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Synthesis and characterization of chlorhexidine acetate drug–montmorillonite intercalates for antibacterial applications
Applied Clay Science 101 (2014) 477–483
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Applied Clay Science journal homepage: www.elsevier.com/locate/clay
Research paper
Synthesis and characterization of chlorhexidine acetate drug–montmorillonite intercalates for antibacterial applications Kasturi Saha, B.S. Butola, Mangala Joshi ⁎ Department of Textile Technology, Indian Institute of Technology Delhi, New Delhi 110016, India
a r t i c l e
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Article history: Received 11 June 2013 Received in revised form 8 September 2014 Accepted 10 September 2014 Available online 27 September 2014 Keywords: Intercalation Chlorhexidine acetate Sustained release Montmorillonite Antibacterial activity
a b s t r a c t In this study, a drug intercalated montmorillonite (Mt) has been prepared which can be useful in designing novel topical drug delivery system. The drug–Mt intercalates were synthesized by ion exchange route where interlayer cations i.e., K+, Na+ etc. of Na+–Mt exchange with the cation of the drug, chlorhexidine acetate (Ca++). The characterization of drug–Mt intercalates has been done using X-ray diffraction, Fourier transform infrared spectroscopy, thermogravimetric technique and energy dispersive X-ray analysis; all of which indicate successful intercalation of drug into the interlayer space. These drug–Mt intercalates strongly inhibited the growth of a wide range of microorganisms including both Staphylococcus aureus and Escherichia coli. In vitro release study of the antibacterial drug– Mt intercalates in phosphate buffer saline (pH 7.4) media at 37 °C was investigated. The pattern was found to be initially burst release followed by sustained release. The Ca++–Mt intercalates with a wide range of bioactivity against microbes and controlled release characteristics have the potential for application in the area of topical drug delivery. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Montmorillonite (Na+–Mt), a bioinert clay mineral is one of the important members of the smectite family. The interlayer space of Mt is generally occupied by various exchangeable cations such as Na+, K+, Ca2+, Mg2+ and water molecules, explaining high cation exchange capacity (CEC: 70–120 mequiv/100 g) for this class of clay minerals. Unique crystalline structure of Na+–Mt allows it to expand and contract the interlayer space while retaining the octahedral and/or tetrahedral two dimensional crystallographic integrity via substitution with various organic and inorganic cations to form the intercalates (Lagaly et al., 2013; Lin et al., 2002). Na+–Mt also has large specific surface area and colloidal properties, good absorbability, adhesive ability and drug carrying capability (Iliescu et al., 2011; Seema and Datta, 2013; Van Olphen, 1963). Thus, Mt is one of the most extensively used minerals both as the excipient as well as the active substance in pharmaceutical products (Deshamane et al., 2007; Koerner et al., 2006; Wang et al., 2008; Yuan et al., 2008). Chlorhexidine acetate (Ca++) is a cationic antibiotic acting as a bacteriostatic as well as a bactericidal agent at higher concentrations (Russell, 1986). At low concentrations, the mechanism of action of this biguanide drug is ATPase inactivation whereas at higher bactericidal concentrations, it induces damage of cytoplasmic membrane by precipitating essential protein and nucleic acids. This antibiotic drug has established application for treating the nosocomial transmission (Wang et al., 2008) of infections ⁎ Corresponding author. Tel.: +91 11 26596623; fax: +91 11 26581103. E-mail address: [email protected] (M. Joshi).
http://dx.doi.org/10.1016/j.clay.2014.09.010 0169-1317/© 2014 Elsevier B.V. All rights reserved.
caused by the bacteria. It is also applicable for topical applications such as antiseptic, pharmaceutical and cosmetic preservatives and also as an antiplaque agent (Hugo and Russell, 1982; Walihauser, 1984). Nanotechnology is actually focusing on drug delivery through clay minerals (McGinity and Lach, 1976; Wai and Banker, 1966) to give protection of the drug in the systematic circulation, to provide restricted accession of the drug to the affected site and also to deliver the drug entity to the action site at a controlled manner. The current literature reports various types of release profile of modified Mt. For example Na+–Mt (Zheng et al., 2007) has been shown to act as a sustained release drug carrier after intercalation of ibuprofen (IBU) drug. The release rate is pH dependent and IBU–Mt intercalate is useful for oral administration. Another in vitro release study of Ca++ intercalated in Mt (Menga et al., 2009) reveals that this system is useful as an advanced drug delivery carrier with controlled release characteristics. In another study, amido cationic drug, acyclovir–Mt intercalate (Junping et al., 2007) leads to the development of a pH dependent controlled release system which could be used for oral applications. The present paper focuses on the preparation and characterization of a controlled drug release system based on the intercalation of Ca++ drug into the interlayer space of Na+–Mt via cation exchange route. The drug loaded Mt (Ca++–Mt) was characterized by X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, thermogravimetric (TGA) technique and energy dispersive X-ray (EDX) analysis. The antimicrobial activity of Ca++–Mt intercalates was determined against both gram-positive and gram-negative bacteria. In vitro release study of the antibacterial intercalates was investigated in phosphate buffer saline (PBS, pH 7.4) media at 37 °C. These bioactive Ca++–Mt
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intercalates have the potential for application as efficient antibacterial agents in the area of medical textiles. They can also act as a precursor for the preparation of clay polymer nanocomposite with potential application for topical drug delivery systems. 2. Experimental details 2.1. Materials Sodium montmorillonite (Na+–Mt) with a cation exchange capacity of 120 mequiv/100 g was procured from Southern Clay Products, Inc. (Japan) and used without further treatment. Chlorhexidine acetate (Ca++; white powder; M.W. 625.55; M.P. 155 °C; solubility: 19 mg/ml of water at 25 °C) was obtained from Sigma-Aldrich Company Ltd. (Dorset, UK) and used as received. The chemical structure (C22H30Cl2N10·2C2H4O2) of the model drug is shown in Fig. 1. Orthophosphoric acid (H3PO4; liquid; M.W. 98; density: 1.69 g/ml; B.P. 158 °C) was procured from Thermo Fisher Scientific India Pvt. Ltd. (Mumbai, India). Dialysis membranes (molecular weight cut-off ≤ 12,400) were obtained from Sigma-Aldrich, Bangalore. All other chemicals and solvents were of reagent grade and used without further purification. 2.2. Synthesis of drug loaded clay mineral The Ca++–Mt intercalates were prepared through cation exchange reaction of Ca++ drug with Na+–Mt. 1.0 g of Na+–Mt was dispersed in 30 ml of distilled water with vigorous stirring for 0.5 h at room temperature. Fixed amount of Ca++ drug was also dispersed in desired amount of distilled water separately. The concentrations of Ca++ used were 0.2, 0.5, 1.0 and 2.0 times CEC of Mt, respectively. These two solutions were mixed together at optimized conditions (pH value adjusted to 4.1 with H3PO4) using a magnetic stirrer at 500 rpm for about 3 h at 80 °C. After cooling, the mixture was centrifuged at 5000 rpm for 0.5 h. The supernatant solution was decanted and the residue was collected. All products were washed with water, dried at 90 °C and ground into fine powder form suitable for further characterization. The different product codes (drug–Mt intercalates) are listed in Table 1. 2.3. Characterization of drug loaded clay mineral XRD studies of Na+–Mt and Ca++–Mt intercalates were carried out on a PANalytical X-ray Powder Diffractometer using Ni-filtered CuKα radiation of wavelength 1.5418 Å, working voltage 40 kV and working current 30 mA. Scanning was carried out in the range 2θ values from 2 to 20° at a scanning rate of 2°/min for all the samples.
