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Clay–polymer nanocomposites as a novel drug carrier: Synthesis, characterization and controlled release study of Propranolol Hydrochloride
Applied Clay Science 80–81 (2013) 85–92
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Research paper
Clay–polymer nanocomposites as a novel drug carrier: Synthesis, characterization and controlled release study of Propranolol Hydrochloride Seema Monika Datta ⁎ Analytical Research Laboratory, Department of Chemistry, University of Delhi, Delhi 110007, India
a r t i c l e
i n f o
Article history: Received 25 May 2012 Received in revised form 17 May 2013 Accepted 1 June 2013 Available online xxxx Keywords: Clay–polymer nanocomposites, Montmorillonite Controlled drug delivery Antihypertensive drug
a b s t r a c t Short half life of Propranolol Hydrochloride (PPN), an antihypertensive drug is a prime requirement to develop a formulation which could extend the release of PPN in the human body and also eliminate daily multiple dosage of PPN. In this study, a system of PPN loaded Montmorillonite–Poly lactic-co-glycolic acid (Mt–PLGA) nanocomposites has been developed. PPN incorporated PLGA nanoparticles have been compared with Mt–PPN–PLGA nanocomposites. Mt was used as sustained release carrier for PPN with addition of biodegradable polymer PLGA by preparing Mt–PPN–PLGA nanocomposites by double emulsion solvent evaporation method. The drug encapsulation efficiency and drug loading capacity of synthesized products were estimated with HPLC including suitable analytical techniques to confirm the formation of clay–polymer nanocomposites (CPN). The release profile of encapsulated PPN in CPN shows pH dependent release in simulated gastrointestinal fluid for a period of 8 h. This study suggests that the methodologies used are suitable for the synthesis of Mt based PLGA nanocomposites with high drug encapsulation efficiency and controlled drug release characteristics and indicates that the Mt–PPN–PLGA nanocomposites are supposed to be better oral controlled drug delivery system, for a highly hydrophilic low molecular weight antihypertensive drug PPN to minimize the drug dosing frequency and hence improving the patient compliance. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Drug delivery systems have been of great interest for the past few decades to realize the effective and controlled drug delivery and minimize the side effects in the field of pharmaceutics. Oral controlled drug delivery system is an essential part of the development of new medicines. The carriers used for control drug release were mainly biodegradable polymers (Langer et al., 1999) and porous inorganic matrix (Suresh et al., 2010; Aguzzi et al., 2007). In recent years, drug intercalated smectite, especially Montmorillonite (Mt) pharmaceutical grade clay mineral has attracted great interest of researchers (Joshi et al., 2009a,b). Mt has large specific surface area, exhibits good adsorption ability, cation exchange capacity, and drug-carrying capability. Mt is hydrophilic and highly dispersible in water and can accommodate various protonated and hydrophilic organic molecules along the (001) planes which can be released in controlled manner by replacement with other kind of cations in the release media (Bergaya et al., 2006; Chen et al., 2010; Iliescu et al., 2011). Therefore the Mt is suggested to be a good delivery carrier of the hydrophilic drugs. Mt is a potent detoxifier with excellent adsorbent properties due to its high aspect ratio. It can adsorb excess water from feces and thus act as anti-diarrhoeic. Mt can also provide ⁎ Corresponding author. Tel.: +91 9811487825; fax: +91 11 27666605. E-mail address: [email protected]. 0169-1317/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.clay.2013.06.009
mucoadhesive capability for the nanoparticles to cross the gastrointestinal barrier (Dong and Feng, 2005; Feng et al., 2009). It has also been used as a controlled release system. Mt has been proved to be nontoxic by hematological, biochemical and histopathological analyses in rat models (Lee et al., 2005). Mt is utilized as a sustained release carrier for various therapeutic molecules, such as 5 Fluorouracil (Lin et al., 2002), sertraline (Nunes et al., 2007), vitamin B1 (Joshi et al., 2009a,b), promethazine chloride (Seki and Kadir, 2006) and buspiron hydrochloride (Joshi et al., 2010). Propranolol Hydrochloride [(2RS)-1-(1-Methylethyl) amino-3(naphthalen-1-yloxy) propan-2-ol monohydrochloride] an antihypertensive drug is a nonselective, beta-adrenergic receptor-blocking agent (Dollery, 1991). It is a white crystalline solid, highly soluble in water. The dose of Propranolol Hydrochloride (PPN) ranges from 40 to 80 mg/day. Due to shorter half life (3.9 h) the drug has to be administrated 2 or 3 times daily so as to maintain adequate plasma levels of the drug (Chaturvedi et al., 2010). Thus, the development of controlled release dosage forms would clearly be advantageous (Sahoo et al., 2008). Sanghavi et al. (1998), prepared matrix tablets of PPN using hydroxypropyl methylcellulose which exhibited first order release kinetics. Velasco-De-Paola et al., 1999, described dissolution kinetics of controlled release tablets containing PPN prepared using eudragit. Some other researchers have also formulated oral controlled release products of PPN by various techniques (Gil et al., 2006; Mohammadi-Samani et al.,
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2000; Paker-Leggs and Neau, 2009; Patel et al., 2010; Patra et al., 2007). However, many researchers in the development of PPN sustained release dosage forms were met with problems, such as the difficulty to control the release of the drug due to the high aqueous solubility of PPN. Sánchez-Martin et al. (1981) and Rojtanatanya and Pongjanyakul (2010) have reported the interaction of PPN with Mt and magnesiumaluminium-silicate mineral (MAS, a mixture of Mt and saponite) respectively. PPN can intercalate into the interlayer space of MAS and the obtained complexes showed control of the release of PPN. However no report has been available in the literature for the combination of a biodegradable polymer Poly lactic-co-glycolic acid (PLGA) and Mt for controlled release of PPN. In this study we have tried to obtain the synergism of biodegradable and biocompatible polymer which has already been widely explored for controlled drug release properties, with pharmaceutical grade Mt to produce the oral and controlled drug delivery formulations for PPN. Being a highly hydrophilic drug molecule, it is very difficult to encapsulate a high amount of PPN within the hydrophobic polymer matrix. Therefore, in the present study a modified double emulsion solvent evaporation technique has been developed to entrap a substantial amount of drug in the synthesized formulations. PPN–PLGA nanoparticles and Mt–PPN–PLGA nanocomposites were prepared by w/o/w double emulsion/solvent evaporation method by using biodegradable polymer PLGA and non-ionic Pluronic F68 (a triblock co-polymer selected as an emulsifier and stabilizing agent for the formation of PPN–PLGA nanoparticles and Mt–PPN–PLGA nanocomposites). The synthesized products were characterized for interlayer structural changes in the solid by XRD, surface morphology and particle size by SEM and TEM with EDX, physical status of the drug and Mt by thermal studies and drug loading by HPLC technique. The drug release profile of the synthesized PPN–PLGA nanoparticles and Mt–PPN–PLGA nanocomposites was investigated in simulated gastrointestinal fluid. The Mt–PPN–PLGA nanocomposites obtained were intercalated and partially exfoliated in nature, spherical in shape with about 50–300 nm in size, the favorable size range for intestinal mucosal membrane uptake. DSC results clearly indicate the degradation of the drug encased within synthesized PPN–PLGA nanoparticles and Mt–PPN–PLGA nanocomposites. The drug release profile of Mt–PPN–PLGA nanocomposites shows up to 14% of the drug was released in simulated gastric fluid whereas in simulated intestinal fluid it shows up to 72% of drug release in a period 8 h. Thus we can suggest that Mt–PLGA nanocomposites can be used as a potential drug carrier for the controlled drug delivery of the low molecular weight cationic hydrophilic drugs like PPN.
