International Journal of Pharmaceutics 468 (2014) 214–222
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Studies on drug-polymer interaction, in vitro release and cytotoxicity from chitosan particles excipient Thandapani Gomathi a , C. Govindarajan b , Maximas H. Rose H.R. c , P.N. Sudha a, ** , P.K. Mohamed Imran d, Jayachandran Venkatesan e, * , Se-Kwon Kim e, * a
Department of Chemistry, D.K.M. College for Women, Vellore, Tamil Nadu, India Indian Institute of Chromatography and Mass Spectrometry, Chennai, Tamil Nadu, India c Manonmaniam Sundaranar University, Tirunelveli, Tamil Nadu, India d Department of Chemistry, Islamiah College, Vaniyambadi, Tamil Nadu, India e Department of Marine Bio Convergence Science and Marine Bioprocess Research Center, Pukyong National University, Busan 608-737, South Korea b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 January 2014 Received in revised form 24 March 2014 Accepted 10 April 2014 Available online 15 April 2014
Nonobvious controlled polymeric pharmaceutical excipient, chitosan nanoparticles (CS-NPs) for lenalidomide encapsulation were geared up by the simple ionic cross linking method to get better bioavailability and to reduce under as well as overloading of hydrophobic and sparingly soluble drug lenalidomide towards cancer cells. Lenalidomide loaded chitosan nanoparticles (LND-CS-NPs) were in the size range of 220–295 nm and characterized by DLS, TEM, FT-IR, TGA and XRD. Encapsulation of lenalidomide over chitosan nanoparticles was observed about 99.35% using UV spectrophotometry method. In vitro release and the cytotoxic studies were performed using LND-CS-NPs. This study implies the new drug delivery route for lenalidomide and illustrates that the CS-NPs serves as the effective pharmaceutical carrier for sustained delivery of lenalidomide. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Pharmaceutical carrier Biodegradable polymer New drug delivery route Nanoparticles Nanoencapsulation Lenalidomide
1. Introduction Biopolymers with attractive characteristic properties acquire more attention in biomedical applications. Chitosan, the second most abundant biopolymer (cationic polysaccharide) is obtained by partial deacetylation of chitin. Chitosan is an most advantageous material with many promising applications. Biomedical application is one of them. Evidently, literature is galore with concern about their safety, toxicology and biodegradable consideration. Chitosan and its derivatives have been used as gene carriers due to their less toxic nature (Lee et al., 1998) and carriers for directly compressed tablets (Kristmundsdottir et al., 1995). They have been
Abbreviations: LND, Lenalidomide; C-NPs, Chitosan nanoparticles; LND-CS-NPs, Lenalidomide loaded chitosan nanoparticles; DLS, Dynamic light scattering; PBS, Phosphate buffer solution; MTT, 3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyl tetrazolium bromide. * Corresponding authors. Tel.: +82 51 629 7094; fax: +82 51 629 7099. ** Corresponding author at: PG and Research Department of Chemistry, DKM College for Women, Vellore, Tamil Nadu, India. Tel.: +91 9842910157. E-mail addresses: [email protected], [email protected] (P.N. Sudha), [email protected] (J. Venkatesan), [email protected] (S.-K. Kim). http://dx.doi.org/10.1016/j.ijpharm.2014.04.026 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
used as blood anticoagulants as well as hypocholesterolemic agents (Agnihotri et al., 2004). Due to their good muco adhesive character, chitosan and its derivatives are used as pharmaceutical carriers for sustained drug release (Hirano, 1996). Chitosan derivatives are used in tissue engineering, wound healing (Jayakumar et al., 2005; Madhumathi et al., 2010) and to enhancing the bioavailability and dissolution rates of hydrophobic drugs (Miyazaki et al., 1981). Nowadays chitosan based micro and nanoparticles are playing a vital role in sustained and targeted drug delivery (Dev et al., 2010; Sanoj Rejinold et al., 2011a,b). Lenalidomide, a thalidomide analog is an IMiDs immunomodulatory compound used for the treatment of myelodysplastic syndromes, with pleiotropic activities including induction of apoptosis, inhibition of angiogenesis and broad immunomodulatory effects (Kastritis and Dimopoulos, 2007; Richardson et al., 2006). The introduction of lenalidomide and other new anticancer agents, such as thalidomide and bortezomib, has a major impact on outcomes in patients with multiple myeloma (MM), significantly improving 5–10 years of survival rates (Brenner et al., 2009). Lenalidomide has dual mode action including tumoricidal and immunomodulatory effects (Morgan, 2010). Shaji Kumar and Vincent Rajkumar (2006), gave a detailed clinical response of
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thalidomide and lenalidomide in the treatment of multiple myeloma. Lenalidomide is an off-white to pale-yellow solid powder. As lenalidomide is an effective derivative of thalidomide in medical oncology therapeutics, lower solubility may limit its effectiveness. In general, the formulation of a drug in soluble form is much essential and more challenging. Many researchers extended their research to increase drug solubility, where the solubility renders stability of the compound. Nanoscience and nanotechnology are the focal points in recent years and propelled to the forefront by researchers from both academia and industry (Park, 2000; Hughes, 2005; Whitesides, 2005). In general the nanoparticulate acts as a promising drug delivery system, by bringing about substantial changes in drug biodistribution, minimizing toxicity and other adverse effects associated with important drugs. When the drug delivery device with a particle size about >5 mm is used, it accumulates in the lungs (Sato et al., 1996). Therefore, nanoencapsulation glitters with remediating advantages on these aspects; they also include the enhanced stability of labile drugs, enhanced drug bioavailability and controlled drug release owing to the fact that particles in the nanosize ranges are efficient in crossing permeability barriers (Sharma et al., 2004). In recent days, biopolymeric nanoparticles have been focused as potential drug carriers which led to the development of chemotherapeutic oncology. From literature, as we have understood, chitosan nanoparticles play a vital role as good pharmaceutical excipients. Chitosan nanocarrier is a system, having the capacity to cross biological barriers, to protect macromolecules like proteins, oligonucleotides and genes from degradation in biological media, and to deliver drugs or macromolecules to a target site with controlled release (Lopez-Leon et al., 2005). Thus with indispensable interest, chitosan nanoparticulates were prepared and used as a vehicle for sustained release of lenalidomide, where chitosan nanocarriers enhance the water solubility of lenalidomide and the bioavailability by enhancing their permeability across physiological barriers. Moreover, the chitosan nanoparticulate has high drug-loading capacity, a controlled release profile for the incorporated drug and good compatibility between the core-forming polymeric block and the incorporated drug. In the present study, an attempt was made to prepare the novel chitosan nanoparticles as carriers for the COOH CH3
COOCH3 CH3
a
NO2
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hydrophobic drug lenalidomide. Lenalidomide was synthesized, encapsulated in chitosan nanoparticles and characterized using FTIR, TGA, XRD, TEM and DLS. In vitro sustained release study was also performed to see the effectiveness of the carrier. 2. Materials and methods 2.1. Materials Chitosan (CS) with the degree of deacetylation of 92% was procured from India Sea Foods, Cochin, Kerala. Lenalidomide (LND) was in house synthesized (Kapoor et al., 2011). Sodium hexametaphosphate (SHMP) was purchased from Fischer Scientific, Mumbai and used without further purification. All other chemicals and reagents used are analytical grade. 2.2. Preparation of lenalidomide 2-Methyl-3-nitro benzoic acid was esterified using thionyl chloride in the presence of methanol to give methyl ester of 2methyl-3-nitro benzoic acid. Prepared methyl ester was brominated using N-bromo succinimide yielding bromo compound. This bromo compound was coupled with 3-amino piperidine-2,6-dione hydrochloride using triethylamine in the mixture of acetonitrile and dimethylformamide which formed 3-(4-nitro-1-oxoisoindoline-2-yl)piperidine-2,6-dione. Reduction of 3-amino piperidine2,6-dione hydrochloride using triethylamine in the mixture of acetonitrile and dimethylformamide gave 3-(4-nitro-1-oxoisoindoline-2-yl)piperidine-2,6-dione. In the presence of palladium, 3(4-nitro-1-oxoisoindoline-2-yl)piperidine-2,6-dione was reduced to get crude lenalidomide, and it was recrystalized using methanol to obtain a pure lenalidomide as shown in Scheme 1 (Kapoor et al., 2011). HPLC purity: >99.00% and the structure was confirmed by 1 HNMR and IR spectroscopy. 2.3. Preparation of chitosan nanoparticles 50 mg of chitosan was dispersed in 10 ml of aqueous acetic acid (2% v/v) solution and continuously stirred for about 20 min at 600 rpm to obtain the homogeneous solution. 5 ml (0.8% w/v) of SHMP solution was used as a crosslinker (Calvo et al., 1997). The
O
COOCH3 b
NH2.HCl
Br
NO2
NH O
NO2
c
NH2
O NH O
NO2
O d
N O
NH O
N O
Reagents and conditions: (a) Thionyl chloride, Methanol, 4h, 650 C; (b) N- Bromosuccinimide, Azoisobutyronitrile, Carbon tetrachloride, 3h, 80oC; (c) Triethylamine, Dimethylformamide, Acetonitrile, 10h, 55oC; (d) 10% Palladium on charcoal, Acetonitrile, Methanol, H2, 1h, 25oC. Scheme 1. Synthesis of lenalidomide.