Fig. 1. Chemical structure of chlorhexidine acetate drug.
Table 1 Product codes for drug–montmorillonite intercalates. Sample codes
Concentration of Ca++ used (Fraction of CEC of Na+–Mt)
0.2Ca++–Mt 0.5Ca++–Mt 1.0Ca++–Mt 2.0Ca++–Mt
0.2 0.5 1.0 2.0
Perkin Elmer, Spectrum BX FT-IR system was used for determining the functional groups present in Na+–Mt, Ca++ drug and Ca++–Mt intercalates. FT−IR samples were prepared by KBr pressed disk technique applying 400 kg/cm2 pressure. Analyses were performed in the transmission mode between 4000 and 400 cm−1, with a resolution of 2 cm−1. Perkin Elmer, TGA-7 system was used to determine degradation behavior of Na+–Mt, Ca++ drug and Ca++–Mt intercalates. The amount of drug loading (Ca++ in mass%) was determined from the TGA analysis. Testing was carried out at a heating rate of 20 °C/min up to 900 °C in nitrogen atmosphere. ZEISS EVO 50 Scanning Electron Microscope working with EDX attachment (detection limits typically 0.1–100 mass%) was used to determine the elemental composition of the Na+–Mt, Ca++ drug and various Ca++–Mt intercalates. EDX spectra based on the silicon drift detector (SDD) technology with an energy resolution of 127 eV at MnKα were recorded. EDX samples were coated with carbon to prevent charging during exposure to the electron beam prior to the experiment. 2.4. Antibacterial activity assay 2.4.1. Colony counting method Minimum inhibitory concentration (MIC) of the Ca++ drug and various Ca++–Mt intercalates was determined by colony counting method (AATCC 100). AATCC stands for American Association of Textile Chemists and Colorists. AATCC 100 is an antimicrobial test method which provides a quantitative evaluation of the antimicrobial activity of the test material. The MIC is defined as the lowest concentration of antimicrobial agent which inhibits a visible growth of bacterial colony formation. Luria broth solution was freshly prepared by dispersing 2 g of Luria broth in 100 ml of distilled water. This solution was sterilized by autoclaving at 15 lb pressure for 15 min. Neat Na+–Mt (control), Ca++ and Ca++–Mt powders at different concentration levels ranging from 0.1 to 10 ppm were dispersed in conical flask containing Luria broth solution and then inoculated with 10 μl of Staphylococcus aureus (106 CFU/ml) and Escherichia coli (106 CFU/ml) bacterial solution and kept at 37 °C for 24 h. Serial dilution of these solutions was made in sterilized DI water. Dilutions of 10−4 and 10− 5 were used for colony counting. 10 μl of diluted solutions was spread on to the agar plate and plates were incubated at 37 °C for 24 h. After incubation bacterial colonies were counted using Yorco digital colony counter on the surface of agar plate. The minimum concentration at which 99% reduction in number of colonies in neat drug takes place as compared to the control clay mineral is indicated as MIC of Ca++ drug. 2.4.2. Disk diffusion test Antibacterial activity of the Ca++–Mt intercalates was tested against both gram-positive (S. aureus) and gram-negative bacteria (E. coli) by the disk diffusion test (AATCC 90). AATCC 90 is an antimicrobial test method which provides a qualitative assessment of the bacteriostatic activity of the test material that is treated with antimicrobials and is capable of producing a zone of inhibition. Homogeneous pastes of Na+–Mt and Ca++–Mt were prepared by mixing 0.1 g of each in 0.2 ml of DI water. Pastes were uniformly applied on the paper disks of about 15 mm diameter. The disks were placed in UV chamber for 30 min for sterilization. Nutrient agar solution was made by suspending 20 g of Luria broth in 1000 ml of DI water and 15 g of
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agar–agar was also added in the solution as a solidifying agent. After sterilization, about 25–30 ml of nutrient agar solution was uniformly spread on petri dishes. 10 μl of bacterial solution was evenly spread over the nutrient agar solution. S. aureus and E. coli concentrations of around 1.2 × 106 and 1.5 × 10 6 CFU/ml respectively were used for conducting these experiments. The paper disks prepared earlier were then placed onto the nutrient agar plates. The agar plates were kept for incubation at 37 °C for 24 h. The zone of inhibition was measured after 24 h incubation period.
2.5. In vitro drug release In vitro drug release study was performed by dispersing dialysis membrane bag containing 10 mg of Ca++ drug or 40 mg of 2.0Ca++–Mt separately in 5 ml PBS media of pH 7.4 at 37 °C. Dialysis bag was dipped into the receptor compartment containing 100 ml PBS dissolution medium under shaking at 37 °C at 200 rpm. At suitable intervals, 3 ml of dissolution medium was withdrawn and analyzed for Ca++ content by taking absorbance value at 254 nm using UV spectrophotometer. The same volume of dissolution medium was replaced with a fresh dissolution medium and the total volume was maintained at 100 ml. Tests were performed in triplicate and the results were recorded as an average for further analysis.