form a w/o/w-emulsion. The middle organic phase separated the internal water droplets from each other as well as from the external aqueous continuous phase. After solvent evaporation the PPN–PLGA nanoparticles were isolated by centrifugation and washed with double distilled water before freeze-drying. 2.1.2. Synthesis of PPN–PLGA–MMT nanocomposites The synthesis of Mt–PPN–PLGA nanocomposites involved the emulsification of first w/o emulsion in Pluronic F-68 and Mt aqueous dispersion (Fig. 1) followed by the same procedure as discussed in Section 2.1.1. 2.2. Characterizations Powder X-ray diffraction (PXRD) measurements of samples were performed on a powder X-ray diffractometer (XPERT PRO Pananlytical, model (PW3040160, Netherland) the measurement conditions were a Cu K α radiation, generated at 40 kV and 30 mA as X-ray source 2–40° (2θ) and step angle 0.01°/s. The differential scanning calorimetric studies were conducted on a DSC instrument (DSC Q200 V23.10 Build 79). The samples were purged with dry nitrogen at a flow rate of 10 ml/min and the temperature was raised at 10 °C/min. The effect of Mt content on thermal stability of the Mt–PPN–PLGA nanocomposites was assessed by the thermogravimetric analyzer (TGA 2050 Thermal gravimetric Analyzer). The surface morphology and particle size of the synthesized products were examined with the Scanning Electron Microscope (Zeiss EVO 40) and high resolution transmission electron microscope (TECNAI G2 T30, U-TWIN) with an accelerating voltage of 300 kV. 2.3. Estimation of drug loading and encapsulation efficiency with high pressure liquid chromatography (HPLC technique) 2.3.1. HPLC apparatus and conditions The HPLC system consisted of a Shimadzu Model DGU 20 A5 HPLC pump, a Shimadzu-M20A Diode Array Detector, Shimadzu column oven
2. Materials and methods 2.1. Materials Mt KSF, PLGA 50:50 (molecular weight 40–75,000), Pluronic F-68 and drug PPN (purity >98%) were ordered from Sigma Aldrich St. Louise USA. HCl, KCl, NaOH, potassium dihydrogen phosphate of analytical grade for simulated gastric fluid HCl (pH 1.2) and simulated intestinal fluid (PBS, pH 7.4) preparation were ordered from MERCK (Germany). HPLC grade methanol and water were used for drug estimation by HPLC technique. All other reagents whether specified or not were of analytical grade. Double distilled water was used throughout the experimental work. 2.1.1. Synthesis of PPN–PLGA nanoparticles In this study, the water/oil/water (w/o/w) double emulsion solvent evaporation method has been selected to encapsulate highly hydrophilic drug PPN in the nanoparticles. PPN–PLGA nanoparticles were synthesized in two steps. First, PPN was dissolved in water and emulsified in a solution of methylene chloride containing PLGA under magnetic stirring followed by sonication. In the second step, the primary w/o emulsion was emulsified in the external aqueous phase of Pluronic F68 (0.2%, w/v) to
Fig. 1. Schematic representation of clay–polymer–drug nanocomposite synthesis.
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CTO-10AS governed by a LC Solution software. The detector wavelength was set at 289 nm. Separation was achieved by low pressure gradient elution by modifying the reported literature (El-Saharty, 2003) on mobile phase (40:60 ratio v/v) delivered at a flow rate of 1.0 ml/min at ambient temperature through a C18 analytical column Luna 5μ (250 × 4.6 mm i.d., 5 μm particle size). 2.3.2. Stock solutions and standards Stock solutions of PPN were prepared by dissolving 2.50 mg PPN in 25 ml HPLC water, resulting in a solutions containing 100 μg/ml. This solution was diluted to give working standard solutions in concentration range of 0.5 to 50 μg/ml. Standards were prepared with the following concentrations of 0.5, 1, 2, 4, 6, 8, 10, 15, 20, 25, 30 and 50 μg/ml for PPN. 2.3.3. Preparation of sample solutions Supernatants recovered after centrifugation of the synthesized samples were used for the estimation of unloaded drug resulting in indirect estimation of drug encapsulation efficiency. 200 μl of supernatant was diluted up to 10 ml in a standard volumetric flask, filtered by 0.22 μm milipore filters. The filtered solutions were injected to HPLC. The HPLC studies indicate that the calibration curve for known PPN solutions was linear in the concentration range of 0.5 to 100.0 μg/ml in water with a correlation coefficient of 0.99. 2.4. In vitro drug release studies In vitro drug release studies of PPN were conducted in a constant temperature bath with the dialysis bag technique (Joshi et al., 2009a,b). Buffer solution of pH 1.2 (simulated gastric fluid) was prepared by mixing 250 ml of 0.2 M HCL and 147 ml of 0.2 M KCL. Phosphate buffer solution (PBS) of pH 7.4 (simulated intestinal fluid) was prepared by mixing 250 ml of 0.1 M KH2PO4 and 195.5 ml of 0.1 M NaOH. In vitro release studies were carried out in simulated intestinal fluid at pH 7.4 and simulated gastric fluid at pH 1.2 using the dialysis bag technique. Dialysis sacs were overnight equilibrated with the dissolution medium prior to experiments. Weighed amount of the synthesized products was taken in 5 ml of buffer solution in the dialysis bag. The dialysis bag was dipped into the receptor compartment containing 100 ml dissolution medium, which was stirred at 100 rpm at 37 ± 0.5 °C. The receptor compartment was closed to prevent the evaporation losses from the dissolution medium. The stirring speed was kept at 100 rpm. 5 ml of the sample was withdrawn at regular time intervals and the same volume was replaced with a fresh dissolution medium. Samples were analyzed for drug PPN content by UV spectrophotometer at λmax = 289 nm.
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Table 1 Formulations of PPN–PLGA nanoparticles and Mt–PPN–PLGA nanocomposites. S.No.
PPN-01 PPN-02 PPN-03 PPN-04 PPN-05
PPN (mg)
20 20 20 40 60
Mt (mg)
– – 20 20 20
P-F68 (mg)
50 50 50 50 50
PLGA (mg)
50 100 50 50 50
Composite (mg)
27.16 79.97 30.57 40.00 76.48
A amount of PPN in the composite mg
D.L. (%)
E.E. (%)
2.389 2.23 3.540 14.072 46.385
08.6 02.82 11.4 35.17 60.69
11.69 12.16 17.69 35.17 77.30
encapsulated in the Mt–PPN–PLGA nanocomposites increases from 18 to 35% in a linear manner (Table 1) with increase in drug to Mt ratio from 1:1 to 2:1, however with further increase in drug to Mt ratio up to 3:1, an increase in encapsulation efficiency up to 77.30% was obtained. This excessive increase of encapsulation may be attributed to the cationic nature of PPN in the nanocomposites, which could enhance the interaction of PPN with negatively charged Mt and polymer resulting in high encapsulation efficiency. The increase in drug loading can be attributed to the increase in final yield obtained which is directly involved in the calculation of drug loading percentage as per Eq. (1).
Drug loading % ¼ ðDrug amount within the nanoparticles=Total weight of nanoparticlesÞ X 100…
ð1Þ
Encapsulation efficiency% ¼ ðDrug amount within the nanoparticles=initial drug amountÞ X 100…
ð2Þ
3. Results and discussion In the series of experiment, the concentration of stabilizing agent Pluronic F-68 was kept constant (0.2% wt/v) as per the reported critical micelle concentration (Schmolka, 1977). In the case of PPN–PLGA nanoparticles PPN-01, the amount of drug encapsulated was found to be about 12% (Table 1) and with further increase in polymer content (50 mg) the encapsulation efficiency was found to increase by about 0.5%. Therefore, in order to avoid the presence of excess non-emulsified polymer the amount of PLGA was fixed at 50 mg for all formulations. It has also been observed that in the case of Mt–PPN–PLGA nanocomposites, the maximum amount of drug (PPN-05) retained was 77%. Variations in the composition of drug polymer–clay nanocomposites were further studied in detail. 3.1. Effect of PPN content on drug loading and encapsulation efficiency The drug loading and extent of drug encapsulation in Mt–PPN–PLGA as function of PPN content were studied, The amount of PPN
Fig. 2. XRD patterns of a — pristine Mt, b —Mt–PPN–PLGA nanocomposites (PPN-03), c — Mt–PPN–PLGA nanocomposites (PPN-04), d — Mt–PPN–PLGA nanocomposites (PPN-05), and e — pure PPN.
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Fig. 3. Diagrammatic representation of I — intercalated, II — partially exfoliated, and III — exfoliated Mt layers.
3.2. XRD studies The physical status of Mt and PPN in the synthesized Mt–PPN–PLGA nanocomposites was investigated with the help of XRD. The XRD pattern of pristine Mt shows characteristic diffraction peak at 2θ value of 6.4° corresponding to 001 plane with d spacing of 13.6 A° (Fig. 2a). In the case of Mt–PPN–PLGA nanocomposites, PPN–03 (1:1 drug to clay ratio) an increase in intensity of the 001 plane along with the shift in the 2θ value, from 6.4° to 4.04°, was observed (Fig. 2b), the hump in the background from 2θ values of 12° to 28° is due to the presence of a polymer within the Mt–PPN–PLGA nanocomposites. According to Bragg's law, a shift in 2θ value from higher diffraction angle to lower diffraction angle is indicative of an increase in d spacing i.e., from 13.6 A° to 21.4 A° (Joshi et al., 2009a,b; Liu et al., 2006; Lin et al., 2002) and an increase of 8 A° has been attributed to the intercalation of polymer– drug moiety (Fig. 3, case-I) and is further supported by the HRTEM image (Fig. 7b) by the presence of expanded and uniformly spaced Mt layers in the PPN-03 Mt–PPN–PLGA nanocomposites. However, presence of a minor component of a population cannot be ruled out because of the broad nature of 001 reflection at 2θ value of 4.04°. With the increase in drug to Mt ratio from 1:1 to 2:1 in the case of (PPN–04) and 3:1 in the case of (PPN-05), exfoliation of Mt layers
Fig. 4. TGA curves of, a — Pure Mt, b — PPN–PLGA nanoparticles (PPN-01), c — Mt–PPN– PLGA nanocomposites (PPN-03), and d — Mt–PPN–PLGA nanocomposites (PPN-05).
