresman, M. Bilotas, V. Abello, Effects of aromatase inhibitors on proliferation and apoptosis in eutopic endometrial cell cultures from patients with endometriosis. Fertil. Steril., 84 (2005) 459-463.
[7]
L.L. Amsterdam, W. Gentry, S. Jobanputra, Anastrazole and oral contraceptives: a novel treatment for endometriosis, Fertil. Steril. 84 (2005) 300-304.
[8]
H.M. Fatemi, H.A. Turki, E.G. Papanikolaou, Successful treatment of an aggressive recurrent post-menopausal endometriosis with an aromatase inhibitor, Reprod. Biomed. 11 (2005) 455-457.
[9]
M.R. Saboktakin, R.M. Tabatabaie, A. Maharramov, M.A. Ramazanov, Synthesis and in vitro studies of biodegradable thiolated chitosan hydrogels for breast cancer therapy, Int. J. Biol. Macromol. 48 (2011) 747-752.
[10] N. Mondal, T.K. Pal, S.K. Ghosal, Development, physical characterization, micromeritics and in vitro release kinetics of letrozole loaded biodegradable nanoparticles, Pharmazie 63 (2008) 361-365. [11] J. L. Scott, J.S. Keam, Letrozole : in postmenopausal hormone-responsive early-stage breast cancer, Drugs 66 (2006) 353. [12] P. Furet, C. Batzl, A. Bhatnagar, E. Francotte, G. Rihs, M. Lang, Aromatase inhibitors: synthesis, biological activity, and binding mode of azole-type compounds, J. Med. Chem., 36 (1993) 1393–1400. [13] M. Lang, C. Batzl, P. Furet, R. Bowman, A. Hausler, A.S. Bhatnagar, Structure-activity relationships and binding model of novel aromatase inhibitors, J. Steroid Biochem. Mol. Biol. 44 (1993) 421–428.
22
[14] I. Brigger, C. Dubernet, P. Couvreur, Nanoparticles in cancer therapy and diagnosis. Adv Drug Del Rev. 54 (2002) 631–51. [15] C. Vauthier, C. Dubernet, C. Chauvierre, I. Brigger, P. Couvreur, Drug delivery to resistant tumors: the potential of poly(alkyl cyanoacrylate) nanoparticles, J Controlled Release. 93 (2003) 151–160. [16] K. Kataoka, A. Harada, Y. Nagasaki, Block copolymer micelles for drug delivery Design, characterization and biological significance, Advance Drug Delivery Review 47 (2001) 113-131. [17] S.H. Kim, J.P. Tan, F. Nederberg, K. Fukushima, J. Colson, C. Yang, A. Nelson, Y.Y. Yang, J.L. Hedrick, Hydrogen bonding-enhanced micelle assemblies for drug delivery, Biomaterials, 31 (2010) 8063-71. [18]
A.G. Cuenca, H. Jiang, S.N. Hochwald, M. Delano, W.G. Cance, S.R. Grobmyer, Emerging implications of nanotechnology on cancer diagnostics and therapeutics. Cancer, 107 (2006) 459-66.
[19] R.L. Manthe, S.P. Foy, N. Krishnamurthy, B. Sharma, V. Labhasetwar, Tumor ablation and nanotechnology. Mol Pharm, 7 (2010) 1880-98. [20] A.Z. Wang, R.S. Langer, O.C. Farokhzad, Nanoparticle delivery of cancer drugs. Annu Rev Med 2011. [21] P. Sharma, A. Singh, S.C. Brown, N. Bengtsson, G.A. Walter, S.R. Grobmyer, N. Iwakuma, S. Santra, E.W. Scott, B.M. Moudgil, Multimodal nanoparticulate bioimaging contrast agents. Methods Mol Biol. 624(2010) 67-81. [22] P. Calvo, C. Remunan, J.J.L. Vila, M.J. Alonso, Novel hydrophilic chitosan-polyethylene oxide nanoparticles as protein carriers. Journal ofApplied Polymer Science, 63(1997) 125–132.