Fig. 3. FT-IR spectra of montmorillonite and Ca++–Mt intercalates.
3. Results and discussions
3.2. FT-IR spectra analysis
3.1. XRD analysis
In the FT-IR studies of Na+–Mt (Fig. 3), the Si–O stretching vibrations were observed at 624 and 522 cm−1. Al–O–H stretching vibration was observed at 911 cm−1. Band at 1040 cm−1 indicates Si–O–Si stretching vibration. However, a band at 1638 cm−1 in the spectrum of clay suggests (Nayak and Singh, 2007) the possibility of water of hydration in the adsorbent. Strong band at 3414 cm−1 indicates the presence of the hydroxyl linkage and symmetric (O–H) stretching vibration band of adsorbed water. In the FT-IR spectra of Ca++ drug, the reflection at 3380 cm− 1 is attributed to the N–H stretching vibration of the aromatic ring of Ca++ and the peaks at 2934 and 2859 cm−1 correspond to the symmetric and asymmetric stretching vibrations of the methylene groups of Ca++ respectively. The absorption bands in the region of 1600–1200 cm−1 result from C–N and C–C vibrations of Ca++ drug. After the intercalation of Ca++ drug (Fig. 4), the absorption band at 3414 cm−1 corresponding to the symmetric (O–H) stretching vibration band of adsorbed water shifts to higher frequency at 3478 cm−1 which reflects a decrease in amount of adsorbed water in the external surface
XRD patterns of Na+–Mt and Ca++–Mt are shown in Fig. 2. The XRD pattern clearly shows the reflection corresponding to d001 (Ohashi et al., 1998) with interlayer space of 12.655 Å, a characteristic d001 value for Na+–Mt. After cation exchange, the Ca++–Mt intercalates showed that there is a shift in this reflection which indicates that the d001 value of Ca++–Mt increases from 12.655 to 14.82 Å, an increase of 17.10%. The increased d-value indicated that Ca++ was successfully intercalated in the Na+–Mt interlayer space. From XRD, it is also observed that Na+–Mt shows a sharp reflection around 6.985°. The reflection position of Na+–Mt corresponding to d001 is different from the reflection at 6.03° observed for Ca++–Mt intercalates indicating disorder caused due to restacking of silicate layers after drug intercalation into the clay mineral interlayer space.
Fig. 2. X-ray powder diffraction pattern of montmorillonite, Ca++ drug and Ca++ –Mt intercalates.
Fig. 4. FT-IR spectra of Ca++ drug.
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Fig. 5. TGA curves of montmorillonite, Ca++ drug and Ca++–Mt intercalates.
or in the interlayer space of drug–Mt intercalates. This suggests that the hydrophilicity of Na+–Mt surface decreases after the intercalation of Ca++ into the clay mineral interlayer space. Therefore, in the FT-IR spectra of Ca++–Mt intercalates the presence of additional absorption reflections at 3380, 2934, 2859 and in the region of 1600–1200 cm−1 due to characteristic groups of Ca++ drug indicates successful intercalation of drug into the Na+–Mt interlayer space. These reflections are absent in FT-IR spectra of Na+–Mt. Also, it can be concluded from the FT-IR spectra that the hydrophilic surface of Na+–Mt turns somewhat hydrophobic in nature after the successful intercalation of Ca++ drug into the Na+–Mt interlayer space.
behavior was also found in the case of the Ca++ drug which degrades completely at 690 °C without leaving any residue. TGA analysis shows that Na+–Mt contains 82.6% residue at 900 °C. Based on the left over residue in the case of various Ca++–Mt intercalates, the Ca++ loading has been deduced from the TGA curves and is summarized in Table 2.
3.3. Thermogravimetric analysis The TGA curves for Na+–Mt, Ca++ drug and Ca++–Mt intercalates are shown in Fig. 5. Decomposition of Na+–Mt occurs in two distinct steps. In the first step desorption of water from the interlayer space occurs in the range of 90–120 °C. In the second step dehydroxylation of the layered crystal lattice structure occurs around 700 °C (Xie et al., 2001). The modification of Na+–Mt with organic drug cation increases the number of decomposition steps. The first step corresponds to the onset of decomposition of organic matter around 220 °C with the maximum decomposition occurring around 330–350 °C. The second step is mainly attributed to the chemical decomposition of the bonded structure of the organic drug around 400 °C. The onset for the third and final decomposition starts at around 550 °C due to dehydroxylation of the layers and proceeds till around 900 °C. Residual carbonaceous product formation occurs above 700 °C (Xi et al., 2004). A similar kind of degradation
Table 2 Loading efficiency of Ca++–Mt intercalates. Sample ++
0.2Ca –Mt 0.5Ca++–Mt 1.0Ca++–Mt 2.0Ca++–Mt
Ca++ loading (mass%) 5 12 17 24
Fig. 6. EDX spectra of montmorillonite, Ca++ drug and Ca++–Mt intercalates.
K. Saha et al. / Applied Clay Science 101 (2014) 477–483 Table 3 Elemental analysis data for Na+–Mt, Ca++ and Ca++–Mt using EDX spectra. Element (mass%) C
Si
O
Al
Fe
Na
Mg
Cl
N
Na+–Mt Ca++ 0.2Ca++–Mt 0.5Ca++–Mt 1.0Ca++–Mt 2.0Ca++–Mt
24.56 – 21.76 17.71 16.45 11.80
55.02 26.25 44.12 42.13 39.84 38.33
11.91 – 11.00 9.40 8.64 4.35
3.67 – 1.80 1.20 1.01 0.55
2.78 – 1.17 0.77 – –
2.06 – 1.93 1.59 1.40 0.85
– 10.05 1.81 3.04 3.27 7.75
– 29.60 7.44 9.91 11.43 13.05
– 34.08 8.96 14.24 17.88 23.28
With the increase in drug concentration (from 0.2 to 2.0 CEC) the loading efficiency of drug into the clay mineral interlayer space increases from 5 to 24% as expected.