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Na O
Na O
P
P
O
O
O HO
OH O
O
O
O *
O
O HO
HO
O P
NaO NH3
NH3
P
*
O Na O
n
Chitosan
O P
O P
O Na O O Sodium hexametaphosphate
Na O
Chitosan nanoparticles (Figure 1) Scheme 2. Preparation of chitosan nanoparticles.
resulting chitosan nanoparticle suspension (Scheme 2) was subsequently centrifuged for about 45 min at 12,000 rpm and re-suspended in water for washing followed by air drying. The sample was used for further investigation. The same procedure was repeated by varying the concentration of SHMP to study the effect of crosslinking towards the particle size (Table 1). 2.4. Preparation of lenalidomide loaded chitosan nanoparticles Lenalidomide loaded chitosan nanoparticles were prepared by ionic geleation using SHMP as the crosslinking agent (Scheme 2). Biopolymer chitosan (50 mg in 5 ml of (2% v/v) acetic acid) was stirred to get the transparent homogeneous solution. Lenalidomide (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 min. SHMP solution (38.00 mg in 5 ml deionised water) was added dropwise to the chitosan/lenalidomide solution over a period of 60 min at a stirring speed in the range of 550–600 rpm (Scheme 3). 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 air drying. 2.5. Characterization of nanoparticles FT-IR spectroscopy was measured for the determination of the types of bonds present in the nanoparticulate. FT-IR spectra of CSNPs, LND and LND-CS-NPs were carried out using KBr tablets (1% w/w of product in KBr) with a resolution of 4 cm1 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 transmission electron microscopy analysis (TEM) was conducted to observe the size, shape and surface morphology of the
Table 1 Particle size distribution. Chitosan (mg)
SHMP (mg)
Lenalidomide (mg)
Particle size (nm)
50 50 50 50 50 50
15 25 40 40 40 40
– – – 5 10 15
455.6 389.4 122.3 125.7 193.4 225.0
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). A 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). 2.6. Lenalidomide loading measurements The quantity of lenalidomide (LND) entrapped in the nanoparticulate system was determined indirectly by measuring the quantity of lenalidomide remaining in the supernatant based on the absorbance of the samples at 250 nm (Albertoni et al., 1994). The standard curve was obtained, and the sample absorbance was measured in 3 ml quartz cuvettes using Shimadzu UV-1700 Pharma spec UV–visible 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: EE ¼
Total lenalidomide ðmgÞ free lenalidomide ðmgÞ 100 Total lenalidomide ðmgÞ
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. 2.7. In vitro drug release studies In vitro drug release profile of lenalidomide from drug loaded chitosan nanoparticles were done by direct dispersion method as explained in literature (Bisht et al., 2007; Anitha et al., 2011; Müller et al., 2001). In vitro drug release studies were done for a period of one week at pH 7.4. 10 mg of LND loaded CS-NPs were taken in 50 ml 10 mM PBS in a beaker under magnetic stirring at 100 rpm. The receptor phase was stirred and thermally controlled at 37 C. The base absorbance of the release media was accounted by using the release medium as the solution in the UV-
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217
O HO
OH
O
O O *
O HO NH3
*
HO NH3
O
N NH
n
Chitosan
O
NH2
Lenalidomide
SHMP
Lenalidomide loaded chitosan nanoparticles (LND-CS-NPs) (Figure 2) Scheme 3. Preparation of lenalidomide loaded chitosan nanoparticles.
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 at the lmax value of 250 nm. The cumulative percentage amount of drug release was calculated and plotted against time. 2.8. Cytotoxicity studies Cells lines representing the most common human cancers were obtained from the King Institute of Preventive Medicine, Chennai. These included human breast adenocarcinoma (MCF7) and human multiples myeloma cell lines (U266) cells were cultured in minimal essential medium (MEM) supplemented with 10% heat inactivated foetal bovine serum (FBS), 3% L-glutamine, 100 U/ml penicillin G and 100 mg/ml streptomycin (Hi media) grown at 37 C in a humidified atmosphere of 5% CO2 in air. Cytotoxicity assays were carried out using cell suspension, containing one lakh cells per millilitre seeded in each well of a 96well microtitre plate. Fresh medium containing different concentrations of the test sample was added after 24 h of seeding. 0.5 mg of drug is dissolved in 4.5 ml of dimethyl sulfoxide (DMSO) to obtain working concentration of 10 mg/ml. The working concentration was prepared fresh and filtered through 0.45 m filter before each assay. Control cells were incubated without the test sample and with DMSO. The small percentage of DMSO present in the wells (maximal 0.2%) was found not to affect the experiment.
3.2. Preparation of lenalidomide loaded chitosan nanoparticles LND loaded CS-NPs were synthesized by simple ionic crosslinking method with SHMP due to the coacervation reaction between two oppositely charged species. The interaction is mostly through hydrogen bonding. The prepared nanoparticles were dried and used for further characterization. The expected loading chemistry is shown in Fig. 2. NH2, the reactive functional group of chitosan gets protonated and interacts with the carbonyl (CQO) group of lenalidomide. The amine functionality of lenalidomide also interacts with the hydroxyl group of chitosan. The hydrogen bond interaction is enough to hold lenalidomide into/on chitosan nanoparticles. The formation of LND-CS-NPs with SHMP was confirmed by FT-IR analysis. 3.3. FT-IR studies FT-IR spectroscopy is an important tool for predicting the reaction progress. It gives details about the covalent bonds and hydrogen bonding present in the system. Chitosan, a biopolymer, when crosslinked with SHMP gives chitosan nanoparticles. The ratio between chitosan and SHMP is critical and does control the size and PDI of the nanoparticles. The formation of chitosan nanoparticles by the efficient crosslinking with SHMP was
HO
OH
O
O
NH3+
NH3+
3. Results and discussion
-
3.1. Preparation of chitosan nanoparticles The cationic nature of chitosan made a notable achievement for the development of drug delivery systems. These cations contact with polyanions to form a gel through inter- and intramolecular crosslinkages. Ionic gelation (Vila et al., 2004) is a simple and straightforward technique 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 SHMP and NH2 group of chitosan (Fig. 1). The nanoparticles with different characteristics were prepared by varying concentrations of chitosan and SHMP (1:0.3; 1:0.5; 1:0.8).
*
O HO
O HO
O
O P
O
O
O
n
-
O
P
O O
P
O
P
HO
OH O
O +
P
O
O
P O
O-
O
H 3N
+
HO
H3N
HO
*
O
OH
O n* OH
Fig. 1. Crosslinking between chitosan and sodium hexametaphosphate.
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O
O
O
O
O
O N
O
N
O
NH
NH
NH
O
O P
O O
O
O
O
O
P
O
O
N
P
HO
O
OH O
HN
P
O
O
P
O
N +
O-
O
H3N
HO *
+
O
NH
O
-
O
NH3+ n
NH3+
O
O
O
O HO
NH3+
P
O
O
O
O HO
OH
OH
OH
O
NH2
NH2
NH2
NH2
N
NH3+
H3N
HO O
OH
HO
OH
H2N
O
NH2
O n
OH
H2N
H2N O N
O O
N HN
N
O O
O HN
HN
O
O O
Hydrogen bonding
Ionic interaction
Fig. 2. Proposed loading chemistry for lenalidomide with chitosan nanoparticles.
confirmed by FT-IR spectra. Also the drug–polymer interaction during encapsulation was confirmed by this study. The IR spectra of chitosan nanoparticles (CS-NPs) (Fig. 3) showed strong absorption band at 3446 cm1, indicating the presence of OH group and NH stretching vibrations. The wider peak indicates that hydrogen bonding has been enhanced in nanochitosan (Jia-hui et al., 1999). Primary amines also show a sharp peak between 3500 and 3400 cm1 which could be attributed to the asymmetric and symmetric stretching of N H bonds. The peaks at 2924.09 and 2852.72 cm1 were due to asymmetric and symmetric CH2 stretching vibration attributed to the pyranose ring. Asymmetric CQO stretching was observed at 1641.46 cm1 indicating the presence of amide I band (Qi et al., 2004). The peak at 1411 cm1 is due to CQO symmetric stretching in chitosan nanoparticles (Safee et al., 2010) and also the peak indicating PQO stretching at around 1200 cm1 (Wang and Wang, 2007). The peaks at 1382.96, 1271.09, 1157.20 cm1 were due to the presence of C O stretching, OH in plane bending and C O C
Fig. 3. FT-IR spectra of chitosan nanoparticles (CS-NPs) and lenalidomide loaded chitosan nanoparticles (LND-CS-NPs).
