International Journal of Biological Macromolecules 78 (2015) 173–179
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Optimization and evaluation of bioactive drug-loaded polymeric nanoparticles for drug delivery Ruma Rani a , Neeraj Dilbaghi a , Dinesh Dhingra b , Sandeep Kumar a,∗ a b
Department of Bio & Nanotechnology, Guru Jambheshwar University of Science & Technology, Hisar 125001, India Department of Pharmaceutical Science, Guru Jambheshwar University of Science & Technology, Hisar 125001, India
a r t i c l e
i n f o
Article history: Received 16 December 2014 Received in revised form 24 March 2015 Accepted 30 March 2015 Available online 13 April 2015 Keywords: Glycyrrhizin Antibacterial Nanoformulation Sustained release
a b s t r a c t The premise of the present study was to suitably select or modify the constitution of the polymer matrix to achieve significantly high entrapment of hydrophilic drugs within polymeric nanoparticles (NPs). Glycyrrhizin (GL), the bioactive drug was selected as a representative hydrophilic drug. Ionotropic gelation technique was used for the preparation of glycyrrhizin-loaded NPs. Concentration of polymers were optimized by 3-level factorial design which affected the particle size and encapsulation efficiency. The formulation was subjected to morphological, physiochemical and in vitro drug release studies. Mean particle size of nanoparticles was around 181 nm as estimated with particle size analyzer. TEM observations revealed spherical shape and size in the range of 140–200 nm. Fourier transform-infrared analysis did not reveal any chemical interaction among the drug and polymers used for the nano-formulation. A release study conducted in vitro over a period of 24 h indicated primarily burst release after that controlled release of glycyrrhizin from the formulation. Antibacterial activities of glycyrrhizin, blank chitosan–gum arabic NPs and glycyrrhizin-loaded chitosan–gum arabic NPs were tested against two Gram negative and two Gram positive bacteria. The study demonstrates the benefit of excipient screening techniques in improving entrapment efficiency of a hydrophilic drug. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Transport and delivery of the drug molecules specifically, gradually or securely to the site of action with the help of effective drug delivery systems (DDS) is becoming a highly important research area for the pharmaceutical researchers. Nanotechnology provides an effective DDS for delivering at the targeted sites and release of therapeutic compound in a controlled manner. Nanoencapsulation is the formation of drug-loaded NPs that increase the efficiency, efficacy, protect drug from degradation and maintains functional activity of encapsulated drugs [1]. Unique features of nanosized particles expand the scope of nanotechnology to many fields, especially in nanomedicine which namely include diagnosis, prevention and treatment of disease. Several nanoscale DDS have been used like liposomes, solid-lipid nanoparticles, micelles, hydrogels and dendrimers. Therefore NPs are fabricated from natural and synthetic macro-molecules for the fulfillments of drug delivery purpose [2,3]. Polysaccharides have gained attention due to mucoadhesive properties, biodegradability, sustainability, lack
∗ Corresponding author. Tel.: +91 1662-263378. E-mail address: [email protected] (S. Kumar). http://dx.doi.org/10.1016/j.ijbiomac.2015.03.070 0141-8130/© 2015 Elsevier B.V. All rights reserved.
of toxicity and non-antigenicity, therefore they are the most abundant industrial raw material for pharmaceutical applications [4]. For polymeric nanoparticulate system, several natural polymers like chitosan, starch, dextran, albumin, gelatin, alginate, gums and synthetic polymers like polylactic acid (PLA), poly-(lactide-coglycolide) (PLGA), polyanhydrides, poly--caprolactone (PCL) have been used [5]. The polymeric materials that are to be engulfed, injected or implanted in the body should have the properties of biodegradability and biocompatibility. Several reports exist in the literature wherein polymeric NPs have been used for delivery of formulation to the oral [6], parental [7], and ocular purpose [8]. The polymeric material can protect the labile molecule from enzymatic and hydrolytic degradation in the gastrointestinal tract and enhances the transport across the systemic circulation [5,9]. Some researchers have used polymer in conjugation with other polymers [10–12] so as to enhance the solubility, encapsulation efficiency, reducing the degradation of drug and prolonging the residence time by increasing the contact time between therapeutic molecule and biomembrane at the target or absorption site. Ionotropic-gelation is a method for the encapsulation of macromolecules such as insulin [11], using natural polysaccharides; chitosan (CS) and gum arabic (GA). Chitosan and gum arabic are naturally occurring polysaccharides, basically polycationic and