(Paul and Robeson, 2008) is being proposed (Fig. 2c & d). This is also supported by the substantial suppression of 001 reflection at 2θ value of 4.5° and corresponding increase in the intensity of broad hump between 2θ values of 12°–28°, which is indicative of the release of polymeric material from the interlayer gallery (Fig. 3, cases II and III). This fact is further supported by the presence of exfoliated Mt layers in the HRTEM image of PPN-05 Mt–PPN–PLGA nanocomposites (Fig. 7c). Excessive amount of polymer–drug moiety within the interlayers can no longer hold the Mt platelets together. Increase in drug encapsulation efficiency from 18% (PPN-03) to 35% (PPN-04) and 77% (PPN-05) can be attributed to the change in the nature of Mt– PPN–PLGA nanocomposite from intercalation of the drug polymer moiety (to a small extent) to adsorption of the exfoliated negatively charged Mt platelets on the drug–polymer moiety. The extent of drug encapsulation seems to be proportional to the extent of exfoliation in the case of PPN-04 and PPN-05. Further increase in drug content in the Mt–PPN–PLGA nanocomposites did not show further enhancement in the encapsulation efficiency indicating complete saturation of negative sites on Mt platelets. The pure crystalline drug PPN shows intense peaks at 14.56°, 17.32°, 22.84°, 23.47°, 31.19°, 32.30°, 35.05°, 36.23° and 37.58° and the observation is in good agreement with the reported values in the literature (Wang et al., 2002). Mt–PPN–PLGA nanocomposites reveal the
Fig. 5. DSC curves of, a — Pure PPN, b — PPN–PLGA nanoparticles (PPN-01), c — Mt–PPN– PLGA nanocomposites (PPN-03), and d — Mt–PPN–PLGA nanocomposites (PPN-05).
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Fig. 6. SEM–EDX images of a — PPN–PLGA nanoparticles and b — Mt–PPN–PLGA nanocomposites.
presence of crystalline PPN in the nanocomposites (Fig. 2), with increase in drug to Mt ratio, the appearance of characteristic peaks of PPN becomes more evident.
3.3. TGA studies The Mt shows weight loss of 10% from 30 to 140 °C and is attributed to the loss of adsorbed and interlayer water (Fig. 4a). In the case of Mt–PPN–PLGA nanocomposites (PPN-03) due to the replacement of surface and interlayer water by organic moiety no such weight loss was observed in this region. Hence, the stability was observed in this region. In the case of Mt–PPN–PLGA nanocomposites (PPN-05) due to exfoliation of Mt no such weight loss was observed. Hence, the stability was observed in this region. In the temperature range of 30 to 425 °C PPN-PLGA nanoparticles (PPN-01), Mt–PPN–PLGA nanocomposites (PPN-03) and (PPN-05) show 100%, 43.2% and 89.6% weight loss respectively (Fig. 4). It could be clearly understood that weight loss observed in the case of Mt–PPN–PLGA
nanocomposites (PPN-03 and PPN05) is because of the thermal degradation of PPN, PF68 and PLGA contents present in the formulation (Dong and Feng, 2005). Mt content in the nanocomposites (PPN-03) and (PPN-05) is about 56.8% and 10.4% respectively. The Mt–PPN–PLGA nanocomposite (PPN-03) shows less weight loss as compared to the Mt–PPN–PLGA nanocomposite (PPN-05) because in the case of the intercalated sample (PPN-03) the polymer–drug moiety is present within the ordered lattice whereas in the case of PPN-05 the high weight loss is due to the complete degradation of polymer–drug moiety which is out of the order lattice due to the exfoliation.
3.4. DSC studies The appearance of small endothermic peak about 48–50 °C followed by a broad endothermic peak in the temperature region of 360 °C corresponds to the glass transition temperature (Mukherjee and Vishwanatha, 2009) and thermal decomposition of the polymer PLGA in PPN–PLGA nanoparticles and Mt–PPN–PLGA nanocomposites indicates no change in the polymer chain structure.
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of PPN encapsulated within the PPN–PLGA nanoparticles and Mt–PPN– PLGA nanoparticles and nanocomposites (Fig. 5).
Table 2 SEM–EDX analysis report. Element
Series
C norm (wt.%)
C Atom (at.%)
C error (%)
48.77 7.29 0.04 43.90 100.0
55.42 7.11 0.02 37.45 100.0
14.7 2.2 0.0 13.2
nanocomposites K series 47.48 K series 45.31 K series 1.34 K series 1.26 K series 0.42 K series 2.98 100.0
57.09 40.90 0.69 0.67 0.42 0.22 100.0
14.5 13.9 0.1 0.1 0.1 0.1
a, PPN–PLGA nanoparticles Carbon K series Nitrogen K series Silicon K series Oxygen K series Total b, Mt–PPN–PLGA Carbon Oxygen Silicon Aluminium Iron Gold Total
3.5. Scanning electron microscopic and EDX studies PPN–PLGA nanoparticles (PPN-01) and Mt–PPN–PLGA nanocomposites (PPN-03 to 05) appear to be 100–300 nm spherical particles (inset Fig. 6), the former has a smooth surface and the latter has a rough surface because of the presence of Mt nano platelets on the surface which has been further confirmed by SEM–EDX studies (Fig. 6a & b, Table 2). Due to the presence of carbon polymer chain and organic drug moiety in case of PPN-01, high content of carbon, oxygen and nitrogen was seen on the surface (Fig. 6, a) whereas, samples containing Mt, PPN-03 to 05, additional peaks of silicon, aluminium and iron confirms the presence of Mt nano platelets on the surface of the Mt–PPN–PLGA nanocomposites (Fig. 6b). 3.6. Transmission electron microscopic and EDX studies
Pure PPN reveals a sharp endothermic peak at 166 ºC and a broad endothermic peak at 292 ºC followed by an exothermic peak at 300 °C corresponding to the melting point and the decomposition of PPN (Rojtanatanya and Pongjanyakul, 2010). The short broad endothermic region in the temperature range of 276 °C to 296 °C has been observed in the case of PPN–PLGA nanoparticles and Mt– PPN–PLGA nanocomposites prepared and is due to the decomposition
PPN–PLGA nanoparticles (PPN-01) are spherical particles of 50–200 nm in size (inset Fig. 7a). In the TEM micrograph of PPN03 (Fig. 7b) uniformly spaced Mt layers are in support of intercalation (Table 3). In the TEM micrograph of PPN-05 (Fig. 7c), the presence of exfoliated Mt layers is distinct. Both micrographs are also supported by their corresponding XRD data.
Fig. 7. TEM-EDX images of a — PPN–PLGA nanoparticles (PPN-01), b — Mt–PPN–PLGA nanocomposites (PPN-03) and c — Mt–PPN–PLGA nanocomposites (PPN-05).
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As has been observed in the case of SEM–EDX studies, the TEM–EDX studies (Fig. 7a b and c; Table 3) also indicate the presence of high content of carbon, oxygen and nitrogen in PPN-01 and additional peaks of silicon, aluminium and iron in samples PPN-03 and PPN-05 which confirms the presence of Mt in the Mt–PPN–PLGA nanocomposites. 4. Drug release profile PPN-01 and the samples containing Mt, PPN-03 & 05, have been selected for the study of drug release kinetics, in simulated intestinal fluid PBS (pH 7.4) (Fig. 8). As per the XRD data, encapsulation of drug has been found in two kinds of Mt–PPN–PLGA nanocomposites in which the Mt particles were intercalated (PPN-03) and exfoliated (PPN-04 to PPN-05). In the latter category PPN-05 has been selected because of its high yield, loading and encapsulation efficiency. Release of pure drug (PPN) in simulated gastric (HCl, pH 1.2) and intestinal fluid (PBS, pH 7.4) was observed to be 86% (Fig. 9) and 89% (Fig. 8) over a period of 4 h respectively and in both the cases release pattern was not in a controlled manner (~36% release per minute). With PPN-01 only 28% of the encapsulated drug was released over a period of 8 h whereas, the Mt–PPN–PLGA nanocomposites PPN-03 and PPN-05 demonstrated a slow controlled cumulative drug release of 59% and 72% in the PBS (pH 7.4) over a period of 8 h (Fig. 8). The increase in the amount of drug release with respect to the PPN-01 is related to the presence of Mt platelets in the bulk and on the surface (further confirmed by SEM–EDX and TEM–EDX results). The presence of these platelets imparts porosity to the Mt–PPN–PLGA nanocomposites which results in higher ease of passage through the Mt–PPN–PLGA nanocomposite because of which the PPN-05 Mt–PPN–PLGA nanocomposites with higher extent of exfoliation also shows higher release of PPN in a controlled manner as represented by Fig. 10. It is well understood that if a drug delivery system is able to retain high amount of drug in the stomach fluid, it would be able to release
Table 3 TEM–EDX analysis report. Element
Fig. 8. Drug release profile of a — pure PPN, b — PPN–PLGA nanoparticles (PPN-01), c — intercalated Mt–PPN–PLGA nanocomposites (PPN-03) and d — exfoliated Mt–PPN– PLGA nanocomposites, (PPN-05) in simulated intestinal fluid (PBS, pH 7.4) at 37 °C.