3.4. EDX analysis A standard EDX spectrum recorded for the elemental analysis of Na+–Mt, Ca++ drug and Ca++–Mt intercalates is shown in Fig. 6. In the case of Na+–Mt, sharp maxima for carbon (used for coating), oxygen, silicon, sodium, aluminum and iron (as impurity) were observed. These reflections are directly related to the Na+–Mt characteristic line K. Another low intensity reflection for magnesium was also observed in clay mineral EDX analysis. In the case of Ca++ drug, two sharp characteristic maxima for nitrogen (located between 0 and 1 keV) and chlorine (located between 2 and 3 keV) were examined. These sharp reflections of Ca++ drug were also observed in EDX spectrum of drug–Mt intercalates in addition to characteristic K line of Na+–Mt indicating successful loading of Ca++ drug into the clay mineral interlayer space. Amount of various elements (in mass%) present in Na+–Mt, Ca++ drug and Ca++–Mt intercalates is summarized in Table 3. From the table it is obvious that the amount of various elements such as silicon, aluminum, iron and magnesium decreases with the increase in Ca++ concentration (from 0.2 to 2.0 CEC) in Ca++–Mt intercalates. Also, the absence of sodium was observed in the elemental analysis of 1.0Ca++–Mt and 2.0Ca++–Mt. Furthermore, an increasing trend in mass percentage of elements like chlorine and nitrogen was observed
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as the CEC of drug–Mt intercalates increased from 0.2 to 2.0. Therefore, it can be concluded that the drug has been successfully intercalated into the Na+–Mt interlayer space after ion exchange. 3.5. Antibacterial activity assay 3.5.1. Colony counting method Minimum inhibitory concentration (MIC) of Ca++ drug against S. aureus was found by varying the concentration of Ca++ in the range of 0.1 to 10 ppm (0.2, 0.5, 1 and 5 ppm). The MIC of Ca++ drug was found to be 1 ppm. Based on this, three different concentrations such as 1, 2 and 5 ppm were chosen for determining the MIC of 2.0Ca++–Mt intercalates. It was found that the MIC of Ca++–Mt intercalates was 2 ppm which is higher than MIC of Ca++ drug. This is obvious as the drug content in the Ca++–Mt intercalates is only 24% by mass. 3.5.2. Disk diffusion test Disk diffusion test results of Na+–Mt and Ca++–Mt intercalates are shown in Figs. 7 and 8. Neat Na+–Mt showed no inhibitory zone for S. aureus and E. coli, reflecting no antibacterial activity for neat clay mineral. However, Ca++ drug and various Ca++–Mt intercalates showed a very clear inhibition zone around the specimen. The results from the zone of inhibition tests are given in Table 4. These results indicate that Ca++–Mt has a strong activity against both gram-positive and gram-negative bacteria. Only 0.2Ca++–Mt did not exhibit a clear inhibition zone due to very low Ca++ content (only 5%) compared to other Ca++–Mt intercalates. The presence of zone of inhibition is an indication of the diffusion of the antibacterial drug CA from Ca++–Mt intercalates into the agar media. It can also be concluded that with increasing drug loading concentration (5 to 24% by mass) in the Ca++–Mt intercalates, the size of inhibition zone increases. The mechanism responsible for the observed antibacterial activity of Ca++ (Kuyyakanond and Quesnel, 1992) is electrostatic attraction between the chlorhexidine (cation) and the negatively charged bacterial cells. After the adsorption onto the microorganism's cell wall, drug molecule disrupts the integrity of the cell membrane and causes the leakage of intracellular components of the organisms. As a result of this the microorganisms gradually die.
Fig. 7. Photograph showing zone of inhibition of Na+–Mt and Ca++–Mt against S. aureus.
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Fig. 8. Photograph showing zone of inhibition of Na+–Mt and Ca++–Mt against E. coli.
3.6. Drug release study
4. Conclusions
The release profile of Ca++ drug and intercalated drug from 2.0Ca++–Mt in PBS media of pH 7.4 at 37 °C is shown in Fig. 9. From the figure it is clear that, Ca++ drug is continuously released for over 180 h whereas the initial burst release happens within the initial 18 h. Based on the release profile at pH 7.4, the maximum release of 84% of neat Ca++ drug was observed over 180 h. But 100% release of Ca++ drug was never attained due to the equilibrium characteristics of ion exchange reaction, i.e. intercalated cations cannot be exchanged completely. In the case of 2.0Ca++–Mt, initial burst release lasted for up to 12 h perhaps due to the presence of drug adsorbed on the surface of Na+–Mt which is advantageous for treating bacterial colonization and infection related diseases. However, the total amount of drug released (20%) within 12 h is much less in Ca++–Mt as compared to 60% in the case of neat Ca++ drug. After the initial burst release, the drug released slowly and in 24 h only 24% of the total drug was released. After that the drug release rate remains continuous for about 180 h in a sustained manner compared to the Ca++ drug. The total release of drug from Ca++–Mt was observed to be around 54%. This kind of sustained release occurs due to the presence of bulky and hydrophobic drug cation into the interlayer space which cannot be de-intercalated easily (Jung et al., 2008) by ion exchange reaction with the small Na+ and K+ cations. This sustained release characteristics are beneficial for application as a drug delivery carrier for topical applications where long term activity is necessary.
Ca++–Mt intercalates have been synthesized using Na+–Mt and antibacterial drug, Ca++. XRD patterns of Ca++–Mt show an increase in the d-value confirming the intercalation of Ca++ into the interlayer space of Na+–Mt. FT-IR and EDX studies further support successful intercalation of Ca++ drug into the Na+–Mt interlayer space. The antibacterial assay showed that Ca++–Mt intercalates had a broad spectrum antibacterial capability against both gram-positive and gram-negative bacteria. The drug release profile of intercalated species (Ca++–Mt) into PBS media indicates burst release followed by sustained release activity in comparison with neat drug (Ca++) which initially shows a very high amount (60%) of burst release followed by a slow release. Such intercalated species have the potential to be used in topical drug delivery in the form of fibrous implant polymeric materials such as sutures and wound dressings.
Table 4 Inhibition zone of Na+–Mt and Ca++–Mt against S. aureus and E. coli bacteria. Zone of inhibition (in mm)
Na+–Mt Ca++ 0.2Ca++–Mt 0.5Ca++–Mt 1.0Ca++–Mt 2.0Ca++–Mt Initial diameter: 15 mm; no inhibition.
S. aureus
E. coli
Absent 35 Uneven 25 28 34
Absent 36 17 25 30 32 Fig. 9. Release profile of neat drug and Ca++ from 2.0Ca++–Mt in PBS media at 37 °C.