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linkage respectively. The peaks at 1070.49 and 1030.34 cm1 were for P O for PO43 and COH stretching and C N vibrations (Nagini Maganti et al., 2011) and peak at 877.61 cm1 was due to C C stretching vibration. The peak at 793.60 cm1 was indicative of N H wagging vibration. The IR spectrum of LND-CS-NPs (Fig. 3) showed the prominent peaks at 3415.93, 2926.10, 1631.78, 1529.55, and 1382.96 cm1. The broad peak at 3415.93 cm1 corresponds to OH and NH stretching vibration, peak broadening indicates the presence of intermolecular hydrogen bonding. The peak at 2926.10 cm1 is due to C H stretching vibration. Three characteristics peaks appeared at 1631.78, 1529.55 and 1382 cm1 due to the amide I, amide II and amide III bands, respectively. The possible LND-CS-NPs interaction is shown in Fig. 2. The peak in the region of around 1022–1153 cm1 shows the presence of PQO and P O stretching of PO4 of SHMP. On comparing the FT-IR spectrum of LND-CS-NPs with CS-NPs, the OH and NH peak reduction from 3146.79 to 3415.93 cm1 and also broadening of this peak indicate the participation of OH and NH groups of LND-CS-NPs in increased intermolecular hydrogen bond interaction between chitosan, crosslinking agent as well as with lenalidomide. The intensity of C H stretching peak at around 2926 cm1 and CQO stretching peak at 1631 cm1 gets increased due to the introduction of lenalidomide in chitosan nanoparticles. The peak for CQO stretching reducing to lowers wave number shows that the carbonyl group is involved in Van der Waals interaction. The NH2 group is the main functional group which is responsible for all the changes in the chitosan molecule. The lone pair of electron present in this amine group interacts with a proton to form an effective hydrogen bond formation. The spectrum clearly tells the finger print of lenalidomide encapsulation by chitosan nanocarrier. 3.4. Thermal analysis The physical process like evaporation and the chemical process like loss of volatile gases due to degradation are studied thoroughly using thermogravimetry. The thermogram shows the relationship between a sample’s mass and its temperature. Where polymers
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have different thermal stabilities, TG afforded the qualitative fingerprint in terms of temperature range, extent and kinetics of decomposition provides a rapid means to distinguish one polymer from another using only by a milligram quantity of the material (Price Duncan et al., 2000). The TGA of CS-NPs and LND-CS-NPs were studied (Fig. 4a and b). The results indicate that the degradation of both CS-NPs and LNDCS-NPs were started after 200 C. The initial loss is due to the elimination of moisture. From 200 to 240 C, the sudden decrease was observed which may be due to the loss of small molecules like NH3. After that the noticeable weight loss was at 250–300 C, due to the breaking of crosslinking and degradation of the polymer matrix may take place. Till 400–500 C, 30% of CS-NPs and 43% of LND-CSNPs were lost. At the end of the experiment at 870 C, 54.703% of CSNPs and 48.582% of LND-CS-NPs remained as a residue showing that the samples are having high thermal stabilities, which confirms the slower degradation rate. The DTA of the same materials were analyzed which supported the TG data. As per DTA, the characteristic endothermic peak was not observed which confirms the amorphous nature of the sample, high amorphous nature and thereby, better stability. The exothermic peak was found at around 230 C; also it was shifted to lower temperature ranges, and there is an increase in the amorphous area of the peak, i.e., the exothermic peak is broadened, which again confirmed the loaded LND-CS-NPs having high amorphous nature, which is evident from Fig. 4a and b. The more amorphous the therapeutic system, the higher would be its delivery efficiency (Abdelwahed et al., 2006). 3.5. XRD studies X-ray diffraction analysis was conducted in order to compare the physical nature of lenalidomide loaded chitosan nanoparticles (LND-CS-NPs) with the bare lenalidomide (LND) and its nanocarrier. Pure lenalidomide was crystalline in nature. Where LNDCS-NPs are amorphous in nature, the broadening of the peak confirms it (Fig. 5). When compared with the XRD of chitosan (figure not given), the 2u values at 10 and 19.5 of chitosan were disappearing/merging to give the broad peak at 18.35 during the
Fig. 4. TGA thermogram of (a) chitosan nanoparticles (CS-NPs); (b) lenalidomide loaded chitosan nanoparticles (LND-CS-NPs).
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there was a change in physical properties of nanoparticles (Cheng et al., 2008). The increase in drug concentration from 5 to 15 mg, increases the size of the nanoparticles, confirming the loading of drug into chitosan nanoparticles. The bare chitosan nanoparticles with ratio (1:0.8) showed a size range of 105.7–122.4 nm (Fig. 7a). This ratio has a comparatively small size in nanorange. Thus, this ratio was chosen to load the anticancer drug lenalidomide. While the lenalidomide loaded chitosan nanoparticles showed a size range of 220–295 nm (Fig. 7b), 122.4 nm is the optimum size of bare chitosan nanoparticles with 99% intensity, and 225 nm is for lenalidomide loaded chitosan nanoparticles with 71.4% intensity. The results showed that the SHMP crosslinker drastically reduce the size of chitosan to the nanolevel, and the encapsulation of lenalidomide into chitosan nanoparticles slightly increases the particle size. 3.8. Drug encapsulation Fig. 5. XRD of chitosan nanoparticles (CS-NPs), lenalidomide loaded chitosan nanoparticles (LND-CS-NPs) and lenalidomide.
formation of chitosan nanoparticles and lenalidomide loaded chitosan nanoparticles. The amorphous nature can be attributed by the crosslinking mechanism between the reactive functional groups of chitosan and sodium hexametaphosphate as well as with lenalidomide through hydrogen bonding. 3.6. TEM analysis The transmission electron microscope (TEM) images (Fig. 6a and b) show the morphology and size of chitosan nanoparticles and LND-CS-NPs. The TEM images confirms that the particles are in nanorange. The unloaded chitosan nanoparticles and lenalidomide loaded chitosan nanoparticles are in the size range of 120 and 220 nm. The image (Fig. 6b) shows that the drug was adsorbed on the surface as well as encapsulated in the matrix of nanocarrier. Looking at the image of a pure chitosan nanoparticle, the reflection of signal on the surface of the nanoparticle indicated that the surface was relatively smooth in the scale of observation. Lenalidomide encapsulated chitosan nanoparticles has a relatively rough surface confirming the surface adsorption. Also, it can be deduced that the dimple or porosity were not too deep. This suggested that the primary drug release mechanism would be diffusion through the material matrix, channels and pores. 3.7. Particle size analysis Particle size and size distribution are the key parameters used for evaluating the physical stability and activity of nanoparticles. The physical properties are an important driving force for efficient loading and releasing mechanism. The effect of crosslinking due to the concentration of SHMP was exhibited by the particle size of distribution (Table 1). During incorporation of hydrophobic drug,
Encapsulation efficiency (EE) is one of the key criteria for evaluating nanoencapsulation process. It can be seen from the literature that formulations with high EE ranges from 90.1% to 100% will have high loading capacity, which is the essential criteria to reduce the quantity of the carrier required for administration. Drug loading can be accomplished by two methods: (i) incorporating the drug during nanoparticle production, (ii) adsorbing the drug after the formation of nanoparticles by incubating in the drug solution. Thus, in the present work the drug was incorporated at the time of nanoparticle formation. The nanoencapsulation efficiency of lenalidomide was measured by UV-spectrum, and it was found to be 99.35%. It is thus evident that a large amount of drug can be entrapped by the incorporation method when compared to the adsorption (Alenso et al., 1991; Ueda et al., 1998). 3.9. Drug release The drug release study was carried out in triplicate to control the homogeneity of the drug release, and the results were averaged. Initially lenalidomide was rapidly released from the nanoparticles followed by slow release. The initial fast release could be due to the rapid dissolution of surface adhered/entrapped lenalidomide. The later slow release was due to the penetration of release medium into the nanoparticles and dissolves the entrapped drug. When SHMP was used, it led to higher crosslinking which reduces the free volume of the matrix, thereby hindering the easy transport of drug molecules from the matrix (Mi et al., 2003). The drug release curve is shown in Fig. 8. Normally, three basic mechanisms, namely swelling/erosion, diffusion, and degradation are present for the release of the loaded drug from polymeric particles (Schwendeman et al., 1996; Langer, 1990). In general, drug release mechanism may occur by following any one of the method or all the methods given above. In our case, the degradation of nanocarrier was slow; therefore, the release
Fig. 6. TEM of (a) chitosan nanoparticles (CS-NPs); (b) lenalidomide loaded chitosan nanoparticles (LND-CS-NPs).