more drug in the intestine (Chaturvedi et al., 2010) which is the desired site of drug absorption. In this study we found that over a period of 8 h in simulated gastric fluid (HCl, pH 1.2), PPN-01 and Mt containing samples PPN-03 and PPN-05 show 18%, 14% and 28% drug release respectively (Fig. 9). This low release of PPN from PPN-03 is probably related with the stability of Mt in acidic media which prevents the release of drug in acidic media (Junping et al., 2006). In the case of the formulation PPN-05, collapse of Mt layered structure was observed which comes out to be less effective in the acidic media as compared to the PPN-03. The controlled behavior of PPN release could also be explained by the barrier properties or hindrance in the path offered by high amount of Mt layers to release the drug in both the releasing media (Fig. 10). 5. Conclusion
Weight%
Atomic%
a — PPN–PLGA nanoparticles (PPN-01) CK 58.6 NK 2.7 OK 11.3 FK 2.2 Ca K 2.9 Cu K 22.3 Total 100.0
77.3 3.1 11.1 1.8 1.1 5.6 100.0
b — Mt–PPN–PLGA nanocomposites (PPN-03) CK 20.9 NK 6.7 OK 35.7 Mg K 4.1 Al K 4.0 Si K 14.5 KK 0.9 Fe K 3.2 Cu K 9.9 Total 100.0
31.5 8.7 40.4 3.1 2.7 9.4 0.4 1.0 2.8 100.0
c — Mt–PPN–PLGA nanocomposites (PPN-05) CK 27.8 NK 6.9 OK 35.2 Mg K 0.2 Al K 6.2 Si K 12.7 KK 0.5 Fe K 2.1 Co K 0.3 Cu K 11.1 Total 100.0
31.5 8.7 40.4 3.1 2.7 9.4 0.4 1.0 0.1 3.0 100.0
In the present study an oral controlled drug delivery system for PPN loaded PLGA nanoparticles and Mt–PPN–PLGA nanocomposites was developed by w/o/w double emulsion solvent evaporation technique. About 77% entrapment efficiency and 72% release were achieved for the highly hydrophilic drug, PPN. Two types of Mt–PPN–PLGA
Fig. 9. Drug release profile of a — pure PPN, b — PPN–PLGA nanoparticles (PPN-01), c — intercalated Mt–PPN–PLGA nanocomposites (PPN-03) and d — exfoliated Mt–PPN– PLGA nanocomposites, (PPN-05) in simulated gastric fluid (HCl, pH 1.2) at 37 °C.
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Fig. 10. Diagrammatic representation of drug path within a — intercalated and b — exfoliated Mt–PPN–PLGA nanocomposites.
nanocomposites (intercalated and exfoliated Mt) were obtained. The presence of drug within the PPN–PLGA nanoparticles and Mt–PPN– PLGA nanocomposites nanoparticles and nanocomposites was also confirmed by DSC data. 50–300 nm spherical PPN–PLGA nanoparticles and Mt–PPN–PLGA nanocomposites was obtained with the Mt platelets on the surface (confirms by SEM–EDX and TEM–EDX data). The drug release profile of PPN was found to be pH dependent, the presence of Mt platelets within the PPN–PLGA formulations results in controlled and higher % release of drug. Therefore, it can be said that the synthesized formulations have high potential as a controlled drug delivery system for PPN. Disclosure The authors report no conflicts of interest in this work. Acknowledgments We sincerely express our thanks to the director, USIC, University of Delhi for instrumentation facilities, Director, AIRF, JNU for providing SEM facilities and UGC/RGNF for providing financial assistance for this research work under the project of sch/rgnf/srf/f-10/2007-08. The authors are thankful to Dr. R Nagarajan for his valuable suggestions regarding the interpretation of XRD and TGA data. References Aguzzi, C., Cerezo, P., Viseras, C., Caramella, C., 2007. Use of clays as drug delivery systems: possibilities and limitations. Applied Clay Science 36, 22–36. Bergaya, F., Theng, B.K.G., Lagaly, G., 2006. First edition Handbook of Clay Science. Elsevier. Chaturvedi, K., Umadevi, S., Vaghani, S., 2010. Floating matrix dosage form for propranolol hydrochloride based on gas formation technique: development and in vitro evaluation. Scientia Pharmaceutica 78, 927–939. Chen, Y., Zhou, A., Liu, B., Liang, J., 2010. Tramadol hydrochloride/montmorillonite composite: Preparation and controlled drug release. Applied Clay Science 49, 108–112. Dollery, S.C., 1991. Therapeutic Drugs. Churchill Livingstone, Edinburgh P272–P278. Dong, Y., Feng, S.S., 2005. Poly (D, L-lactide-co-lycolide)/montmorillonite nanoparticles for oral delivery of anticancer drugs. Biomaterials 26, 6068–6076. El-Saharty, Y.S., 2003. Simultaneous high-performance liquid chromatographic assay of furosemide and propranolol HCL and its application in a pharmacokinetic study. Journal of Pharmaceutical and Biomedical Analysis 33, 699–709. Feng, S.S., Mei, L., Anitha, P., Gan, C.W., Zhou, W., 2009. Poly(lactide)–vitamin E derivative/ montmorillonite nanoparticle formulations for the oral delivery of Docetaxel. Biomaterials 30, 3297–3306. Gil, E.C., Colarte, A.I., Bataille, B., Pedraz, J.L., Rodríguez, F., Heinämäki, J., 2006. Development and optimization of a novel sustained-release dextran tablet formulation for propranolol hydrochloride. International Journal of Pharmaceutics 2006 (317), 32–39. Iliescu, R.I., Andronescu, E., Voicu, G., Ficai, A., Covaliu, C.I., 2011. Hybrid materials based on montmorillonite and citostatic drugs: preparation and characterization. Applied Clay Science 52, 62–68. Joshi, G.V., Kevadiya, B.D., Patel, H.A., Bajaj, H.C., Jasra, R.V., 2009a. Montmorillonite as a drug delivery system: intercalation and in vitro release of Timolol maleate. International Journal of Pharmaceutics 374, 53–57.