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Applied Clay Science journal homepage: www.elsevier.com/locate/clay
Research paper
Synthesis and characterization of chlorhexidine acetate drug–montmorillonite intercalates for antibacterial applications Kasturi Saha, B.S. Butola, Mangala Joshi ⁎ Department of Textile Technology, Indian Institute of Technology Delhi, New Delhi 110016, India
a r t i c l e
i n f o
Article history: Received 11 June 2013 Received in revised form 8 September 2014 Accepted 10 September 2014 Available online 27 September 2014 Keywords: Intercalation Chlorhexidine acetate Sustained release Montmorillonite Antibacterial activity
a b s t r a c t In this study, a drug intercalated montmorillonite (Mt) has been prepared which can be useful in designing novel topical drug delivery system. The drug–Mt intercalates were synthesized by ion exchange route where interlayer cations i.e., K+, Na+ etc. of Na+–Mt exchange with the cation of the drug, chlorhexidine acetate (Ca++). The characterization of drug–Mt intercalates has been done using X-ray diffraction, Fourier transform infrared spectroscopy, thermogravimetric technique and energy dispersive X-ray analysis; all of which indicate successful intercalation of drug into the interlayer space. These drug–Mt intercalates strongly inhibited the growth of a wide range of microorganisms including both Staphylococcus aureus and Escherichia coli. In vitro release study of the antibacterial drug– Mt intercalates in phosphate buffer saline (pH 7.4) media at 37 °C was investigated. The pattern was found to be initially burst release followed by sustained release. The Ca++–Mt intercalates with a wide range of bioactivity against microbes and controlled release characteristics have the potential for application in the area of topical drug delivery. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Montmorillonite (Na+–Mt), a bioinert clay mineral is one of the important members of the smectite family. The interlayer space of Mt is generally occupied by various exchangeable cations such as Na+, K+, Ca2+, Mg2+ and water molecules, explaining high cation exchange capacity (CEC: 70–120 mequiv/100 g) for this class of clay minerals. Unique crystalline structure of Na+–Mt allows it to expand and contract the interlayer space while retaining the octahedral and/or tetrahedral two dimensional crystallographic integrity via substitution with various organic and inorganic cations to form the intercalates (Lagaly et al., 2013; Lin et al., 2002). Na+–Mt also has large specific surface area and colloidal properties, good absorbability, adhesive ability and drug carrying capability (Iliescu et al., 2011; Seema and Datta, 2013; Van Olphen, 1963). Thus, Mt is one of the most extensively used minerals both as the excipient as well as the active substance in pharmaceutical products (Deshamane et al., 2007; Koerner et al., 2006; Wang et al., 2008; Yuan et al., 2008). Chlorhexidine acetate (Ca++) is a cationic antibiotic acting as a bacteriostatic as well as a bactericidal agent at higher concentrations (Russell, 1986). At low concentrations, the mechanism of action of this biguanide drug is ATPase inactivation whereas at higher bactericidal concentrations, it induces damage of cytoplasmic membrane by precipitating essential protein and nucleic acids. This antibiotic drug has established application for treating the nosocomial transmission (Wang et al., 2008) of infections ⁎ Corresponding author. Tel.: +91 11 26596623; fax: +91 11 26581103. E-mail address: [email protected] (M. Joshi).
http://dx.doi.org/10.1016/j.clay.2014.09.010 0169-1317/© 2014 Elsevier B.V. All rights reserved.
caused by the bacteria. It is also applicable for topical applications such as antiseptic, pharmaceutical and cosmetic preservatives and also as an antiplaque agent (Hugo and Russell, 1982; Walihauser, 1984). Nanotechnology is actually focusing on drug delivery through clay minerals (McGinity and Lach, 1976; Wai and Banker, 1966) to give protection of the drug in the systematic circulation, to provide restricted accession of the drug to the affected site and also to deliver the drug entity to the action site at a controlled manner. The current literature reports various types of release profile of modified Mt. For example Na+–Mt (Zheng et al., 2007) has been shown to act as a sustained release drug carrier after intercalation of ibuprofen (IBU) drug. The release rate is pH dependent and IBU–Mt intercalate is useful for oral administration. Another in vitro release study of Ca++ intercalated in Mt (Menga et al., 2009) reveals that this system is useful as an advanced drug delivery carrier with controlled release characteristics. In another study, amido cationic drug, acyclovir–Mt intercalate (Junping et al., 2007) leads to the development of a pH dependent controlled release system which could be used for oral applications. The present paper focuses on the preparation and characterization of a controlled drug release system based on the intercalation of Ca++ drug into the interlayer space of Na+–Mt via cation exchange route. The drug loaded Mt (Ca++–Mt) was characterized by X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, thermogravimetric (TGA) technique and energy dispersive X-ray (EDX) analysis. The antimicrobial activity of Ca++–Mt intercalates was determined against both gram-positive and gram-negative bacteria. In vitro release study of the antibacterial intercalates was investigated in phosphate buffer saline (PBS, pH 7.4) media at 37 °C. These bioactive Ca++–Mt
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intercalates have the potential for application as efficient antibacterial agents in the area of medical textiles. They can also act as a precursor for the preparation of clay polymer nanocomposite with potential application for topical drug delivery systems. 2. Experimental details 2.1. Materials Sodium montmorillonite (Na+–Mt) with a cation exchange capacity of 120 mequiv/100 g was procured from Southern Clay Products, Inc. (Japan) and used without further treatment. Chlorhexidine acetate (Ca++; white powder; M.W. 625.55; M.P. 155 °C; solubility: 19 mg/ml of water at 25 °C) was obtained from Sigma-Aldrich Company Ltd. (Dorset, UK) and used as received. The chemical structure (C22H30Cl2N10·2C2H4O2) of the model drug is shown in Fig. 1. Orthophosphoric acid (H3PO4; liquid; M.W. 98; density: 1.69 g/ml; B.P. 158 °C) was procured from Thermo Fisher Scientific India Pvt. Ltd. (Mumbai, India). Dialysis membranes (molecular weight cut-off ≤ 12,400) were obtained from Sigma-Aldrich, Bangalore. All other chemicals and solvents were of reagent grade and used without further purification. 2.2. Synthesis of drug loaded clay mineral The Ca++–Mt intercalates were prepared through cation exchange reaction of Ca++ drug with Na+–Mt. 1.0 g of Na+–Mt was dispersed in 30 ml of distilled water with vigorous stirring for 0.5 h at room temperature. Fixed amount of Ca++ drug was also dispersed in desired amount of distilled water separately. The concentrations of Ca++ used were 0.2, 0.5, 1.0 and 2.0 times CEC of Mt, respectively. These two solutions were mixed together at optimized conditions (pH value adjusted to 4.1 with H3PO4) using a magnetic stirrer at 500 rpm for about 3 h at 80 °C. After cooling, the mixture was centrifuged at 5000 rpm for 0.5 h. The supernatant solution was decanted and the residue was collected. All products were washed with water, dried at 90 °C and ground into fine powder form suitable for further characterization. The different product codes (drug–Mt intercalates) are listed in Table 1. 2.3. Characterization of drug loaded clay mineral XRD studies of Na+–Mt and Ca++–Mt intercalates were carried out on a PANalytical X-ray Powder Diffractometer using Ni-filtered CuKα radiation of wavelength 1.5418 Å, working voltage 40 kV and working current 30 mA. Scanning was carried out in the range 2θ values from 2 to 20° at a scanning rate of 2°/min for all the samples.