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221
Fig. 7. Particle size plot of (a) chitosan nanoparticles (CS-NPs); (b) lenalidomide loaded chitosan nanoparticles (LND-CS-NPs).
mechanism of lenalidomide from LND-CS-NPs may depend on the drug diffusion or the nanosurface. In the present typical matrix delivery system, the polymer, drug, and additive had been mixed to form a homogeneous system, in which the diffusion occurred when the drug passes from the uniform blending matrix onto the external environment. As the release continued, the rate of drug release was decreased with this type of system, as the drug had progressively a longer distance to travel and thus, required a longer diffusion time to release. Usually slow release would be considered for in vivo studies and applications. 3.10. Cytotoxicity studies
cells) and U266 (multiple myeloma cells) cell lines. Earlier studies proved that the prepared CS-NPs were working as a carrier, and it was found nontoxic to MCF7 and U266 cell lines in various concentrations (Prabaharan and Jayakumar, 2009). LND-CS-NPs were prepared in eight different concentrations (varying from 1.00 to 100.00 mg/ml) and tested against MCF7 and U266 cell lines; results imply (Fig. 9) that LND-CS-NPs has showed specific toxicity against both MCF7 and U266 cell lines. Comparatively LND-CS-NPs are more toxic towards U266 cell lines than MCF7 cell lines. Inhibitory concentration 50 (IC50) values were calculated from concentration and response, IC50 value of LND-CS-NPs against MCF7 cell lines was 48.95 mg/ml. On the other hand, the same for U266 cell
Percentage cell viability and the cytotoxicity of the LND-CS-NPs were tested by MTT assay method against MCF7 (breast cancer
Fig. 8. Cumulative percentage of drug release.
Fig. 9. MTT assay of LND-CS-NPs.
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line was 24.56 mg/ml, while comparing the IC50 values of both cell lines, LND-CS-NPs shows good potency against U266 cell lines. 4. Conclusion Biodegradable polymeric nanoparticle of chitosan was incorporated with the highly potent and sparingly soluble anticancer drug lenalidomide. Prepared CS-NPs and LND-CS-NPs were characterized by FT-IR, TGA, DLS and TEM. TEM results confirmed that the prepared LND-CS-NPs were nanosize in range. The size distribution of CS-NPs and LND-CS-NPs was confirmed by DLS study. The drug encapsulation and release studies conclude that the novel LND-CS-NPs are showing sustained and effective drug release towards the target (cancer cell). Cytotoxic study reveals that LND-CS-NPs are toxic towards cancer cell lines such as MCF7 and U266. The drug incorporation and release profile tell that the chitosan nanoparticles were a novel, successful drug delivery system for chemotherapeutic lenalidomide. References Abdelwahed, W., Degobert, G., Stainmesse, S., Fessi, H., 2006. Freeze-drying of nanoparticles: formulation, process and storage considerations. Advanced Drug Delivery Reviews 58, 1688–1713. Agnihotri, S.A., Mallikarjuna, N.N., Aminabhavi, T.M., 2004. Recent advances on chitosan-based micro- and nanoparticles in drug delivery. Journal of Controlled Release 100, 5–28. Albertoni, F., Pettersson, B., Reichelova, V., Juliusson, G., Liliemark, J., 1994. Therapeutic Drug Monitoring 16, 413–418. Alenso, M.J., Losa, C., Calvo, P., Vila-jato, J.L., 1991. Approaches to improve the association of amikacinsulphate to poly-(cyanoacrylate) nanoparticles. International Journal of Pharmaceutics 68, 69–76. Anitha, A., Maya, S., Deepa, N., Chennazhi, K.P., Nair, S.V., Jayakumar, R., 2011. Curcumin-loaded N,O-carboxymethyl chitosan nanoparticles for cancer drug delivery. Journal of Biomaterials Science, Polymer Edition [Epub ahead of print]. Bisht, S., Feldman, G., Soni, S., Ravi, R., Karikar, C., Maitra, A., Maitra, A., 2007. Polymeric nanoparticle-encapsulated curcumin (“nanocurcumin") a novel strategy for human cancer therapy. Journal of Nanobiotechnology 5, 1–18. Brenner, H., Gondos, A., Pulte, D., 2009. Expected long-term survival of patients diagnosis with multiple myeloma in 2006–2010. Haematologica 94, 270–275. Calvo, P., Remunan-Lopez, C., Vila-jato, J.L., Alonso, M.J., 1997. Novel hydrophilic chitosan-polyethylene oxide nanoparticles as protein carriers. Journal of Applied Polymer Science 63, 125–132. Cheng, Y., Samia, A.C., Meyers, J.D., Panagopoulos, I., Fei, B., Burda, C., 2008. Highly efficient drug delivery with gold nanoparticles vectors for in vivo photodynamic therapy of cancer. Journal of the American Chemical Society 32, 10643–10647. Dev, A., Binulal, N.S., Anitha, A., Nair, S.V., Furuike, T., Tamura, H., Jayakumar, R., 2010. Preparation of poly(lactic acid)/chitosan nanoparticles for anti-HIV drug delivery applications. Carbohydrate Polymers 80, 833–838. Hirano, S., 1996. Chitin biotechnology applications. Biotechnology Annual Review 2, 237–258. Hughes, G.A., 2005. Nanostructure-mediated drug delivery. Nanomedicine: Nanotechnology Biology and Medicine 1, 22–30. Jayakumar, R., Prabakaran, M., Reis, R.L., Mano, J.F., 2005. Graft copolymerized chitosan – present status and applications. Carbohydrate Polymers 62, 142–158. Jia-hui, Y., Yu-min, D., Hua, Z., 1999. Blend films of chitosan–gelatin. Journal of Wuhan University 45, 440–444. Kapoor, M., Kumar, S., Murugesan, B., Sathyanarayana, S., Thaper, R., Prasad, M., 2011. Process for the Preparation of Lenalidomide. WO 2011/027326 A1. Kastritis, E., Dimopoulos, M.A., 2007. The evolving role of lenalidomide in the treatment of hematologic malignancies. Expert Opinion on Pharmacotherapy 8, 497–509. Kristmundsdottir, T., Ingvarsdottir, K., Saemundsdottir, G., 1995. Chitosan matrix tablets: the influence of excipients on drug release. Drug Development and Industrial Pharmacy 21, 1591–1598. Langer, R., 1990. New methods of drug delivery. Science 249, 1527–1533. Lee, K.Y., Kwon, I.C., Kim, Y.H., 1998. Preparation of chitosan self-aggregates as a gene delivery system. Journal of Control Release 51, 213–220.
Lopez-Leon, T., Carvalho, E.L.S., Seijo, B., Ortego-Vinuesa, J.L., Basttos-Gonzalez, D., 2005. Physicochemical characterization of chitosan nanoparticles: electrokinetic and stability behavior. Journal of Colloid and Interface Science 283, 344– 351. Madhumathi, K., Sudheeshkumar, P.T., Abilash, S., Sreeja, V., Tamura, H., Manzoor, K., 2010. Development of novel chitin/nanosilver composite scaffolds for wound dressing applications. Journal of Materials Science: Materials in Medicine 21, 807–813. Mi, F.L., Sung, H.W., Shyu, S.S., Su, C.C., Peng, C.K., 2003. Synthesis and characterization of biodegradable TPP/genipin co-crosslinked chitosan gel beads. Polymer 44, 6521–6530. Miyazaki, S., Ishii, K., Nadai, T., 1981. The use of chitin and chitosan as drug carriers. Chemical and Pharmaceutical Bulletin 29, 3067–3069. Morgan, G., 2010. Future drug developments in multiple myeloma: an overview of novel lenalidomide-based combination therapies. Blood Reviews 24, S27–S32. Müller, R.H., Jacobs, C., Kayser, O., 2001. Nanosuspensions as particulate drug formulations in therapy. Rationale for development and what we can expect for the future. Advanced Drug Delivery Reviews 47, 3–19. Nagini Maganti, Pavan, K.C., Venkat Surya, Wah Wah Thein-Han, Thomas Pesacreta, C., Devesh, R., Misra, K., 2011. Structure–process–property relationship of biomimetic chitosan-based nanocomposite scaffolds for tissue engineering: biological, physico-chemical, and mechanical functions. Advanced Engineering Materials 13, B108–B122. Park, K., 2000. Nanotechnology: What it can do for drug delivery. Journal of Controlled Release 120, 1–3. Prabaharan, M., Jayakumar, R., 2009. Chitosan-graft-[beta]-cyclodextrin scaffolds with controlled drug release capability for tissue engineering applications. International Journal of Biological Macromolecules 44, 320–325. Price Duncan, M., Hourston Douglas, J., Fabrice Dumont, 2000. Encyclopedia of Analytical Chemistry, Thermogravimetry of Polymers. In: Meyers, R.A. (Ed.), John Wiley & Sons Ltd., Chichester, pp. 8094–8105. Qi, X., Li, T.G., Hao, J., Hu, J., Wang, J., Simmons, H., Miura, S., Mishina, Y., Zhao, G.Q., 2004. BMP4 supports self-renewal of embryonic stem cells by inhibiting mitogen-activated protein kinase pathways. Proceedings of the National Academy of Sciences of the United States of America 101, 6027–6032. Richardson, P.G., Mitsiades, C., Hideshima, T., Anderson, K.C., 2006. Lenalidomide in multiple myeloma. Expert Review of Anticancer Therapy 6, 1165–1173. Nurul Hidayah Ahmad Safee, Pauzi Absullah, Mohammed Rozali Othman, 2010. Carboxymethyl chitosan-Fe3O4 nanoparticles: synthesis and characterization. Malaysian Journal of Analytical Sciences 14, 63–68. Sanoj Rejinold, N., Muthunarayanan, M., Divyarani, V.V., Sreerekha, P.R., Chennazhi, K.P., Nair, S.V., Tamura, H., Jayakumar, R., 2011a. Curcumin-loaded biocompatible thermoresponsive polymeric nanoparticles for cancer drug delivery. Journal of Colloid and Interface Science 360, 39–51. Sanoj Rejinold, N., Muthunarayanan, M., Chennazhi, K.P., Tamura, H., Jayakumar, R., 