23
[23] M.
Prabaharan,
R.L.
Reis,
J.F.
Mano,
Carboxymethyl
chitosan-graft-
phosphotidylethanolamine: amphiphilic matrices for controlled drug delivery.”- Reactive and Functional Polymers, 67 (2007) 43 – 52. [24] F.Albertoni, B. Pettersson, V. Reichelova, G. Juliusson, J. Liliemark, Analysis of 2Chloro-2'-Deoxyadenosine in Human Blood Plasma and Urine by High-Performance Liquid Chromatography Using Solid-Phase Extraction. Therapeutic Drug Monitoring, 16 (1994) 413-418. [25] S. Bisht, G. Feldman, S. Soni, R. Ravi, C. Karikar, A. Maitra, A. Maitra, Polymeric nanoparticle-encapsulated curcumin (“nanocurcumin") a novel strategy for human cancer therapy. Journal of Nanobiotechnology, 5 (2007) 1–18. [26] A. Anitha, S. Maya, N. Deepa, K.P. Chennazhi, S.V. Nair, R. Jayakumar, Curcuminloaded N,O-carboxymethyl chitosan nanoparticles for cancer drug delivery. Journal of Biomaterials Science, 23(11) (2012) 1381-1400. [27] T.P. Hadjiioannou, G.D. Christian, M.A. Koupparis, P.E.
Macheras, Quantitative
Calculations in Pharmaceutical Practice and Research, VCH Publishers Inc., New York, (1993) 345-348. [28] D.W.A. Bourne, Pharmacokinetics. In: Banker GS, Rhodes CT, Modern Pharmaceutics, 4th ed., Marcel Dekker Inc., New York, (2002) 67-92. [29] T.J. Higuchi, Mechanism of sustained medication. Theoretical analysis of rate of release of solid drugs dispersed in solid matrices., Pharm Sci.,84 (1963) 1464-1477. [30] A.W. Hixson, J.H. Crowell, Dependence of reaction velocity upon surface and agitation (I) theoretical consideration. Ind. Eng. Chem., 23 (1931) 923-931.
24
[31] R.W. Korsmeyer, R. Gurny, E. Doelker, P. Buri, N.A. Peppas, Mechanisms of solute release from porous hydrophilic polymers. Int. J. Pharm., 15 (1983) 25-35. [32] J. Venkatesan, I. Bhatnagar, S.K. Kim, Chitosan-Alginate Biocomposite Containing Fucoidan for Bone Tissue Engineering Chitosan-Alginate Biocomposite Containing Fucoidan for Bone Tissue Engineering. Mar. Drugs, 12 (2014) 300-316. [33] Q. Gan, T. Wang, Chitosan nanoparticle as protein delivery carrier-Systematic examination of fabrication conditions for efficient loading and release. Colloids and Surfaces B:Biointerfaces, 59 (2007) 24-34. [34] Q. Gan, T. Wang, C. Cochrane, P. McCarron, Modulation of surface charge, particle size and morphological properties of chitosan-TPP nanoparticles intended for gene delivery, Colloids and Surfaces B, 44 (2005) 65–73. [35] L. Qi, Z. Xu, X. Jiang, C. Hu, X. Zou, Preparation and antibacterial activity of chitosan nanoparticles. Carbohydrate Research, 339(16) (2004) 2693–2700. [36] M. Prabaharan, and J.F. Mano, Hydroxypropyl chitosan bearing β-cyclodextrin cavities: synthesis and slow release of its inclusion complex with a model hydrophobic drug”. – Macromolecular Bioscience, 5 (2005) 965 – 973. [37] Y.J. Liu, Y. Jiang, Y.F. Feng, D.L. Han, Study on the chitosan hydrolysis catalyzed by special cellulase and preparation of chitooligosaccharide. J. Func. Polym., 18 (2005) 325– 329. [38] Y. Xu, Y. Du, Effect of molecular structure of chitosan on protein delivery properties of chitosan nanoparticles. International Journal of Pharmaceutics, 250(1) (2003) 215–226.