Joshi, G.V., Patel, H.A., Kevadiya, B.D., Bajaj, H.C., 2009b. Montmorillonite intercalated with Vitamin B1 as drug carrier. Applied Clay Science 45, 248–253. Joshi, G.V., Kevadiya, B.D., Bajaj, H.C., 2010. Design and evaluation of controlled drug delivery system of buspirone using inorganic layered clay mineral. Microporous and Mesoporous Materials 132, 526–530. Junping, Z., Hongyan, W., Hong, Z., Lifei, X., Kangde, Y., 2006. Intercalation of amido cationic drug with montmorillonite. Journal of Wuhan University of Technology-Materials Science Edition 22, 250–252. Langer, R.S., Kathryn, E.U., Cannizzaro, S.M., Shakesheff, K.M., 1999. Polymeric systems for controlled drug release. Chemical Reviews 99, 3181–3198. Lee, Y.H., Kuo, T.F., Chen, B.Y., Feng, Y.K., Wen, Y.R., Lin, W.C., Lin, F.H., 2005. Toxicity assessment of montmorillonite as a drug carrier for pharmaceutical applications: yeast and rats model. Biomedical Engineering-Applications Basis Communications 17, 72–78. Lin, F.H., Lee, Y.H., Jian, C.H., Wong, J.M., Shieh, M.J., Wang, C.Y., 2002. A study of purified montmorillonite intercalated with 5-fluorouracil as drug carrier. Biomaterials 23, 1981–1987. Liu, J., Boo, W.J., Clearfield, A., Sue, H.J., 2006. Intercalation and exfoliation: a review on morphology of polymer nanocomposites reinforced by inorganic layer structures. Materials and Manufacturing Processes 20, 143–151. Mohammadi-Samani, S., Adrangui, M., Siahi-Shadbad, M.R., Nokhodchi, A., 2000. An approach to controlled-release dosage form of propranolol hydrochloride. Drug Development and Industrial Pharmacy 26 (1), 91–94. Mukherjee, A., Vishwanatha, J.K., 2009. Formulation, characterization and evaluation of curcumin-loaded PLGA nanospheres for cancer therapy. Anticancer Research 29, 3867–3876. Nunes, C.D., Vaz, P.D., Fernandes, A.C., Ferreira, P., Romão, C.C., Calhorda, M.J., 2007. Loading and delivery of sertraline using inorganic micro and mesoporous materials. European Journal of Pharmaceutics and Biopharmaceutics 66, 357–365. Paker-Leggs, S., Neau, S.H., 2009. Pellet characteristics and drug release when the form of propranolol is fixed as moles or mass in formulations for extruded and spheronized Carbopol-containing pellets. International Journal of Pharmaceutics 369, 96–104. Patel, J., Patel, D., Raval, J., 2010. Formulation and evaluation of propranolol hydrochlorideloaded carbopol-934p/ethyl cellulose mucoadhesive microspheres. Iranian Journal of Pharmaceutical Research 9, 221–232. Patra, C.N., Kumar, A.B., Pandit, H.K., Singh, S.K., Devi, M.V., 2007. Design and evaluation of sustained release bilayer tablets of propranolol hydrochloride. Acta Pharmaceutica 57, 479–489. Paul, D.R., Robeson, L.M., 2008. Polymer nanotechnology: nanocomposites. Polymer 49, 3187–3204. Rojtanatanya, S., Pongjanyakul, T., 2010. Propranolol–magnesium aluminum silicate complex dispersions and particles: characterization and factors influencing drug release. International Journal of Pharmaceutics 383 (1-2), 106–115. Sahoo, J., Murthy, P.N., Biswal, S., Sahoo, S.K., Mahapatra, A.K., 2008. Comparative study of propranolol hydrochloride release from matrix tablets with Kollidon®SR or hydroxy propyl methyl cellulose. AAPS PharmSciTechnol 9, 577–582. Sánchez-Martin, M.J., Sánchez-Camazano, M., Vicente-Hernández, T., Dominguez-Gil, A., 1981. Interaction of propranolol hydrochloride with montmorillonite. Journal of Pharmacy and Pharmacology 33, 408–410. Sanghavi, N.M., Sarawade, V.B., Kamath, P.R., Bijilani, C.P., 1998. Matrix tablets of propranolol hydrochloride. Indian Drugs 26 (8), 404–407. Schmolka, I.R., 1977. A review of block polymer surfactants. Journal of American Oil Chemists' society 54, 110–116. Seki, Y.S., Kadir, Y.C., 2006. Adsorption of promethazine hydrochloride with KSF montmorillonite. Adsorption 12, 89. Suresh, R., Borkar, S.N., Sawant, V.A., Shende, V.S., Dimble, S.K., 2010. Nanoclay drug delivery system. International Journal of Pharmaceutical Sciences and Nanotechnology 3, 901–905. Velasco-De-Paola, M.V.R., Santoro, M.I.R.M., Gai, M.N., 1999. Dissolution kinetics evaluation of controlled-release tablets containing propranolol hydrochloride. Drug Development and Industrial Pharmacy 25 (4), 535–541. Wang, X., Wang, X.J., Ching, C.B., 2002. Solubility, metastable zone width and racemic character of Propranolol hydrochloride. Chirality 14, 318–324.
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Research paper
Clay–polymer nanocomposites as a novel drug carrier: Synthesis, characterization and controlled release study of Propranolol Hydrochloride Seema Monika Datta ⁎ Analytical Research Laboratory, Department of Chemistry, University of Delhi, Delhi 110007, India
a r t i c l e
i n f o
Article history: Received 25 May 2012 Received in revised form 17 May 2013 Accepted 1 June 2013 Available online xxxx Keywords: Clay–polymer nanocomposites, Montmorillonite Controlled drug delivery Antihypertensive drug
a b s t r a c t Short half life of Propranolol Hydrochloride (PPN), an antihypertensive drug is a prime requirement to develop a formulation which could extend the release of PPN in the human body and also eliminate daily multiple dosage of PPN. In this study, a system of PPN loaded Montmorillonite–Poly lactic-co-glycolic acid (Mt–PLGA) nanocomposites has been developed. PPN incorporated PLGA nanoparticles have been compared with Mt–PPN–PLGA nanocomposites. Mt was used as sustained release carrier for PPN with addition of biodegradable polymer PLGA by preparing Mt–PPN–PLGA nanocomposites by double emulsion solvent evaporation method. The drug encapsulation efficiency and drug loading capacity of synthesized products were estimated with HPLC including suitable analytical techniques to confirm the formation of clay–polymer nanocomposites (CPN). The release profile of encapsulated PPN in CPN shows pH dependent release in simulated gastrointestinal fluid for a period of 8 h. This study suggests that the methodologies used are suitable for the synthesis of Mt based PLGA nanocomposites with high drug encapsulation efficiency and controlled drug release characteristics and indicates that the Mt–PPN–PLGA nanocomposites are supposed to be better oral controlled drug delivery system, for a highly hydrophilic low molecular weight antihypertensive drug PPN to minimize the drug dosing frequency and hence improving the patient compliance. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Drug delivery systems have been of great interest for the past few decades to realize the effective and controlled drug delivery and minimize the side effects in the field of pharmaceutics. Oral controlled drug delivery system is an essential part of the development of new medicines. The carriers used for control drug release were mainly biodegradable polymers (Langer et al., 1999) and porous inorganic matrix (Suresh et al., 2010; Aguzzi et al., 2007). In recent years, drug intercalated smectite, especially Montmorillonite (Mt) pharmaceutical grade clay mineral has attracted great interest of researchers (Joshi et al., 2009a,b). Mt has large specific surface area, exhibits good adsorption ability, cation exchange capacity, and drug-carrying capability. Mt is hydrophilic and highly dispersible in water and can accommodate various protonated and hydrophilic organic molecules along the (001) planes which can be released in controlled manner by replacement with other kind of cations in the release media (Bergaya et al., 2006; Chen et al., 2010; Iliescu et al., 2011). Therefore the Mt is suggested to be a good delivery carrier of the hydrophilic drugs. Mt is a potent detoxifier with excellent adsorbent properties due to its high aspect ratio. It can adsorb excess water from feces and thus act as anti-diarrhoeic. Mt can also provide ⁎ Corresponding author. Tel.: +91 9811487825; fax: +91 11 27666605. E-mail address: [email protected]. 0169-1317/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.clay.2013.06.009
mucoadhesive capability for the nanoparticles to cross the gastrointestinal barrier (Dong and Feng, 2005; Feng et al., 2009). It has also been used as a controlled release system. Mt has been proved to be nontoxic by hematological, biochemical and histopathological analyses in rat models (Lee et al., 2005). Mt is utilized as a sustained release carrier for various therapeutic molecules, such as 5 Fluorouracil (Lin et al., 2002), sertraline (Nunes et al., 2007), vitamin B1 (Joshi et al., 2009a,b), promethazine chloride (Seki and Kadir, 2006) and buspiron hydrochloride (Joshi et al., 2010). Propranolol Hydrochloride [(2RS)-1-(1-Methylethyl) amino-3(naphthalen-1-yloxy) propan-2-ol monohydrochloride] an antihypertensive drug is a nonselective, beta-adrenergic receptor-blocking agent (Dollery, 1991). It is a white crystalline solid, highly soluble in water. The dose of Propranolol Hydrochloride (PPN) ranges from 40 to 80 mg/day. Due to shorter half life (3.9 h) the drug has to be administrated 2 or 3 times daily so as to maintain adequate plasma levels of the drug (Chaturvedi et al., 2010). Thus, the development of controlled release dosage forms would clearly be advantageous (Sahoo et al., 2008). Sanghavi et al. (1998), prepared matrix tablets of PPN using hydroxypropyl methylcellulose which exhibited first order release kinetics. Velasco-De-Paola et al., 1999, described dissolution kinetics of controlled release tablets containing PPN prepared using eudragit. Some other researchers have also formulated oral controlled release products of PPN by various techniques (Gil et al., 2006; Mohammadi-Samani et al.,
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2000; Paker-Leggs and Neau, 2009; Patel et al., 2010; Patra et al., 2007). However, many researchers in the development of PPN sustained release dosage forms were met with problems, such as the difficulty to control the release of the drug due to the high aqueous solubility of PPN. Sánchez-Martin et al. (1981) and Rojtanatanya and Pongjanyakul (2010) have reported the interaction of PPN with Mt and magnesiumaluminium-silicate mineral (MAS, a mixture of Mt and saponite) respectively. PPN can intercalate into the interlayer space of MAS and the obtained complexes showed control of the release of PPN. However no report has been available in the literature for the combination of a biodegradable polymer Poly lactic-co-glycolic acid (PLGA) and Mt for controlled release of PPN. In this study we have tried to obtain the synergism of biodegradable and biocompatible polymer which has already been widely explored for controlled drug release properties, with pharmaceutical grade Mt to produce the oral and controlled drug delivery formulations for PPN. Being a highly hydrophilic drug molecule, it is very difficult to encapsulate a high amount of PPN within the hydrophobic polymer matrix. Therefore, in the present study a modified double emulsion solvent evaporation technique has been developed to entrap a substantial amount of drug in the synthesized formulations. PPN–PLGA nanoparticles and Mt–PPN–PLGA nanocomposites were prepared by w/o/w double emulsion/solvent evaporation method by using biodegradable polymer PLGA and non-ionic Pluronic F68 (a triblock co-polymer selected as an emulsifier and stabilizing agent for the formation of PPN–PLGA nanoparticles and Mt–PPN–PLGA nanocomposites). The synthesized products were characterized for interlayer structural changes in the solid by XRD, surface morphology and particle size by SEM and TEM with EDX, physical status of the drug and Mt by thermal studies and drug loading by HPLC technique. The drug release profile of the synthesized PPN–PLGA nanoparticles and Mt–PPN–PLGA nanocomposites was investigated in simulated gastrointestinal fluid. The Mt–PPN–PLGA nanocomposites obtained were intercalated and partially exfoliated in nature, spherical in shape with about 50–300 nm in size, the favorable size range for intestinal mucosal membrane uptake. DSC results clearly indicate the degradation of the drug encased within synthesized PPN–PLGA nanoparticles and Mt–PPN–PLGA nanocomposites. The drug release profile of Mt–PPN–PLGA nanocomposites shows up to 14% of the drug was released in simulated gastric fluid whereas in simulated intestinal fluid it shows up to 72% of drug release in a period 8 h. Thus we can suggest that Mt–PLGA nanocomposites can be used as a potential drug carrier for the controlled drug delivery of the low molecular weight cationic hydrophilic drugs like PPN.