Fig. 1. Chemical structure of chlorhexidine acetate drug.
Table 1 Product codes for drug–montmorillonite intercalates. Sample codes
Concentration of Ca++ used (Fraction of CEC of Na+–Mt)
0.2Ca++–Mt 0.5Ca++–Mt 1.0Ca++–Mt 2.0Ca++–Mt
0.2 0.5 1.0 2.0
Perkin Elmer, Spectrum BX FT-IR system was used for determining the functional groups present in Na+–Mt, Ca++ drug and Ca++–Mt intercalates. FT−IR samples were prepared by KBr pressed disk technique applying 400 kg/cm2 pressure. Analyses were performed in the transmission mode between 4000 and 400 cm−1, with a resolution of 2 cm−1. Perkin Elmer, TGA-7 system was used to determine degradation behavior of Na+–Mt, Ca++ drug and Ca++–Mt intercalates. The amount of drug loading (Ca++ in mass%) was determined from the TGA analysis. Testing was carried out at a heating rate of 20 °C/min up to 900 °C in nitrogen atmosphere. ZEISS EVO 50 Scanning Electron Microscope working with EDX attachment (detection limits typically 0.1–100 mass%) was used to determine the elemental composition of the Na+–Mt, Ca++ drug and various Ca++–Mt intercalates. EDX spectra based on the silicon drift detector (SDD) technology with an energy resolution of 127 eV at MnKα were recorded. EDX samples were coated with carbon to prevent charging during exposure to the electron beam prior to the experiment. 2.4. Antibacterial activity assay 2.4.1. Colony counting method Minimum inhibitory concentration (MIC) of the Ca++ drug and various Ca++–Mt intercalates was determined by colony counting method (AATCC 100). AATCC stands for American Association of Textile Chemists and Colorists. AATCC 100 is an antimicrobial test method which provides a quantitative evaluation of the antimicrobial activity of the test material. The MIC is defined as the lowest concentration of antimicrobial agent which inhibits a visible growth of bacterial colony formation. Luria broth solution was freshly prepared by dispersing 2 g of Luria broth in 100 ml of distilled water. This solution was sterilized by autoclaving at 15 lb pressure for 15 min. Neat Na+–Mt (control), Ca++ and Ca++–Mt powders at different concentration levels ranging from 0.1 to 10 ppm were dispersed in conical flask containing Luria broth solution and then inoculated with 10 μl of Staphylococcus aureus (106 CFU/ml) and Escherichia coli (106 CFU/ml) bacterial solution and kept at 37 °C for 24 h. Serial dilution of these solutions was made in sterilized DI water. Dilutions of 10−4 and 10− 5 were used for colony counting. 10 μl of diluted solutions was spread on to the agar plate and plates were incubated at 37 °C for 24 h. After incubation bacterial colonies were counted using Yorco digital colony counter on the surface of agar plate. The minimum concentration at which 99% reduction in number of colonies in neat drug takes place as compared to the control clay mineral is indicated as MIC of Ca++ drug. 2.4.2. Disk diffusion test Antibacterial activity of the Ca++–Mt intercalates was tested against both gram-positive (S. aureus) and gram-negative bacteria (E. coli) by the disk diffusion test (AATCC 90). AATCC 90 is an antimicrobial test method which provides a qualitative assessment of the bacteriostatic activity of the test material that is treated with antimicrobials and is capable of producing a zone of inhibition. Homogeneous pastes of Na+–Mt and Ca++–Mt were prepared by mixing 0.1 g of each in 0.2 ml of DI water. Pastes were uniformly applied on the paper disks of about 15 mm diameter. The disks were placed in UV chamber for 30 min for sterilization. Nutrient agar solution was made by suspending 20 g of Luria broth in 1000 ml of DI water and 15 g of
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agar–agar was also added in the solution as a solidifying agent. After sterilization, about 25–30 ml of nutrient agar solution was uniformly spread on petri dishes. 10 μl of bacterial solution was evenly spread over the nutrient agar solution. S. aureus and E. coli concentrations of around 1.2 × 106 and 1.5 × 10 6 CFU/ml respectively were used for conducting these experiments. The paper disks prepared earlier were then placed onto the nutrient agar plates. The agar plates were kept for incubation at 37 °C for 24 h. The zone of inhibition was measured after 24 h incubation period.
2.5. In vitro drug release In vitro drug release study was performed by dispersing dialysis membrane bag containing 10 mg of Ca++ drug or 40 mg of 2.0Ca++–Mt separately in 5 ml PBS media of pH 7.4 at 37 °C. Dialysis bag was dipped into the receptor compartment containing 100 ml PBS dissolution medium under shaking at 37 °C at 200 rpm. At suitable intervals, 3 ml of dissolution medium was withdrawn and analyzed for Ca++ content by taking absorbance value at 254 nm using UV spectrophotometer. The same volume of dissolution medium was replaced with a fresh dissolution medium and the total volume was maintained at 100 ml. Tests were performed in triplicate and the results were recorded as an average for further analysis.