2011b. 5-Fluorouracil loaded fibrinogen nanoparticles for cancer drug delivery applications. International Journal of Biological Macromolecules 48, 98–105. Sato, H., Wang, Y.M., Adachi, I., Horikoshi, I., 1996. Pharmacokinetic study of taxolloaded poly(lactic-co-glycolic acid) microspheres containing isopropyl myristate after targeted delivery to the lung in mice. Biological and Pharmaceutical Bulletin 19, 1596–1601. Schwendeman, S.P., Cardamone, M., Brandon, M.R., Klibanov, A., Langer, R., 1996. The stability of proteins and their delivery from biodegradable polymer microspheres. In: Cohen, S., Bernstein, H. (Eds.), Microparticulate Systems for the Delivery of Proteins and Vaccines. Marcel Dekker, New York, pp. 1–49. Shaji Kumar, Vincent Rajkumar, S., 2006. Thalidomide and lenalidomide in the treatment of multiple myeloma. European Journal of Cancer 42, 1612–1622. Sharma, A., Pandey, R., Sharma, S., Khuller, G.K., 2004. Chemotherapeutic efficacy of poly(DL-lactide-co-glycolide) nanoparticle encapsulated antitubercular drugs at sub-therapeutic dose against experimental tuberculosis. International Journal of Antimicrobial Agents 24, 599–604. Ueda, M., Iwara, A., Kreuter, J., 1998. Influence of the preparation methods on the drug release behaviour of loperamide-loaded nanoparticles. Journal of Microencapsulation 15, 361–372. Vila, A., Sanchez, A., Janes, K., Behrens, I., Kissel, T., Vila Jato, J.L., Alonso, M.J., 2004. Low molecular weight chitosan nanoparticles as new carriers for nasal vaccine delivery in mice. European Journal of Pharmaceutics and Biopharmaceutics 57, 123–131. Wang, L., Wang, A.Q., 2007. Adsorption characteristics of congo red onto the chitelsan/montmorillonite nanocomposite. Journal of Hazardous Materials 147, 979–985. Whitesides, G.M., 2005. Nanoscience, nanotechnology and chemistry, nanoscience and chemistry. Small 1, 172–179.
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Studies on drug-polymer interaction, in vitro release and cytotoxicity from chitosan particles excipient Thandapani Gomathi a , C. Govindarajan b , Maximas H. Rose H.R. c , P.N. Sudha a, ** , P.K. Mohamed Imran d, Jayachandran Venkatesan e, * , Se-Kwon Kim e, * a
Department of Chemistry, D.K.M. College for Women, Vellore, Tamil Nadu, India Indian Institute of Chromatography and Mass Spectrometry, Chennai, Tamil Nadu, India c Manonmaniam Sundaranar University, Tirunelveli, Tamil Nadu, India d Department of Chemistry, Islamiah College, Vaniyambadi, Tamil Nadu, India e Department of Marine Bio Convergence Science and Marine Bioprocess Research Center, Pukyong National University, Busan 608-737, South Korea b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 January 2014 Received in revised form 24 March 2014 Accepted 10 April 2014 Available online 15 April 2014
Nonobvious controlled polymeric pharmaceutical excipient, chitosan nanoparticles (CS-NPs) for lenalidomide encapsulation were geared up by the simple ionic cross linking method to get better bioavailability and to reduce under as well as overloading of hydrophobic and sparingly soluble drug lenalidomide towards cancer cells. Lenalidomide loaded chitosan nanoparticles (LND-CS-NPs) were in the size range of 220–295 nm and characterized by DLS, TEM, FT-IR, TGA and XRD. Encapsulation of lenalidomide over chitosan nanoparticles was observed about 99.35% using UV spectrophotometry method. In vitro release and the cytotoxic studies were performed using LND-CS-NPs. This study implies the new drug delivery route for lenalidomide and illustrates that the CS-NPs serves as the effective pharmaceutical carrier for sustained delivery of lenalidomide. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Pharmaceutical carrier Biodegradable polymer New drug delivery route Nanoparticles Nanoencapsulation Lenalidomide
1. Introduction Biopolymers with attractive characteristic properties acquire more attention in biomedical applications. Chitosan, the second most abundant biopolymer (cationic polysaccharide) is obtained by partial deacetylation of chitin. Chitosan is an most advantageous material with many promising applications. Biomedical application is one of them. Evidently, literature is galore with concern about their safety, toxicology and biodegradable consideration. Chitosan and its derivatives have been used as gene carriers due to their less toxic nature (Lee et al., 1998) and carriers for directly compressed tablets (Kristmundsdottir et al., 1995). They have been
Abbreviations: LND, Lenalidomide; C-NPs, Chitosan nanoparticles; LND-CS-NPs, Lenalidomide loaded chitosan nanoparticles; DLS, Dynamic light scattering; PBS, Phosphate buffer solution; MTT, 3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyl tetrazolium bromide. * Corresponding authors. Tel.: +82 51 629 7094; fax: +82 51 629 7099. ** Corresponding author at: PG and Research Department of Chemistry, DKM College for Women, Vellore, Tamil Nadu, India. Tel.: +91 9842910157. E-mail addresses: [email protected], [email protected] (P.N. Sudha), [email protected] (J. Venkatesan), [email protected] (S.-K. Kim). http://dx.doi.org/10.1016/j.ijpharm.2014.04.026 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
used as blood anticoagulants as well as hypocholesterolemic agents (Agnihotri et al., 2004). Due to their good muco adhesive character, chitosan and its derivatives are used as pharmaceutical carriers for sustained drug release (Hirano, 1996). Chitosan derivatives are used in tissue engineering, wound healing (Jayakumar et al., 2005; Madhumathi et al., 2010) and to enhancing the bioavailability and dissolution rates of hydrophobic drugs (Miyazaki et al., 1981). Nowadays chitosan based micro and nanoparticles are playing a vital role in sustained and targeted drug delivery (Dev et al., 2010; Sanoj Rejinold et al., 2011a,b). Lenalidomide, a thalidomide analog is an IMiDs immunomodulatory compound used for the treatment of myelodysplastic syndromes, with pleiotropic activities including induction of apoptosis, inhibition of angiogenesis and broad immunomodulatory effects (Kastritis and Dimopoulos, 2007; Richardson et al., 2006). The introduction of lenalidomide and other new anticancer agents, such as thalidomide and bortezomib, has a major impact on outcomes in patients with multiple myeloma (MM), significantly improving 5–10 years of survival rates (Brenner et al., 2009). Lenalidomide has dual mode action including tumoricidal and immunomodulatory effects (Morgan, 2010). Shaji Kumar and Vincent Rajkumar (2006), gave a detailed clinical response of
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thalidomide and lenalidomide in the treatment of multiple myeloma. Lenalidomide is an off-white to pale-yellow solid powder. As lenalidomide is an effective derivative of thalidomide in medical oncology therapeutics, lower solubility may limit its effectiveness. In general, the formulation of a drug in soluble form is much essential and more challenging. Many researchers extended their research to increase drug solubility, where the solubility renders stability of the compound. Nanoscience and nanotechnology are the focal points in recent years and propelled to the forefront by researchers from both academia and industry (Park, 2000; Hughes, 2005; Whitesides, 2005). In general the nanoparticulate acts as a promising drug delivery system, by bringing about substantial changes in drug biodistribution, minimizing toxicity and other adverse effects associated with important drugs. When the drug delivery device with a particle size about >5 mm is used, it accumulates in the lungs (Sato et al., 1996). Therefore, nanoencapsulation glitters with remediating advantages on these aspects; they also include the enhanced stability of labile drugs, enhanced drug bioavailability and controlled drug release owing to the fact that particles in the nanosize ranges are efficient in crossing permeability barriers (Sharma et al., 2004). In recent days, biopolymeric nanoparticles have been focused as potential drug carriers which led to the development of chemotherapeutic oncology. From literature, as we have understood, chitosan nanoparticles play a vital role as good pharmaceutical excipients. Chitosan nanocarrier is a system, having the capacity to cross biological barriers, to protect macromolecules like proteins, oligonucleotides and genes from degradation in biological media, and to deliver drugs or macromolecules to a target site with controlled release (Lopez-Leon et al., 2005). Thus with indispensable interest, chitosan nanoparticulates were prepared and used as a vehicle for sustained release of lenalidomide, where chitosan nanocarriers enhance the water solubility of lenalidomide and the bioavailability by enhancing their permeability across physiological barriers. Moreover, the chitosan nanoparticulate has high drug-loading capacity, a controlled release profile for the incorporated drug and good compatibility between the core-forming polymeric block and the incorporated drug. In the present study, an attempt was made to prepare the novel chitosan nanoparticles as carriers for the COOH CH3