form a w/o/w-emulsion. The middle organic phase separated the internal water droplets from each other as well as from the external aqueous continuous phase. After solvent evaporation the PPN–PLGA nanoparticles were isolated by centrifugation and washed with double distilled water before freeze-drying. 2.1.2. Synthesis of PPN–PLGA–MMT nanocomposites The synthesis of Mt–PPN–PLGA nanocomposites involved the emulsification of first w/o emulsion in Pluronic F-68 and Mt aqueous dispersion (Fig. 1) followed by the same procedure as discussed in Section 2.1.1. 2.2. Characterizations Powder X-ray diffraction (PXRD) measurements of samples were performed on a powder X-ray diffractometer (XPERT PRO Pananlytical, model (PW3040160, Netherland) the measurement conditions were a Cu K α radiation, generated at 40 kV and 30 mA as X-ray source 2–40° (2θ) and step angle 0.01°/s. The differential scanning calorimetric studies were conducted on a DSC instrument (DSC Q200 V23.10 Build 79). The samples were purged with dry nitrogen at a flow rate of 10 ml/min and the temperature was raised at 10 °C/min. The effect of Mt content on thermal stability of the Mt–PPN–PLGA nanocomposites was assessed by the thermogravimetric analyzer (TGA 2050 Thermal gravimetric Analyzer). The surface morphology and particle size of the synthesized products were examined with the Scanning Electron Microscope (Zeiss EVO 40) and high resolution transmission electron microscope (TECNAI G2 T30, U-TWIN) with an accelerating voltage of 300 kV. 2.3. Estimation of drug loading and encapsulation efficiency with high pressure liquid chromatography (HPLC technique) 2.3.1. HPLC apparatus and conditions The HPLC system consisted of a Shimadzu Model DGU 20 A5 HPLC pump, a Shimadzu-M20A Diode Array Detector, Shimadzu column oven
2. Materials and methods 2.1. Materials Mt KSF, PLGA 50:50 (molecular weight 40–75,000), Pluronic F-68 and drug PPN (purity >98%) were ordered from Sigma Aldrich St. Louise USA. HCl, KCl, NaOH, potassium dihydrogen phosphate of analytical grade for simulated gastric fluid HCl (pH 1.2) and simulated intestinal fluid (PBS, pH 7.4) preparation were ordered from MERCK (Germany). HPLC grade methanol and water were used for drug estimation by HPLC technique. All other reagents whether specified or not were of analytical grade. Double distilled water was used throughout the experimental work. 2.1.1. Synthesis of PPN–PLGA nanoparticles In this study, the water/oil/water (w/o/w) double emulsion solvent evaporation method has been selected to encapsulate highly hydrophilic drug PPN in the nanoparticles. PPN–PLGA nanoparticles were synthesized in two steps. First, PPN was dissolved in water and emulsified in a solution of methylene chloride containing PLGA under magnetic stirring followed by sonication. In the second step, the primary w/o emulsion was emulsified in the external aqueous phase of Pluronic F68 (0.2%, w/v) to
Fig. 1. Schematic representation of clay–polymer–drug nanocomposite synthesis.
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CTO-10AS governed by a LC Solution software. The detector wavelength was set at 289 nm. Separation was achieved by low pressure gradient elution by modifying the reported literature (El-Saharty, 2003) on mobile phase (40:60 ratio v/v) delivered at a flow rate of 1.0 ml/min at ambient temperature through a C18 analytical column Luna 5μ (250 × 4.6 mm i.d., 5 μm particle size). 2.3.2. Stock solutions and standards Stock solutions of PPN were prepared by dissolving 2.50 mg PPN in 25 ml HPLC water, resulting in a solutions containing 100 μg/ml. This solution was diluted to give working standard solutions in concentration range of 0.5 to 50 μg/ml. Standards were prepared with the following concentrations of 0.5, 1, 2, 4, 6, 8, 10, 15, 20, 25, 30 and 50 μg/ml for PPN. 2.3.3. Preparation of sample solutions Supernatants recovered after centrifugation of the synthesized samples were used for the estimation of unloaded drug resulting in indirect estimation of drug encapsulation efficiency. 200 μl of supernatant was diluted up to 10 ml in a standard volumetric flask, filtered by 0.22 μm milipore filters. The filtered solutions were injected to HPLC. The HPLC studies indicate that the calibration curve for known PPN solutions was linear in the concentration range of 0.5 to 100.0 μg/ml in water with a correlation coefficient of 0.99. 2.4. In vitro drug release studies In vitro drug release studies of PPN were conducted in a constant temperature bath with the dialysis bag technique (Joshi et al., 2009a,b). Buffer solution of pH 1.2 (simulated gastric fluid) was prepared by mixing 250 ml of 0.2 M HCL and 147 ml of 0.2 M KCL. Phosphate buffer solution (PBS) of pH 7.4 (simulated intestinal fluid) was prepared by mixing 250 ml of 0.1 M KH2PO4 and 195.5 ml of 0.1 M NaOH. In vitro release studies were carried out in simulated intestinal fluid at pH 7.4 and simulated gastric fluid at pH 1.2 using the dialysis bag technique. Dialysis sacs were overnight equilibrated with the dissolution medium prior to experiments. Weighed amount of the synthesized products was taken in 5 ml of buffer solution in the dialysis bag. The dialysis bag was dipped into the receptor compartment containing 100 ml dissolution medium, which was stirred at 100 rpm at 37 ± 0.5 °C. The receptor compartment was closed to prevent the evaporation losses from the dissolution medium. The stirring speed was kept at 100 rpm. 5 ml of the sample was withdrawn at regular time intervals and the same volume was replaced with a fresh dissolution medium. Samples were analyzed for drug PPN content by UV spectrophotometer at λmax = 289 nm.
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Table 1 Formulations of PPN–PLGA nanoparticles and Mt–PPN–PLGA nanocomposites. S.No.
PPN-01 PPN-02 PPN-03 PPN-04 PPN-05
PPN (mg)
20 20 20 40 60
Mt (mg)
– – 20 20 20
P-F68 (mg)
50 50 50 50 50
PLGA (mg)
50 100 50 50 50
Composite (mg)
27.16 79.97 30.57 40.00 76.48
A amount of PPN in the composite mg
D.L. (%)
E.E. (%)
2.389 2.23 3.540 14.072 46.385
08.6 02.82 11.4 35.17 60.69
11.69 12.16 17.69 35.17 77.30
encapsulated in the Mt–PPN–PLGA nanocomposites increases from 18 to 35% in a linear manner (Table 1) with increase in drug to Mt ratio from 1:1 to 2:1, however with further increase in drug to Mt ratio up to 3:1, an increase in encapsulation efficiency up to 77.30% was obtained. This excessive increase of encapsulation may be attributed to the cationic nature of PPN in the nanocomposites, which could enhance the interaction of PPN with negatively charged Mt and polymer resulting in high encapsulation efficiency. The increase in drug loading can be attributed to the increase in final yield obtained which is directly involved in the calculation of drug loading percentage as per Eq. (1).