Fig. 3. FT-IR spectra of montmorillonite and Ca++–Mt intercalates.
3. Results and discussions
3.2. FT-IR spectra analysis
3.1. XRD analysis
In the FT-IR studies of Na+–Mt (Fig. 3), the Si–O stretching vibrations were observed at 624 and 522 cm−1. Al–O–H stretching vibration was observed at 911 cm−1. Band at 1040 cm−1 indicates Si–O–Si stretching vibration. However, a band at 1638 cm−1 in the spectrum of clay suggests (Nayak and Singh, 2007) the possibility of water of hydration in the adsorbent. Strong band at 3414 cm−1 indicates the presence of the hydroxyl linkage and symmetric (O–H) stretching vibration band of adsorbed water. In the FT-IR spectra of Ca++ drug, the reflection at 3380 cm− 1 is attributed to the N–H stretching vibration of the aromatic ring of Ca++ and the peaks at 2934 and 2859 cm−1 correspond to the symmetric and asymmetric stretching vibrations of the methylene groups of Ca++ respectively. The absorption bands in the region of 1600–1200 cm−1 result from C–N and C–C vibrations of Ca++ drug. After the intercalation of Ca++ drug (Fig. 4), the absorption band at 3414 cm−1 corresponding to the symmetric (O–H) stretching vibration band of adsorbed water shifts to higher frequency at 3478 cm−1 which reflects a decrease in amount of adsorbed water in the external surface
XRD patterns of Na+–Mt and Ca++–Mt are shown in Fig. 2. The XRD pattern clearly shows the reflection corresponding to d001 (Ohashi et al., 1998) with interlayer space of 12.655 Å, a characteristic d001 value for Na+–Mt. After cation exchange, the Ca++–Mt intercalates showed that there is a shift in this reflection which indicates that the d001 value of Ca++–Mt increases from 12.655 to 14.82 Å, an increase of 17.10%. The increased d-value indicated that Ca++ was successfully intercalated in the Na+–Mt interlayer space. From XRD, it is also observed that Na+–Mt shows a sharp reflection around 6.985°. The reflection position of Na+–Mt corresponding to d001 is different from the reflection at 6.03° observed for Ca++–Mt intercalates indicating disorder caused due to restacking of silicate layers after drug intercalation into the clay mineral interlayer space.
Fig. 2. X-ray powder diffraction pattern of montmorillonite, Ca++ drug and Ca++ –Mt intercalates.
Fig. 4. FT-IR spectra of Ca++ drug.
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Fig. 5. TGA curves of montmorillonite, Ca++ drug and Ca++–Mt intercalates.
or in the interlayer space of drug–Mt intercalates. This suggests that the hydrophilicity of Na+–Mt surface decreases after the intercalation of Ca++ into the clay mineral interlayer space. Therefore, in the FT-IR spectra of Ca++–Mt intercalates the presence of additional absorption reflections at 3380, 2934, 2859 and in the region of 1600–1200 cm−1 due to characteristic groups of Ca++ drug indicates successful intercalation of drug into the Na+–Mt interlayer space. These reflections are absent in FT-IR spectra of Na+–Mt. Also, it can be concluded from the FT-IR spectra that the hydrophilic surface of Na+–Mt turns somewhat hydrophobic in nature after the successful intercalation of Ca++ drug into the Na+–Mt interlayer space.
behavior was also found in the case of the Ca++ drug which degrades completely at 690 °C without leaving any residue. TGA analysis shows that Na+–Mt contains 82.6% residue at 900 °C. Based on the left over residue in the case of various Ca++–Mt intercalates, the Ca++ loading has been deduced from the TGA curves and is summarized in Table 2.
3.3. Thermogravimetric analysis The TGA curves for Na+–Mt, Ca++ drug and Ca++–Mt intercalates are shown in Fig. 5. Decomposition of Na+–Mt occurs in two distinct steps. In the first step desorption of water from the interlayer space occurs in the range of 90–120 °C. In the second step dehydroxylation of the layered crystal lattice structure occurs around 700 °C (Xie et al., 2001). The modification of Na+–Mt with organic drug cation increases the number of decomposition steps. The first step corresponds to the onset of decomposition of organic matter around 220 °C with the maximum decomposition occurring around 330–350 °C. The second step is mainly attributed to the chemical decomposition of the bonded structure of the organic drug around 400 °C. The onset for the third and final decomposition starts at around 550 °C due to dehydroxylation of the layers and proceeds till around 900 °C. Residual carbonaceous product formation occurs above 700 °C (Xi et al., 2004). A similar kind of degradation
Table 2 Loading efficiency of Ca++–Mt intercalates. Sample ++
0.2Ca –Mt 0.5Ca++–Mt 1.0Ca++–Mt 2.0Ca++–Mt
Ca++ loading (mass%) 5 12 17 24
Fig. 6. EDX spectra of montmorillonite, Ca++ drug and Ca++–Mt intercalates.
K. Saha et al. / Applied Clay Science 101 (2014) 477–483 Table 3 Elemental analysis data for Na+–Mt, Ca++ and Ca++–Mt using EDX spectra. Element (mass%) C
Si
O
Al
Fe
Na
Mg
Cl
N
Na+–Mt Ca++ 0.2Ca++–Mt 0.5Ca++–Mt 1.0Ca++–Mt 2.0Ca++–Mt
24.56 – 21.76 17.71 16.45 11.80
55.02 26.25 44.12 42.13 39.84 38.33
11.91 – 11.00 9.40 8.64 4.35
3.67 – 1.80 1.20 1.01 0.55
2.78 – 1.17 0.77 – –
2.06 – 1.93 1.59 1.40 0.85
– 10.05 1.81 3.04 3.27 7.75
– 29.60 7.44 9.91 11.43 13.05
– 34.08 8.96 14.24 17.88 23.28
With the increase in drug concentration (from 0.2 to 2.0 CEC) the loading efficiency of drug into the clay mineral interlayer space increases from 5 to 24% as expected.