COOCH3 CH3
a
NO2
215
hydrophobic drug lenalidomide. Lenalidomide was synthesized, encapsulated in chitosan nanoparticles and characterized using FTIR, TGA, XRD, TEM and DLS. In vitro sustained release study was also performed to see the effectiveness of the carrier. 2. Materials and methods 2.1. Materials Chitosan (CS) with the degree of deacetylation of 92% was procured from India Sea Foods, Cochin, Kerala. Lenalidomide (LND) was in house synthesized (Kapoor et al., 2011). Sodium hexametaphosphate (SHMP) was purchased from Fischer Scientific, Mumbai and used without further purification. All other chemicals and reagents used are analytical grade. 2.2. Preparation of lenalidomide 2-Methyl-3-nitro benzoic acid was esterified using thionyl chloride in the presence of methanol to give methyl ester of 2methyl-3-nitro benzoic acid. Prepared methyl ester was brominated using N-bromo succinimide yielding bromo compound. This bromo compound was coupled with 3-amino piperidine-2,6-dione hydrochloride using triethylamine in the mixture of acetonitrile and dimethylformamide which formed 3-(4-nitro-1-oxoisoindoline-2-yl)piperidine-2,6-dione. Reduction of 3-amino piperidine2,6-dione hydrochloride using triethylamine in the mixture of acetonitrile and dimethylformamide gave 3-(4-nitro-1-oxoisoindoline-2-yl)piperidine-2,6-dione. In the presence of palladium, 3(4-nitro-1-oxoisoindoline-2-yl)piperidine-2,6-dione was reduced to get crude lenalidomide, and it was recrystalized using methanol to obtain a pure lenalidomide as shown in Scheme 1 (Kapoor et al., 2011). HPLC purity: >99.00% and the structure was confirmed by 1 HNMR and IR spectroscopy. 2.3. Preparation of chitosan nanoparticles 50 mg of chitosan was dispersed in 10 ml of aqueous acetic acid (2% v/v) solution and continuously stirred for about 20 min at 600 rpm to obtain the homogeneous solution. 5 ml (0.8% w/v) of SHMP solution was used as a crosslinker (Calvo et al., 1997). The
O
COOCH3 b
NH2.HCl
Br
NO2
NH O
NO2
c
NH2
O NH O
NO2
O d
N O
NH O
N O
Reagents and conditions: (a) Thionyl chloride, Methanol, 4h, 650 C; (b) N- Bromosuccinimide, Azoisobutyronitrile, Carbon tetrachloride, 3h, 80oC; (c) Triethylamine, Dimethylformamide, Acetonitrile, 10h, 55oC; (d) 10% Palladium on charcoal, Acetonitrile, Methanol, H2, 1h, 25oC. Scheme 1. Synthesis of lenalidomide.
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Na O
Na O
P
P
O
O
O HO
OH O
O
O
O *
O
O HO
HO
O P
NaO NH3
NH3
P
*
O Na O
n
Chitosan
O P
O P
O Na O O Sodium hexametaphosphate
Na O
Chitosan nanoparticles (Figure 1) Scheme 2. Preparation of chitosan nanoparticles.
resulting chitosan nanoparticle suspension (Scheme 2) was subsequently centrifuged for about 45 min at 12,000 rpm and re-suspended in water for washing followed by air drying. The sample was used for further investigation. The same procedure was repeated by varying the concentration of SHMP to study the effect of crosslinking towards the particle size (Table 1). 2.4. Preparation of lenalidomide loaded chitosan nanoparticles Lenalidomide loaded chitosan nanoparticles were prepared by ionic geleation using SHMP as the crosslinking agent (Scheme 2). Biopolymer chitosan (50 mg in 5 ml of (2% v/v) acetic acid) was stirred to get the transparent homogeneous solution. Lenalidomide (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 min. SHMP solution (38.00 mg in 5 ml deionised water) was added dropwise to the chitosan/lenalidomide solution over a period of 60 min at a stirring speed in the range of 550–600 rpm (Scheme 3). 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 air drying. 2.5. Characterization of nanoparticles FT-IR spectroscopy was measured for the determination of the types of bonds present in the nanoparticulate. FT-IR spectra of CSNPs, LND and LND-CS-NPs were carried out using KBr tablets (1% w/w of product in KBr) with a resolution of 4 cm1 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 transmission electron microscopy analysis (TEM) was conducted to observe the size, shape and surface morphology of the
Table 1 Particle size distribution. Chitosan (mg)
SHMP (mg)
Lenalidomide (mg)
Particle size (nm)
50 50 50 50 50 50
15 25 40 40 40 40
– – – 5 10 15
455.6 389.4 122.3 125.7 193.4 225.0
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). A 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). 2.6. Lenalidomide loading measurements The quantity of lenalidomide (LND) entrapped in the nanoparticulate system was determined indirectly by measuring the quantity of lenalidomide remaining in the supernatant based on the absorbance of the samples at 250 nm (Albertoni et al., 1994). The standard curve was obtained, and the sample absorbance was measured in 3 ml quartz cuvettes using Shimadzu UV-1700 Pharma spec UV–visible 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: EE ¼
Total lenalidomide ðmgÞ free lenalidomide ðmgÞ 100 Total lenalidomide ðmgÞ
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. 2.7. In vitro drug release studies In vitro drug release profile of lenalidomide from drug loaded chitosan nanoparticles were done by direct dispersion method as explained in literature (Bisht et al., 2007; Anitha et al., 2011; Müller et al., 2001). In vitro drug release studies were done for a period of one week at pH 7.4. 10 mg of LND loaded CS-NPs were taken in 50 ml 10 mM PBS in a beaker under magnetic stirring at 100 rpm. The receptor phase was stirred and thermally controlled at 37 C. The base absorbance of the release media was accounted by using the release medium as the solution in the UV-
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217
O HO
OH
O
O O *
O HO NH3
*
HO NH3
O
N NH
n
Chitosan
O
NH2
Lenalidomide
SHMP
Lenalidomide loaded chitosan nanoparticles (LND-CS-NPs) (Figure 2) Scheme 3. Preparation of lenalidomide loaded chitosan nanoparticles.
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 at the lmax value of 250 nm. The cumulative percentage amount of drug release was calculated and plotted against time. 2.8. Cytotoxicity studies Cells lines representing the most common human cancers were obtained from the King Institute of Preventive Medicine, Chennai. These included human breast adenocarcinoma (MCF7) and human multiples myeloma cell lines (U266) cells were cultured in minimal essential medium (MEM) supplemented with 10% heat inactivated foetal bovine serum (FBS), 3% L-glutamine, 100 U/ml penicillin G and 100 mg/ml streptomycin (Hi media) grown at 37 C in a humidified atmosphere of 5% CO2 in air. Cytotoxicity assays were carried out using cell suspension, containing one lakh cells per millilitre seeded in each well of a 96well microtitre plate. Fresh medium containing different concentrations of the test sample was added after 24 h of seeding. 0.5 mg of drug is dissolved in 4.5 ml of dimethyl sulfoxide (DMSO) to obtain working concentration of 10 mg/ml. The working concentration was prepared fresh and filtered through 0.45 m filter before each assay. Control cells were incubated without the test sample and with DMSO. The small percentage of DMSO present in the wells (maximal 0.2%) was found not to affect the experiment.
3.2. Preparation of lenalidomide loaded chitosan nanoparticles LND loaded CS-NPs were synthesized by simple ionic crosslinking method with SHMP due to the coacervation reaction between two oppositely charged species. The interaction is mostly through hydrogen bonding. The prepared nanoparticles were dried and used for further characterization. The expected loading chemistry is shown in Fig. 2. NH2, the reactive functional group of chitosan gets protonated and interacts with the carbonyl (CQO) group of lenalidomide. The amine functionality of lenalidomide also interacts with the hydroxyl group of chitosan. The hydrogen bond interaction is enough to hold lenalidomide into/on chitosan nanoparticles. The formation of LND-CS-NPs with SHMP was confirmed by FT-IR analysis. 3.3. FT-IR studies FT-IR spectroscopy is an important tool for predicting the reaction progress. It gives details about the covalent bonds and hydrogen bonding present in the system. Chitosan, a biopolymer, when crosslinked with SHMP gives chitosan nanoparticles. The ratio between chitosan and SHMP is critical and does control the size and PDI of the nanoparticles. The formation of chitosan nanoparticles by the efficient crosslinking with SHMP was
HO
OH
O
O
NH3+
NH3+
3. Results and discussion
-
3.1. Preparation of chitosan nanoparticles The cationic nature of chitosan made a notable achievement for the development of drug delivery systems. These cations contact with polyanions to form a gel through inter- and intramolecular crosslinkages. Ionic gelation (Vila et al., 2004) is a simple and straightforward technique 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 SHMP and NH2 group of chitosan (Fig. 1). The nanoparticles with different characteristics were prepared by varying concentrations of chitosan and SHMP (1:0.3; 1:0.5; 1:0.8).