Drug loading % ¼ ðDrug amount within the nanoparticles=Total weight of nanoparticlesÞ X 100…
ð1Þ
Encapsulation efficiency% ¼ ðDrug amount within the nanoparticles=initial drug amountÞ X 100…
ð2Þ
3. Results and discussion In the series of experiment, the concentration of stabilizing agent Pluronic F-68 was kept constant (0.2% wt/v) as per the reported critical micelle concentration (Schmolka, 1977). In the case of PPN–PLGA nanoparticles PPN-01, the amount of drug encapsulated was found to be about 12% (Table 1) and with further increase in polymer content (50 mg) the encapsulation efficiency was found to increase by about 0.5%. Therefore, in order to avoid the presence of excess non-emulsified polymer the amount of PLGA was fixed at 50 mg for all formulations. It has also been observed that in the case of Mt–PPN–PLGA nanocomposites, the maximum amount of drug (PPN-05) retained was 77%. Variations in the composition of drug polymer–clay nanocomposites were further studied in detail. 3.1. Effect of PPN content on drug loading and encapsulation efficiency The drug loading and extent of drug encapsulation in Mt–PPN–PLGA as function of PPN content were studied, The amount of PPN
Fig. 2. XRD patterns of a — pristine Mt, b —Mt–PPN–PLGA nanocomposites (PPN-03), c — Mt–PPN–PLGA nanocomposites (PPN-04), d — Mt–PPN–PLGA nanocomposites (PPN-05), and e — pure PPN.
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Fig. 3. Diagrammatic representation of I — intercalated, II — partially exfoliated, and III — exfoliated Mt layers.
3.2. XRD studies The physical status of Mt and PPN in the synthesized Mt–PPN–PLGA nanocomposites was investigated with the help of XRD. The XRD pattern of pristine Mt shows characteristic diffraction peak at 2θ value of 6.4° corresponding to 001 plane with d spacing of 13.6 A° (Fig. 2a). In the case of Mt–PPN–PLGA nanocomposites, PPN–03 (1:1 drug to clay ratio) an increase in intensity of the 001 plane along with the shift in the 2θ value, from 6.4° to 4.04°, was observed (Fig. 2b), the hump in the background from 2θ values of 12° to 28° is due to the presence of a polymer within the Mt–PPN–PLGA nanocomposites. According to Bragg's law, a shift in 2θ value from higher diffraction angle to lower diffraction angle is indicative of an increase in d spacing i.e., from 13.6 A° to 21.4 A° (Joshi et al., 2009a,b; Liu et al., 2006; Lin et al., 2002) and an increase of 8 A° has been attributed to the intercalation of polymer– drug moiety (Fig. 3, case-I) and is further supported by the HRTEM image (Fig. 7b) by the presence of expanded and uniformly spaced Mt layers in the PPN-03 Mt–PPN–PLGA nanocomposites. However, presence of a minor component of a population cannot be ruled out because of the broad nature of 001 reflection at 2θ value of 4.04°. With the increase in drug to Mt ratio from 1:1 to 2:1 in the case of (PPN–04) and 3:1 in the case of (PPN-05), exfoliation of Mt layers
Fig. 4. TGA curves of, a — Pure Mt, b — PPN–PLGA nanoparticles (PPN-01), c — Mt–PPN– PLGA nanocomposites (PPN-03), and d — Mt–PPN–PLGA nanocomposites (PPN-05).
(Paul and Robeson, 2008) is being proposed (Fig. 2c & d). This is also supported by the substantial suppression of 001 reflection at 2θ value of 4.5° and corresponding increase in the intensity of broad hump between 2θ values of 12°–28°, which is indicative of the release of polymeric material from the interlayer gallery (Fig. 3, cases II and III). This fact is further supported by the presence of exfoliated Mt layers in the HRTEM image of PPN-05 Mt–PPN–PLGA nanocomposites (Fig. 7c). Excessive amount of polymer–drug moiety within the interlayers can no longer hold the Mt platelets together. Increase in drug encapsulation efficiency from 18% (PPN-03) to 35% (PPN-04) and 77% (PPN-05) can be attributed to the change in the nature of Mt– PPN–PLGA nanocomposite from intercalation of the drug polymer moiety (to a small extent) to adsorption of the exfoliated negatively charged Mt platelets on the drug–polymer moiety. The extent of drug encapsulation seems to be proportional to the extent of exfoliation in the case of PPN-04 and PPN-05. Further increase in drug content in the Mt–PPN–PLGA nanocomposites did not show further enhancement in the encapsulation efficiency indicating complete saturation of negative sites on Mt platelets. The pure crystalline drug PPN shows intense peaks at 14.56°, 17.32°, 22.84°, 23.47°, 31.19°, 32.30°, 35.05°, 36.23° and 37.58° and the observation is in good agreement with the reported values in the literature (Wang et al., 2002). Mt–PPN–PLGA nanocomposites reveal the
Fig. 5. DSC curves of, a — Pure PPN, b — PPN–PLGA nanoparticles (PPN-01), c — Mt–PPN– PLGA nanocomposites (PPN-03), and d — Mt–PPN–PLGA nanocomposites (PPN-05).
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Fig. 6. SEM–EDX images of a — PPN–PLGA nanoparticles and b — Mt–PPN–PLGA nanocomposites.
presence of crystalline PPN in the nanocomposites (Fig. 2), with increase in drug to Mt ratio, the appearance of characteristic peaks of PPN becomes more evident.
3.3. TGA studies The Mt shows weight loss of 10% from 30 to 140 °C and is attributed to the loss of adsorbed and interlayer water (Fig. 4a). In the case of Mt–PPN–PLGA nanocomposites (PPN-03) due to the replacement of surface and interlayer water by organic moiety no such weight loss was observed in this region. Hence, the stability was observed in this region. In the case of Mt–PPN–PLGA nanocomposites (PPN-05) due to exfoliation of Mt no such weight loss was observed. Hence, the stability was observed in this region. In the temperature range of 30 to 425 °C PPN-PLGA nanoparticles (PPN-01), Mt–PPN–PLGA nanocomposites (PPN-03) and (PPN-05) show 100%, 43.2% and 89.6% weight loss respectively (Fig. 4). It could be clearly understood that weight loss observed in the case of Mt–PPN–PLGA
nanocomposites (PPN-03 and PPN05) is because of the thermal degradation of PPN, PF68 and PLGA contents present in the formulation (Dong and Feng, 2005). Mt content in the nanocomposites (PPN-03) and (PPN-05) is about 56.8% and 10.4% respectively. The Mt–PPN–PLGA nanocomposite (PPN-03) shows less weight loss as compared to the Mt–PPN–PLGA nanocomposite (PPN-05) because in the case of the intercalated sample (PPN-03) the polymer–drug moiety is present within the ordered lattice whereas in the case of PPN-05 the high weight loss is due to the complete degradation of polymer–drug moiety which is out of the order lattice due to the exfoliation.
3.4. DSC studies The appearance of small endothermic peak about 48–50 °C followed by a broad endothermic peak in the temperature region of 360 °C corresponds to the glass transition temperature (Mukherjee and Vishwanatha, 2009) and thermal decomposition of the polymer PLGA in PPN–PLGA nanoparticles and Mt–PPN–PLGA nanocomposites indicates no change in the polymer chain structure.
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of PPN encapsulated within the PPN–PLGA nanoparticles and Mt–PPN– PLGA nanoparticles and nanocomposites (Fig. 5).
Table 2 SEM–EDX analysis report. Element
Series
C norm (wt.%)
C Atom (at.%)
C error (%)
48.77 7.29 0.04 43.90 100.0
55.42 7.11 0.02 37.45 100.0
14.7 2.2 0.0 13.2
nanocomposites K series 47.48 K series 45.31 K series 1.34 K series 1.26 K series 0.42 K series 2.98 100.0
57.09 40.90 0.69 0.67 0.42 0.22 100.0
14.5 13.9 0.1 0.1 0.1 0.1
a, PPN–PLGA nanoparticles Carbon K series Nitrogen K series Silicon K series Oxygen K series Total b, Mt–PPN–PLGA Carbon Oxygen Silicon Aluminium Iron Gold Total
3.5. Scanning electron microscopic and EDX studies PPN–PLGA nanoparticles (PPN-01) and Mt–PPN–PLGA nanocomposites (PPN-03 to 05) appear to be 100–300 nm spherical particles (inset Fig. 6), the former has a smooth surface and the latter has a rough surface because of the presence of Mt nano platelets on the surface which has been further confirmed by SEM–EDX studies (Fig. 6a & b, Table 2). Due to the presence of carbon polymer chain and organic drug moiety in case of PPN-01, high content of carbon, oxygen and nitrogen was seen on the surface (Fig. 6, a) whereas, samples containing Mt, PPN-03 to 05, additional peaks of silicon, aluminium and iron confirms the presence of Mt nano platelets on the surface of the Mt–PPN–PLGA nanocomposites (Fig. 6b). 3.6. Transmission electron microscopic and EDX studies
Pure PPN reveals a sharp endothermic peak at 166 ºC and a broad endothermic peak at 292 ºC followed by an exothermic peak at 300 °C corresponding to the melting point and the decomposition of PPN (Rojtanatanya and Pongjanyakul, 2010). The short broad endothermic region in the temperature range of 276 °C to 296 °C has been observed in the case of PPN–PLGA nanoparticles and Mt– PPN–PLGA nanocomposites prepared and is due to the decomposition
PPN–PLGA nanoparticles (PPN-01) are spherical particles of 50–200 nm in size (inset Fig. 7a). In the TEM micrograph of PPN03 (Fig. 7b) uniformly spaced Mt layers are in support of intercalation (Table 3). In the TEM micrograph of PPN-05 (Fig. 7c), the presence of exfoliated Mt layers is distinct. Both micrographs are also supported by their corresponding XRD data.