25
[39] A. Vila, A. Sánchez, K. Janes, I. Behrens, T. Kissel, J.L. Vila Jato, M.J. Alonso, Low molecular weight chitosan nanoparticles as new carriers for nasal vaccine delivery in mice. Eur J Pharm Biopharm, 57(1) (2004) 123-131. [40] H.C. Yang, M.H. Hon, The effect of the molecular weight of chitosan nanoparticles and its application on drug delivery. Microchemical Journal, 92(1) (2009) 87–91. [41] K.A. Janes, M.P. Fresneau, A. Marazuela, A. Fabra, M.J. Alonso, Chitosan nanoparticles as delivery systems for doxorubicin. Journal of Controlled Release, 73(2-3) (2001) 255– 267. [42] D.D. Ankola, B. Viswanad, V. Bhardwaj, P.R. Rao, M.N.V. Kumar, Development of potent oral nanoparticulate formulation of coenzyme Q10 for treatment of hypertension: can the simple nutritional supplements be used as first line therapeutic agents for prophylaxis/therapy? Eur. J. Pharm. Biopharm., 67 (2007) 361– 369. [43] A. Pawlak, M. Mucha, Thermogravimetric and FTIR studies of chitosan blends. Thermochimica Acta, 396 (2003) 153-166. [44] Y. Dong, C. Xu, J. Wang, Y. Wu, M. Wang, Y. Ruan, Influence of degree of deacetylation on critical concentration of chitosan/dichlorocatic acid liquid crystalline solution. Journal of Applied Polymer Science, 83(6) (2002) 1204–1208. [45] C. Qin, H. Li, Q. Xiao, Y. Liu, J. Zhu, Y. Du, Water solubility of chitosan and its antimicrobial activity. Carbohydrate Polymers, 63(3) (2006) 367–374. [46] A.R. Dudhani, S.L. Kosaraju, Bioadhesive chitosan nanoparticles: preparationand characterization. Carbohydr. Polym., 81 (2010) 243–251.
26
[47] Y. Wu, W. Yang, C. Wang, J. Hu, S. Fu, Chitosan nanoparticles as a novel delivery system for ammonium glycyrrhizinate. International Journal of Pharmaceutics, 295(1–2) (2005) 235–245. [48] Y. Jia-hui, D. Yu-min, Z. Hua,
Blend films of chitosan–gelatin. Journal of Wuhan
University, 45 (1999) 440–444. [49] J.Z. Knaul, S.M. Hudson, K.A.M. Creber, Improved mechanical properties of chitosan fibers. J. Appl. Polym. Sci., 72 (1999) 1721–1731. [50] S. Vimal, S. Abdul Majeed, G. Taju, Nambi, K.S.N. Sundar Raj, N. Madan, M.A. Farook, T. Rajkumar, D. Gopinath, A.S. Sahul Hameed, Chitosan tripolyphosphate (CS/TPP) nanoparticles: Preparation,characterization and application for gene delivery in shrimp. Acta Tropica, 128 (2013) 486– 493. [51] N.S. Rejinold, K.P. Chennazhi, S.V. Nair, H. Tamura, R. Jayakumar, Biodegradable and thermo-sensitive chitosan-g-poly (N-vinylcaprolactam) nanoparticles as a 5-fluorouracil carrier. Carbohydr. Polym., 83 (2011a) 76–786. [52] N.S. Rejinold, M. Muthunarayanan, K.P. Chennazhi, S.V. Nair, R. Jayakumar,5Fluorouracil loaded fibrinogen nanoparticles for cancer