*
O HO
O HO
O
O P
O
O
O
n
-
O
P
O O
P
O
P
HO
OH O
O +
P
O
O
P O
O-
O
H 3N
+
HO
H3N
HO
*
O
OH
O n* OH
Fig. 1. Crosslinking between chitosan and sodium hexametaphosphate.
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O
O
O
O
O
O N
O
N
O
NH
NH
NH
O
O P
O O
O
O
O
O
P
O
O
N
P
HO
O
OH O
HN
P
O
O
P
O
N +
O-
O
H3N
HO *
+
O
NH
O
-
O
NH3+ n
NH3+
O
O
O
O HO
NH3+
P
O
O
O
O HO
OH
OH
OH
O
NH2
NH2
NH2
NH2
N
NH3+
H3N
HO O
OH
HO
OH
H2N
O
NH2
O n
OH
H2N
H2N O N
O O
N HN
N
O O
O HN
HN
O
O O
Hydrogen bonding
Ionic interaction
Fig. 2. Proposed loading chemistry for lenalidomide with chitosan nanoparticles.
confirmed by FT-IR spectra. Also the drug–polymer interaction during encapsulation was confirmed by this study. The IR spectra of chitosan nanoparticles (CS-NPs) (Fig. 3) showed strong absorption band at 3446 cm1, indicating the presence of OH group and NH stretching vibrations. The wider peak indicates that hydrogen bonding has been enhanced in nanochitosan (Jia-hui et al., 1999). Primary amines also show a sharp peak between 3500 and 3400 cm1 which could be attributed to the asymmetric and symmetric stretching of N H bonds. The peaks at 2924.09 and 2852.72 cm1 were due to asymmetric and symmetric CH2 stretching vibration attributed to the pyranose ring. Asymmetric CQO stretching was observed at 1641.46 cm1 indicating the presence of amide I band (Qi et al., 2004). The peak at 1411 cm1 is due to CQO symmetric stretching in chitosan nanoparticles (Safee et al., 2010) and also the peak indicating PQO stretching at around 1200 cm1 (Wang and Wang, 2007). The peaks at 1382.96, 1271.09, 1157.20 cm1 were due to the presence of C O stretching, OH in plane bending and C O C
Fig. 3. FT-IR spectra of chitosan nanoparticles (CS-NPs) and lenalidomide loaded chitosan nanoparticles (LND-CS-NPs).
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linkage respectively. The peaks at 1070.49 and 1030.34 cm1 were for P O for PO43 and COH stretching and C N vibrations (Nagini Maganti et al., 2011) and peak at 877.61 cm1 was due to C C stretching vibration. The peak at 793.60 cm1 was indicative of N H wagging vibration. The IR spectrum of LND-CS-NPs (Fig. 3) showed the prominent peaks at 3415.93, 2926.10, 1631.78, 1529.55, and 1382.96 cm1. The broad peak at 3415.93 cm1 corresponds to OH and NH stretching vibration, peak broadening indicates the presence of intermolecular hydrogen bonding. The peak at 2926.10 cm1 is due to C H stretching vibration. Three characteristics peaks appeared at 1631.78, 1529.55 and 1382 cm1 due to the amide I, amide II and amide III bands, respectively. The possible LND-CS-NPs interaction is shown in Fig. 2. The peak in the region of around 1022–1153 cm1 shows the presence of PQO and P O stretching of PO4 of SHMP. On comparing the FT-IR spectrum of LND-CS-NPs with CS-NPs, the OH and NH peak reduction from 3146.79 to 3415.93 cm1 and also broadening of this peak indicate the participation of OH and NH groups of LND-CS-NPs in increased intermolecular hydrogen bond interaction between chitosan, crosslinking agent as well as with lenalidomide. The intensity of C H stretching peak at around 2926 cm1 and CQO stretching peak at 1631 cm1 gets increased due to the introduction of lenalidomide in chitosan nanoparticles. The peak for CQO stretching reducing to lowers wave number shows that the carbonyl group is involved in Van der Waals interaction. The NH2 group is the main functional group which is responsible for all the changes in the chitosan molecule. The lone pair of electron present in this amine group interacts with a proton to form an effective hydrogen bond formation. The spectrum clearly tells the finger print of lenalidomide encapsulation by chitosan nanocarrier. 3.4. Thermal analysis The physical process like evaporation and the chemical process like loss of volatile gases due to degradation are studied thoroughly using thermogravimetry. The thermogram shows the relationship between a sample’s mass and its temperature. Where polymers
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have different thermal stabilities, TG afforded the qualitative fingerprint in terms of temperature range, extent and kinetics of decomposition provides a rapid means to distinguish one polymer from another using only by a milligram quantity of the material (Price Duncan et al., 2000). The TGA of CS-NPs and LND-CS-NPs were studied (Fig. 4a and b). The results indicate that the degradation of both CS-NPs and LNDCS-NPs were started after 200 C. The initial loss is due to the elimination of moisture. From 200 to 240 C, the sudden decrease was observed which may be due to the loss of small molecules like NH3. After that the noticeable weight loss was at 250–300 C, due to the breaking of crosslinking and degradation of the polymer matrix may take place. Till 400–500 C, 30% of CS-NPs and 43% of LND-CSNPs were lost. At the end of the experiment at 870 C, 54.703% of CSNPs and 48.582% of LND-CS-NPs remained as a residue showing that the samples are having high thermal stabilities, which confirms the slower degradation rate. The DTA of the same materials were analyzed which supported the TG data. As per DTA, the characteristic endothermic peak was not observed which confirms the amorphous nature of the sample, high amorphous nature and thereby, better stability. The exothermic peak was found at around 230 C; also it was shifted to lower temperature ranges, and there is an increase in the amorphous area of the peak, i.e., the exothermic peak is broadened, which again confirmed the loaded LND-CS-NPs having high amorphous nature, which is evident from Fig. 4a and b. The more amorphous the therapeutic system, the higher would be its delivery efficiency (Abdelwahed et al., 2006). 3.5. XRD studies X-ray diffraction analysis was conducted in order to compare the physical nature of lenalidomide loaded chitosan nanoparticles (LND-CS-NPs) with the bare lenalidomide (LND) and its nanocarrier. Pure lenalidomide was crystalline in nature. Where LNDCS-NPs are amorphous in nature, the broadening of the peak confirms it (Fig. 5). When compared with the XRD of chitosan (figure not given), the 2u values at 10 and 19.5 of chitosan were disappearing/merging to give the broad peak at 18.35 during the
Fig. 4. TGA thermogram of (a) chitosan nanoparticles (CS-NPs); (b) lenalidomide loaded chitosan nanoparticles (LND-CS-NPs).
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there was a change in physical properties of nanoparticles (Cheng et al., 2008). The increase in drug concentration from 5 to 15 mg, increases the size of the nanoparticles, confirming the loading of drug into chitosan nanoparticles. The bare chitosan nanoparticles with ratio (1:0.8) showed a size range of 105.7–122.4 nm (Fig. 7a). This ratio has a comparatively small size in nanorange. Thus, this ratio was chosen to load the anticancer drug lenalidomide. While the lenalidomide loaded chitosan nanoparticles showed a size range of 220–295 nm (Fig. 7b), 122.4 nm is the optimum size of bare chitosan nanoparticles with 99% intensity, and 225 nm is for lenalidomide loaded chitosan nanoparticles with 71.4% intensity. The results showed that the SHMP crosslinker drastically reduce the size of chitosan to the nanolevel, and the encapsulation of lenalidomide into chitosan nanoparticles slightly increases the particle size. 3.8. Drug encapsulation Fig. 5. XRD of chitosan nanoparticles (CS-NPs), lenalidomide loaded chitosan nanoparticles (LND-CS-NPs) and lenalidomide.