Fig. 7. TEM-EDX images of a — PPN–PLGA nanoparticles (PPN-01), b — Mt–PPN–PLGA nanocomposites (PPN-03) and c — Mt–PPN–PLGA nanocomposites (PPN-05).
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As has been observed in the case of SEM–EDX studies, the TEM–EDX studies (Fig. 7a b and c; Table 3) also indicate the presence of high content of carbon, oxygen and nitrogen in PPN-01 and additional peaks of silicon, aluminium and iron in samples PPN-03 and PPN-05 which confirms the presence of Mt in the Mt–PPN–PLGA nanocomposites. 4. Drug release profile PPN-01 and the samples containing Mt, PPN-03 & 05, have been selected for the study of drug release kinetics, in simulated intestinal fluid PBS (pH 7.4) (Fig. 8). As per the XRD data, encapsulation of drug has been found in two kinds of Mt–PPN–PLGA nanocomposites in which the Mt particles were intercalated (PPN-03) and exfoliated (PPN-04 to PPN-05). In the latter category PPN-05 has been selected because of its high yield, loading and encapsulation efficiency. Release of pure drug (PPN) in simulated gastric (HCl, pH 1.2) and intestinal fluid (PBS, pH 7.4) was observed to be 86% (Fig. 9) and 89% (Fig. 8) over a period of 4 h respectively and in both the cases release pattern was not in a controlled manner (~36% release per minute). With PPN-01 only 28% of the encapsulated drug was released over a period of 8 h whereas, the Mt–PPN–PLGA nanocomposites PPN-03 and PPN-05 demonstrated a slow controlled cumulative drug release of 59% and 72% in the PBS (pH 7.4) over a period of 8 h (Fig. 8). The increase in the amount of drug release with respect to the PPN-01 is related to the presence of Mt platelets in the bulk and on the surface (further confirmed by SEM–EDX and TEM–EDX results). The presence of these platelets imparts porosity to the Mt–PPN–PLGA nanocomposites which results in higher ease of passage through the Mt–PPN–PLGA nanocomposite because of which the PPN-05 Mt–PPN–PLGA nanocomposites with higher extent of exfoliation also shows higher release of PPN in a controlled manner as represented by Fig. 10. It is well understood that if a drug delivery system is able to retain high amount of drug in the stomach fluid, it would be able to release
Table 3 TEM–EDX analysis report. Element
Fig. 8. Drug release profile of a — pure PPN, b — PPN–PLGA nanoparticles (PPN-01), c — intercalated Mt–PPN–PLGA nanocomposites (PPN-03) and d — exfoliated Mt–PPN– PLGA nanocomposites, (PPN-05) in simulated intestinal fluid (PBS, pH 7.4) at 37 °C.
more drug in the intestine (Chaturvedi et al., 2010) which is the desired site of drug absorption. In this study we found that over a period of 8 h in simulated gastric fluid (HCl, pH 1.2), PPN-01 and Mt containing samples PPN-03 and PPN-05 show 18%, 14% and 28% drug release respectively (Fig. 9). This low release of PPN from PPN-03 is probably related with the stability of Mt in acidic media which prevents the release of drug in acidic media (Junping et al., 2006). In the case of the formulation PPN-05, collapse of Mt layered structure was observed which comes out to be less effective in the acidic media as compared to the PPN-03. The controlled behavior of PPN release could also be explained by the barrier properties or hindrance in the path offered by high amount of Mt layers to release the drug in both the releasing media (Fig. 10). 5. Conclusion
Weight%
Atomic%
a — PPN–PLGA nanoparticles (PPN-01) CK 58.6 NK 2.7 OK 11.3 FK 2.2 Ca K 2.9 Cu K 22.3 Total 100.0
77.3 3.1 11.1 1.8 1.1 5.6 100.0
b — Mt–PPN–PLGA nanocomposites (PPN-03) CK 20.9 NK 6.7 OK 35.7 Mg K 4.1 Al K 4.0 Si K 14.5 KK 0.9 Fe K 3.2 Cu K 9.9 Total 100.0
31.5 8.7 40.4 3.1 2.7 9.4 0.4 1.0 2.8 100.0
c — Mt–PPN–PLGA nanocomposites (PPN-05) CK 27.8 NK 6.9 OK 35.2 Mg K 0.2 Al K 6.2 Si K 12.7 KK 0.5 Fe K 2.1 Co K 0.3 Cu K 11.1 Total 100.0
31.5 8.7 40.4 3.1 2.7 9.4 0.4 1.0 0.1 3.0 100.0
In the present study an oral controlled drug delivery system for PPN loaded PLGA nanoparticles and Mt–PPN–PLGA nanocomposites was developed by w/o/w double emulsion solvent evaporation technique. About 77% entrapment efficiency and 72% release were achieved for the highly hydrophilic drug, PPN. Two types of Mt–PPN–PLGA
Fig. 9. Drug release profile of a — pure PPN, b — PPN–PLGA nanoparticles (PPN-01), c — intercalated Mt–PPN–PLGA nanocomposites (PPN-03) and d — exfoliated Mt–PPN– PLGA nanocomposites, (PPN-05) in simulated gastric fluid (HCl, pH 1.2) at 37 °C.
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Fig. 10. Diagrammatic representation of drug path within a — intercalated and b — exfoliated Mt–PPN–PLGA nanocomposites.
nanocomposites (intercalated and exfoliated Mt) were obtained. The presence of drug within the PPN–PLGA nanoparticles and Mt–PPN– PLGA nanocomposites nanoparticles and nanocomposites was also confirmed by DSC data. 50–300 nm spherical PPN–PLGA nanoparticles and Mt–PPN–PLGA nanocomposites was obtained with the Mt platelets on the surface (confirms by SEM–EDX and TEM–EDX data). The drug release profile of PPN was found to be pH dependent, the presence of Mt platelets within the PPN–PLGA formulations results in controlled and higher % release of drug. Therefore, it can be said that the synthesized formulations have high potential as a controlled drug delivery system for PPN. Disclosure The authors report no conflicts of interest in this work. Acknowledgments We sincerely express our thanks to the director, USIC, University of Delhi for instrumentation facilities, Director, AIRF, JNU for providing SEM facilities and UGC/RGNF for providing financial assistance for this research work under the project of sch/rgnf/srf/f-10/2007-08. The authors are thankful to Dr. R Nagarajan for his valuable suggestions regarding the interpretation of XRD and TGA data. References Aguzzi, C., Cerezo, P., Viseras, C., Caramella, C., 2007. Use of clays as drug delivery systems: possibilities and limitations. Applied Clay Science 36, 22–36. Bergaya, F., Theng, B.K.G., Lagaly, G., 2006. First edition Handbook of Clay Science. Elsevier. Chaturvedi, K., Umadevi, S., Vaghani, S., 2010. Floating matrix dosage form for propranolol hydrochloride based on gas formation technique: development and in vitro evaluation. Scientia Pharmaceutica 78, 927–939. Chen, Y., Zhou, A., Liu, B., Liang, J., 2010. Tramadol hydrochloride/montmorillonite composite: Preparation and controlled drug release. Applied Clay Science 49, 108–112. Dollery, S.C., 1991. Therapeutic Drugs. Churchill Livingstone, Edinburgh P272–P278. Dong, Y., Feng, S.S., 2005. Poly (D, L-lactide-co-lycolide)/montmorillonite nanoparticles for oral delivery of anticancer drugs. Biomaterials 26, 6068–6076. El-Saharty, Y.S., 2003. Simultaneous high-performance liquid chromatographic assay of furosemide and propranolol HCL and its application in a pharmacokinetic study. Journal of Pharmaceutical and Biomedical Analysis 33, 699–709. Feng, S.S., Mei, L., Anitha, P., Gan, C.W., Zhou, W., 2009. Poly(lactide)–vitamin E derivative/ montmorillonite nanoparticle formulations for the oral delivery of Docetaxel. Biomaterials 30, 3297–3306. Gil, E.C., Colarte, A.I., Bataille, B., Pedraz, J.L., Rodríguez, F., Heinämäki, J., 2006. Development and optimization of a novel sustained-release dextran tablet formulation for propranolol hydrochloride. International Journal of Pharmaceutics 2006 (317), 32–39. Iliescu, R.I., Andronescu, E., Voicu, G., Ficai, A., Covaliu, C.I., 2011. Hybrid materials based on montmorillonite and citostatic drugs: preparation and characterization. Applied Clay Science 52, 62–68. Joshi, G.V., Kevadiya, B.D., Patel, H.A., Bajaj, H.C., Jasra, R.V., 2009a. Montmorillonite as a drug delivery system: intercalation and in vitro release of Timolol maleate. International Journal of Pharmaceutics 374, 53–57.
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