drug delivery applications. International Journal of Biological Macromolecules, 48(1) (2011b) 98–105. [53] E.B. Yassin, M.K. Anwer, H.A. Mowafy, I.M. Elbagory, M.A. Bayomi, I.A. Alsarra, Optimization of 5-fluorouracil solid-lipid nanoparticles: a preliminary study to treat colon cancer. Int. J. Med. Sci., 7 (2010) 398–408 [54] H.K. Holme, H. Foros, H. Pettersen, M. Dornish, O. Smidsrod, Thermal depolymerization of chitosan chloride. Carbohydrate polym., 46(3) (2001) 287-294
27
[55] A.P. Martínez-Camacho, M.O. Cortez-Rocha, J.M. Ezquerra-Brauer, A.Z. GracianoVerdugo, F. Rodriguez-Félix, M.M. Castillo-Ortega, M.S. Yépiz-Gómez, M. PlascenciaJatomea, Chitosan composite films: Thermal, structural, mechanical and antifungal properties. Carbohydr. Polym., 82 (2010) 305–315. [56] A. Casariego, B.W.S. Souza, M.A. Cerqueira, J.A. Teixeira, L. Cruz, R. Díaz, A.A. Vicente, Chitosan/clay films' properties as affected by biopolymer and clay micro/nanoparticles' concentrations. Food Hydrocolloids, 23(2009) 1895–1902. [57] E. Mansfield, A. Kar, S.A. Hooker, Applications of TGA in quality control of SWCNTs. Analytical and Bioanalytical Chemistry, 396(3) (2010) 1071–1077. [58] C.Y. Ou, C.H. Zhang, S.D. Li, L. Yang, J.J. Dong, X.L. Mo, Mu-Ting Zeng, Thermal degradation kinetics of chitosan–cobalt complex as studied by thermogravimetric analysis. Carbohydrate Polymers, 82(4) (2010) 1284–1289. [59] J. Zawadzki, H. Kaczmarek, Thermal treatment of chitosan in various conditions. Carbohydrate Polymers, 80(2) (2010) 394–400. [60] P.A. Sandford, In G. Skjak-Braek, T. Anthonsen, & P. A. Sandford (Eds.), Chitin/chitosan: Sources, chemistry, biochemistry, physical properties, and applications. Amsterdam: Elsevier, (1990). [61] M.T. Viciosa, M. Dionisio, R.M. Silva, R.L. Reis, J.E. Mano, Molecular motions of chitosan studied by dielectric relaxation spectroscopy. Biomacromolecules, 5 (2004) 2073–2078. [62] M.E. Mathew, J.C. Mohan, K. Manzoor, S.V. Nair, H. Tamura, R. Jayakumar, Folate conjugated carboxymethyl chitosan–manganese doped zinc sulphide nanoparticles for
28
targeted drug delivery and imaging of cancer cells. Carbohyd. Polym., 80 (2010) 442– 448. [63] Y.M. Hao, F.L. Zhao, N. Li, Y.H. Yang, K.A. Li, Studies on a high encapsulation of colchicine by a noisome system. Int. J. Pharm., 244 (2002) 73–80. [64] S.T.R. Aydm, M. Pulat, 5-Fluorouracil Encapsulated Chitosan Nanoparticles for pHStimulated Drug Delivery: Evaluation of Controlled Release Kinetics. Journal of Nanomaterials, (2012) 1-10. [65] C. Lourenco, M. Teixeira, S. Simoes, R. Gaspar, Steric stabilization of nanoparticles: size and surface properties. Int. J. Pharm., 138 (1996) 1–12.