formation of chitosan nanoparticles and lenalidomide loaded chitosan nanoparticles. The amorphous nature can be attributed by the crosslinking mechanism between the reactive functional groups of chitosan and sodium hexametaphosphate as well as with lenalidomide through hydrogen bonding. 3.6. TEM analysis The transmission electron microscope (TEM) images (Fig. 6a and b) show the morphology and size of chitosan nanoparticles and LND-CS-NPs. The TEM images confirms that the particles are in nanorange. The unloaded chitosan nanoparticles and lenalidomide loaded chitosan nanoparticles are in the size range of 120 and 220 nm. The image (Fig. 6b) shows that the drug was adsorbed on the surface as well as encapsulated in the matrix of nanocarrier. Looking at the image of a pure chitosan nanoparticle, the reflection of signal on the surface of the nanoparticle indicated that the surface was relatively smooth in the scale of observation. Lenalidomide encapsulated chitosan nanoparticles has a relatively rough surface confirming the surface adsorption. Also, it can be deduced that the dimple or porosity were not too deep. This suggested that the primary drug release mechanism would be diffusion through the material matrix, channels and pores. 3.7. Particle size analysis Particle size and size distribution are the key parameters used for evaluating the physical stability and activity of nanoparticles. The physical properties are an important driving force for efficient loading and releasing mechanism. The effect of crosslinking due to the concentration of SHMP was exhibited by the particle size of distribution (Table 1). During incorporation of hydrophobic drug,
Encapsulation efficiency (EE) is one of the key criteria for evaluating nanoencapsulation process. It can be seen from the literature that formulations with high EE ranges from 90.1% to 100% will have high loading capacity, which is the essential criteria to reduce the quantity of the carrier required for administration. Drug loading can be accomplished by two methods: (i) incorporating the drug during nanoparticle production, (ii) adsorbing the drug after the formation of nanoparticles by incubating in the drug solution. Thus, in the present work the drug was incorporated at the time of nanoparticle formation. The nanoencapsulation efficiency of lenalidomide was measured by UV-spectrum, and it was found to be 99.35%. It is thus evident that a large amount of drug can be entrapped by the incorporation method when compared to the adsorption (Alenso et al., 1991; Ueda et al., 1998). 3.9. Drug release The drug release study was carried out in triplicate to control the homogeneity of the drug release, and the results were averaged. Initially lenalidomide was rapidly released from the nanoparticles followed by slow release. The initial fast release could be due to the rapid dissolution of surface adhered/entrapped lenalidomide. The later slow release was due to the penetration of release medium into the nanoparticles and dissolves the entrapped drug. When SHMP was used, it led to higher crosslinking which reduces the free volume of the matrix, thereby hindering the easy transport of drug molecules from the matrix (Mi et al., 2003). The drug release curve is shown in Fig. 8. Normally, three basic mechanisms, namely swelling/erosion, diffusion, and degradation are present for the release of the loaded drug from polymeric particles (Schwendeman et al., 1996; Langer, 1990). In general, drug release mechanism may occur by following any one of the method or all the methods given above. In our case, the degradation of nanocarrier was slow; therefore, the release
Fig. 6. TEM of (a) chitosan nanoparticles (CS-NPs); (b) lenalidomide loaded chitosan nanoparticles (LND-CS-NPs).
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Fig. 7. Particle size plot of (a) chitosan nanoparticles (CS-NPs); (b) lenalidomide loaded chitosan nanoparticles (LND-CS-NPs).
mechanism of lenalidomide from LND-CS-NPs may depend on the drug diffusion or the nanosurface. In the present typical matrix delivery system, the polymer, drug, and additive had been mixed to form a homogeneous system, in which the diffusion occurred when the drug passes from the uniform blending matrix onto the external environment. As the release continued, the rate of drug release was decreased with this type of system, as the drug had progressively a longer distance to travel and thus, required a longer diffusion time to release. Usually slow release would be considered for in vivo studies and applications. 3.10. Cytotoxicity studies
cells) and U266 (multiple myeloma cells) cell lines. Earlier studies proved that the prepared CS-NPs were working as a carrier, and it was found nontoxic to MCF7 and U266 cell lines in various concentrations (Prabaharan and Jayakumar, 2009). LND-CS-NPs were prepared in eight different concentrations (varying from 1.00 to 100.00 mg/ml) and tested against MCF7 and U266 cell lines; results imply (Fig. 9) that LND-CS-NPs has showed specific toxicity against both MCF7 and U266 cell lines. Comparatively LND-CS-NPs are more toxic towards U266 cell lines than MCF7 cell lines. Inhibitory concentration 50 (IC50) values were calculated from concentration and response, IC50 value of LND-CS-NPs against MCF7 cell lines was 48.95 mg/ml. On the other hand, the same for U266 cell
Percentage cell viability and the cytotoxicity of the LND-CS-NPs were tested by MTT assay method against MCF7 (breast cancer
Fig. 8. Cumulative percentage of drug release.
Fig. 9. MTT assay of LND-CS-NPs.
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line was 24.56 mg/ml, while comparing the IC50 values of both cell lines, LND-CS-NPs shows good potency against U266 cell lines. 4. Conclusion Biodegradable polymeric nanoparticle of chitosan was incorporated with the highly potent and sparingly soluble anticancer drug lenalidomide. Prepared CS-NPs and LND-CS-NPs were characterized by FT-IR, TGA, DLS and TEM. TEM results confirmed that the prepared LND-CS-NPs were nanosize in range. The size distribution of CS-NPs and LND-CS-NPs was confirmed by DLS study. The drug encapsulation and release studies conclude that the novel LND-CS-NPs are showing sustained and effective drug release towards the target (cancer cell). Cytotoxic study reveals that LND-CS-NPs are toxic towards cancer cell lines such as MCF7 and U266. The drug incorporation and release profile tell that the chitosan nanoparticles were a novel, successful drug delivery system for chemotherapeutic lenalidomide. References Abdelwahed, W., Degobert, G., Stainmesse, S., Fessi, H., 2006. Freeze-drying of nanoparticles: formulation, process and storage considerations. Advanced Drug Delivery Reviews 58, 1688–1713. Agnihotri, S.A., Mallikarjuna, N.N., Aminabhavi, T.M., 2004. Recent advances on chitosan-based micro- and nanoparticles in drug delivery. Journal of Controlled Release 100, 5–28. Albertoni, F., Pettersson, B., Reichelova, V., Juliusson, G., Liliemark, J., 1994. Therapeutic Drug Monitoring 16, 413–418. Alenso, M.J., Losa, C., Calvo, P., Vila-jato, J.L., 1991. Approaches to improve the association of amikacinsulphate to poly-(cyanoacrylate) nanoparticles. International Journal of Pharmaceutics 68, 69–76. Anitha, A., Maya, S., Deepa, N., Chennazhi, K.P., Nair, S.V., Jayakumar, R., 2011. Curcumin-loaded N,O-carboxymethyl chitosan nanoparticles for cancer drug delivery. Journal of Biomaterials Science, Polymer Edition [Epub ahead of print]. Bisht, S., Feldman, G., Soni, S., Ravi, R., Karikar, C., Maitra, A., Maitra, A., 2007. Polymeric nanoparticle-encapsulated curcumin (“nanocurcumin") a novel strategy for human cancer therapy. Journal of Nanobiotechnology 5, 1–18. Brenner, H., Gondos, A., Pulte, D., 2009. Expected long-term survival of patients diagnosis with multiple myeloma in 2006–2010. Haematologica 94, 270–275. Calvo, P., Remunan-Lopez, C., Vila-jato, J.L., Alonso, M.J., 1997. Novel hydrophilic chitosan-polyethylene oxide nanoparticles as protein carriers. Journal of Applied Polymer Science 63, 125–132. Cheng, Y., Samia, A.C., Meyers, J.D., Panagopoulos, I., Fei, B., Burda, C., 2008. Highly efficient drug delivery with gold nanoparticles vectors for in vivo photodynamic therapy of cancer. Journal of the American Chemical Society 32, 10643–10647. Dev, A., Binulal, N.S., Anitha, A., Nair, S.V., Furuike, T., Tamura, H., Jayakumar, R., 2010. Preparation of poly(lactic acid)/chitosan nanoparticles for anti-HIV drug delivery applications. Carbohydrate Polymers 80, 833–838. Hirano, S., 1996. Chitin biotechnology applications. Biotechnology Annual Review 2, 237–258. Hughes, G.A., 2005. Nanostructure-mediated drug delivery. Nanomedicine: Nanotechnology Biology and Medicine 1, 22–30. Jayakumar, R., Prabakaran, M., Reis, R.L., Mano, J.F., 2005. Graft copolymerized chitosan – present status and applications. Carbohydrate Polymers 62, 142–158. Jia-hui, Y., Yu-min, D., Hua, Z., 1999. Blend films of chitosan–gelatin. Journal of Wuhan University 45, 440–444. Kapoor, M., Kumar, S., Murugesan, B., Sathyanarayana, S., Thaper, R., Prasad, M., 2011. Process for the Preparation of Lenalidomide. WO 2011/027326 A1. Kastritis, E., Dimopoulos, M.A., 2007. The evolving role of lenalidomide in the treatment of hematologic malignancies. Expert Opinion on Pharmacotherapy 8, 497–509. Kristmundsdottir, T., Ingvarsdottir, K., Saemundsdottir, G., 1995. Chitosan matrix tablets: the influence of excipients on drug release. Drug Development and Industrial Pharmacy 21, 1591–1598. Langer, R., 1990. New methods of drug delivery. Science 249, 1527–1533. Lee, K.Y., Kwon, I.C., Kim, Y.H., 1998. Preparation of chitosan self-aggregates as a gene delivery system. Journal of Control Release 51, 213–220.
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