3.4. EDX analysis A standard EDX spectrum recorded for the elemental analysis of Na+–Mt, Ca++ drug and Ca++–Mt intercalates is shown in Fig. 6. In the case of Na+–Mt, sharp maxima for carbon (used for coating), oxygen, silicon, sodium, aluminum and iron (as impurity) were observed. These reflections are directly related to the Na+–Mt characteristic line K. Another low intensity reflection for magnesium was also observed in clay mineral EDX analysis. In the case of Ca++ drug, two sharp characteristic maxima for nitrogen (located between 0 and 1 keV) and chlorine (located between 2 and 3 keV) were examined. These sharp reflections of Ca++ drug were also observed in EDX spectrum of drug–Mt intercalates in addition to characteristic K line of Na+–Mt indicating successful loading of Ca++ drug into the clay mineral interlayer space. Amount of various elements (in mass%) present in Na+–Mt, Ca++ drug and Ca++–Mt intercalates is summarized in Table 3. From the table it is obvious that the amount of various elements such as silicon, aluminum, iron and magnesium decreases with the increase in Ca++ concentration (from 0.2 to 2.0 CEC) in Ca++–Mt intercalates. Also, the absence of sodium was observed in the elemental analysis of 1.0Ca++–Mt and 2.0Ca++–Mt. Furthermore, an increasing trend in mass percentage of elements like chlorine and nitrogen was observed
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as the CEC of drug–Mt intercalates increased from 0.2 to 2.0. Therefore, it can be concluded that the drug has been successfully intercalated into the Na+–Mt interlayer space after ion exchange. 3.5. Antibacterial activity assay 3.5.1. Colony counting method Minimum inhibitory concentration (MIC) of Ca++ drug against S. aureus was found by varying the concentration of Ca++ in the range of 0.1 to 10 ppm (0.2, 0.5, 1 and 5 ppm). The MIC of Ca++ drug was found to be 1 ppm. Based on this, three different concentrations such as 1, 2 and 5 ppm were chosen for determining the MIC of 2.0Ca++–Mt intercalates. It was found that the MIC of Ca++–Mt intercalates was 2 ppm which is higher than MIC of Ca++ drug. This is obvious as the drug content in the Ca++–Mt intercalates is only 24% by mass. 3.5.2. Disk diffusion test Disk diffusion test results of Na+–Mt and Ca++–Mt intercalates are shown in Figs. 7 and 8. Neat Na+–Mt showed no inhibitory zone for S. aureus and E. coli, reflecting no antibacterial activity for neat clay mineral. However, Ca++ drug and various Ca++–Mt intercalates showed a very clear inhibition zone around the specimen. The results from the zone of inhibition tests are given in Table 4. These results indicate that Ca++–Mt has a strong activity against both gram-positive and gram-negative bacteria. Only 0.2Ca++–Mt did not exhibit a clear inhibition zone due to very low Ca++ content (only 5%) compared to other Ca++–Mt intercalates. The presence of zone of inhibition is an indication of the diffusion of the antibacterial drug CA from Ca++–Mt intercalates into the agar media. It can also be concluded that with increasing drug loading concentration (5 to 24% by mass) in the Ca++–Mt intercalates, the size of inhibition zone increases. The mechanism responsible for the observed antibacterial activity of Ca++ (Kuyyakanond and Quesnel, 1992) is electrostatic attraction between the chlorhexidine (cation) and the negatively charged bacterial cells. After the adsorption onto the microorganism's cell wall, drug molecule disrupts the integrity of the cell membrane and causes the leakage of intracellular components of the organisms. As a result of this the microorganisms gradually die.
Fig. 7. Photograph showing zone of inhibition of Na+–Mt and Ca++–Mt against S. aureus.
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Fig. 8. Photograph showing zone of inhibition of Na+–Mt and Ca++–Mt against E. coli.
3.6. Drug release study
4. Conclusions
The release profile of Ca++ drug and intercalated drug from 2.0Ca++–Mt in PBS media of pH 7.4 at 37 °C is shown in Fig. 9. From the figure it is clear that, Ca++ drug is continuously released for over 180 h whereas the initial burst release happens within the initial 18 h. Based on the release profile at pH 7.4, the maximum release of 84% of neat Ca++ drug was observed over 180 h. But 100% release of Ca++ drug was never attained due to the equilibrium characteristics of ion exchange reaction, i.e. intercalated cations cannot be exchanged completely. In the case of 2.0Ca++–Mt, initial burst release lasted for up to 12 h perhaps due to the presence of drug adsorbed on the surface of Na+–Mt which is advantageous for treating bacterial colonization and infection related diseases. However, the total amount of drug released (20%) within 12 h is much less in Ca++–Mt as compared to 60% in the case of neat Ca++ drug. After the initial burst release, the drug released slowly and in 24 h only 24% of the total drug was released. After that the drug release rate remains continuous for about 180 h in a sustained manner compared to the Ca++ drug. The total release of drug from Ca++–Mt was observed to be around 54%. This kind of sustained release occurs due to the presence of bulky and hydrophobic drug cation into the interlayer space which cannot be de-intercalated easily (Jung et al., 2008) by ion exchange reaction with the small Na+ and K+ cations. This sustained release characteristics are beneficial for application as a drug delivery carrier for topical applications where long term activity is necessary.
Ca++–Mt intercalates have been synthesized using Na+–Mt and antibacterial drug, Ca++. XRD patterns of Ca++–Mt show an increase in the d-value confirming the intercalation of Ca++ into the interlayer space of Na+–Mt. FT-IR and EDX studies further support successful intercalation of Ca++ drug into the Na+–Mt interlayer space. The antibacterial assay showed that Ca++–Mt intercalates had a broad spectrum antibacterial capability against both gram-positive and gram-negative bacteria. The drug release profile of intercalated species (Ca++–Mt) into PBS media indicates burst release followed by sustained release activity in comparison with neat drug (Ca++) which initially shows a very high amount (60%) of burst release followed by a slow release. Such intercalated species have the potential to be used in topical drug delivery in the form of fibrous implant polymeric materials such as sutures and wound dressings.
Table 4 Inhibition zone of Na+–Mt and Ca++–Mt against S. aureus and E. coli bacteria. Zone of inhibition (in mm)
Na+–Mt Ca++ 0.2Ca++–Mt 0.5Ca++–Mt 1.0Ca++–Mt 2.0Ca++–Mt Initial diameter: 15 mm; no inhibition.
S. aureus
E. coli
Absent 35 Uneven 25 28 34
Absent 36 17 25 30 32 Fig. 9. Release profile of neat drug and Ca++ from 2.0Ca++–Mt in PBS media at 37 °C.
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