Figure 1: Structure of LTZ loaded chitosan nanoparticles using sodium tripolyphosphate Figure 2: FTIR spectrum of (a) CS-NPs-TPP-Air dried; (b) CS-NPs-TPP-Freeze dried; (c) LTZ-CS-NPs-TPP-Air dried; (d) LTZ-CS-NPs-TPP-Freeze dried samples Figure 3: TGA thermogram of (a) CS-NPs-TPP-Air dried; (b) CS-NPs-TPP-Freeze dried; (c) LTZ-CS-NPs-TPP-Air dried; (d) LTZ-CS-NPs-TPP-Freeze dried samples Figure 4: XRD of (a) Letrozole; (b) CS-NPs-TPP-Air dried; (c) CS-NPs-TPP-Freeze dried; (d) LTZ-CS-NPs-TPP-Air dried; (e) LTZ-CS-NPs-TPP-Freeze dried samples Figure 5: SEM images of (a) CS-NPs-TPP-Air dried;(b) CS-NPs-TPP-Freeze dried; (c) LTZ-CS-NPs-TPP-Air dried; (d) LTZ-CS-NPs-TPP-Freeze dried samples Figure 6: TEM images of (a) CS-NPs-TPP-Air dried;(b) CS-NPs-TPP-Freeze dried; (c) LTZ-CS-NPs-TPP-Air dried; (d) LTZ-CS-NPs-TPP-Freeze dried samples Figure 7: Biodegradation assay of unloaded and LTZ loaded chitosan nanoparticles Figure 8: Percentage cumulative drug release of LTZ from chitosan nanoparticles Figure 9: Hemo compatibility studies of LTZ loaded chitosan nanoparticles Figure 10: Serum Stability studies of LTZ loaded chitosan nanoparticles Scheme 1: Preparation of Letrozole loaded chitosan nanoparticles
29
Figure 1
30
(a)
1280.5
3448.1
1653
1183
% T
(b)
1656
3421.88 2377
1234
1223 1171.88
(c) 1657
3448 2230.27
(d)
1220.72 3411.46 1636.30
4000
3500
3000
2500
cm-1
2000
Figure 2
31
1500
1000
500
110
1.6
100
(b)
1.4
90 1.2
(c)
(a)
70
(d)
60
0.8 50
(c) 0.6
40 30
0.4
(b) (d) (a)
20
0.2
10 0 0
200
400
600
Temperature (°C) Figure 3
32
1.0
800
0.0 1000
Deriv. weight (%/°C)
Weight (%)
80
Figure 4
33
(a)
(c)
(b)
(d)
Figure 5
34
(a)
(b)
(c)
(d)
Figure 6
35
Figure 7
36
Figure 8
37
Figure 9
38
Figure 10
Scheme 1
39
Table 1: Effect of drug concentration on the particle size of LTZ loaded CS-NPs Ratio LTZ (mg) CS (mg) TPP (mg) Avg. Particle size (nm) (CS:TPP:LTZ) 5 50 40 1:0.8:0.1 58.77 10 50 40 1:0.8:0.2 78.82 15 50 40 1:0.8:0.3 91.28
Table 2: Effect of drying method on the particle size of LTZ-CS-NPs formulation Ratio Drying method Avg. Particle size (nm) CS TPP LTZ 1 0.8 0.1 58.77 Air 1 0.8 0.1 24.36 Freeze
Table 3: XRD details of letrozole, chitosan nanoparticles and letrozole loaded chitosan nanoparticles Samples 2θ LTZ 11.2°, 13.12°, 14.16°, 16.24°, 17.16°, 19.72°, 21°, 21.44°, 22°, 23.16°, 25.08°, 25.52°, 26.8° CS-NPs-TPP-Air dried 11.36, 20.64 and 22 CS-NPs-TPP-Freeze dried 13.68, 18.4 and 23.38 LTZ-CS-NPs-TPP-Air dried 11.44° and 18.04° LTZ-CS-NPs-TPP-Freeze dried 11.38°, 18.36° and 23.04° Table 4: Statistical parameters of various formulations obtained after fitting the drug release data to various release kinetic models LTZ-CS-NPs Zero First Higuchi HixsonKorsmeyerorder order Crowell Peppas (1:0.8:0.1)-TPP-Air dried (1:0.8:0.1)-TPP-Freeze dried (1:0.8:0.2)- TPP-Air dried (1:0.8:0.3)- TPP-Air dried
R2 0.9833 0.9944 0.9888 0.9967
R2 0.9766 0.9969 0.9829 0.9935
40
R2 0.9159 0.9868 0.9366 0.9598
R2 0.9884 0.9845 0.9918 0.9950
n 0.0941 0.1546 0.1203 0.1063
R2 0.7187 0.9618 0.8304 0.8356