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polyanionic, respectively, and are biodegradable, biocompatible and less toxic [13–15]. Cationic nature of chitosan shows good mucoadhesive and membrane permeability properties [16]. The mechanisms of mucoadhesive and membrane permeation is based on interaction of positive charges with negatively charged cell membrane and prevent structural recognition by membrane associated gate proteins, respectively [16]. Negative charge of GA helps in the interaction with positively charged chitosan polymer leading to the formation of polymer matrix for the purpose of encapsulation. Along with these polymers, surfactant is also used for enhancing the interaction and for reducing the particle size. Glycyrrhizin is bioactive compound extracted from the roots of liquorice (Glycyrrhiza glabra). After hydrolysis glycyrrhizin changed into two molecules of d-glucuronic acid and one molecule of aglycone named 18 -glycyrrhetinic acid. Pharmacological actions, including antiviral activity [17,18], anti-hepatotoxic activity [19], anti-allergic activity [20], anti-inflammatory activity [21], protection against autoimmune disorders [22] and anti-hyperglycaemic effects [23,24] have been reported for glycyrrhizin and 18 glycyrrhetinic acid. Commercial products of glycyrrhizin are available in oral (25 mg/tablet) and intravenous (2 mg/ml glycyrrhizin solution containing glycine and l-cysteine) formulations. The intravenous formulation is administered 2 or 3 times per week over a longer time. Frequent intravenous injections increase patient non-compliances. Therefore, the formulations which are compatible for the patient will be more suitable. The present work is undertaken with an aim to form glycyrrhizin-loaded nanoformulation for drug delivery with increased efficacy and sustained release, using biocompatible polymers. Ionotropic gelation method was used for the preparation of nanoformulations and optimization of concentration of polymers (CS and GA) was done with the help of factorial design with aim of minimum particle size (PS) and maximum encapsulation efficiency (EE). Synthesized glycyrrhizin-loaded CSGA-NPs were further characterized by FTIR ((Fourier Transform Infrared Spectroscopy) for interactive studies and TEM (Transmission Electron Microscope) for microscopic evaluation. Furthermore, in vitro evaluation of antibacterial activity has been conducted against various bacterial strains and in vitro release of the nanoformulation was evaluated in phosphate buffer saline (PBS) at physiological condition (pH 7.4) using dialysis sac method. 2. Experimental 2.1. Materials Chitosan (CS), gum arabic (GA) and polysorbate-60 were obtained from Hi-Media Laboratories Pvt. Ltd. (Mumbia, India). Bioactive compound, glycyrrhizin was procured from SigmaAldrich. The other materials used in experiment were of pharmaceutical and analytical grade. Bacterial cultures i.e. Bacillus ceresus NCDC no. 240, Bacillus polymyxa NCDC no. 068, Pseudomonas aeruginosa NCDC no. 105, Enterobacter aerogenes NCDC no. 106 were procured from National Collection of Dairy Cultures (NCDC), NDRI, Karnal.
Table 1 Actual values of parameters for glycyrrhizin loaded CSGANPs. Coded values
−1 0 1
Actual values Concentration of CS (%)
Concentration of GA (%)
1 1.3 1.5
0.1 0.15 0.2
(1% of the total solution) was added to the mixture and stirring was continued to achieve reduced size of nanoparticles. Glycyrrhizinloaded CSGA-NPs were prepared by gradual addition of aqueous solution of glycyrrhizin in CS solution initially and then same procedure was followed. Polymer–drug ratio was taken 7:1. The obtained nanosuspension was analyzed for PS and % EE. PS was determined by particle size analyzer and % EE was determined by estimating amount of free drug by UV–vis spectrophotometer. The sediment having NPs freeze dried at −80 ◦ C for 4 h followed by lyophilization using a lyopholizer (Alpha 2–4 LD plus, Martin Christ, Germany) for 24 h at −90 ◦ C and 0.0010 mbar, using mannitol (1% w/v) as cryoprotectant. 2.3. Optimization by factorial statistical design The factorial design was applied for the optimization of formulation ingredient to develop a nanoformulation. Based on the preliminary experimental observations, concentrations of polymers were found to be more affective on PS and EE. A 3-factorial design was chosen for optimization of nanoformulation to achieve minimum PS and maximum EE. The concentration parameters for factorial design of glycyrrhizin-loaded CSGA-NPs are depicted in Table 1. Design Expert Software Version 8.0.7.1. was used for the data analysis and for plotting of contour plots and 3-D response surface plots. 3. Characterization 3.1. Particle size (PS) and encapsulation efficiency (EE) The particle size of glycyrrhizin-loaded CSGA-NPs samples were determined by the Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). The % EE was determined by UV–vis spectrophotometer (Shimardzu) by estimating the free amount of drug in the clear supernatant by using supernatant of blank-CSGA-NPs as basic correction. The absorbance was recorded at 258 nm for GL in supernatant. % EE =
(Total glycyrrhizin − Unbound glycyrrhizin) × 100 Total glycyrrhizin
3.2. Zeta potential Zeta potential of the optimized final batch was measured by Zetasizer Nano ZS (Malvern Instruments, Malvern, UK).
2.2. Protocol for nanoparticles synthesis 3.3. Transmission electron microscopy of nanoformulation Glycyrrhizin-loaded chitosan–gum arabic nanoparticles (GLloaded CSGA-NPs) were formulated by ionotropic gelation method using chitosan as primary polymer, gum arabic as secondary polymer i.e. co-polymer and polysorbate-60 as surfactant. CS solution (1–1.5%) was prepared in 2% (v/v) acetic acid solution and GA solution was prepared in distilled water by stirring. Thereafter, GA solution was added to CS solution during stirring. Finally surfactant
TEM (TEM Morgagni 268D, Fei Electron Optics) analysis was used to study the morphology of prepared nanoparticles. The lypholized sample was diluted with distilled water and homogenized using an ultrasonicator. A drop was casted on a copper grid, air dried for 5 min and loaded in the goniometer. The processed sample was observed under microscope and TEM images were recorded.
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Table 2 Factorial design layout for formulation with experimental observations. Exp. no.
Coded values of CS
Coded values of GA
PS (nm) (R1 )
EE (%) (R2 )
1. 2. 3. 4. 5. 6. 7. 8. 9.
0 0 −1 −1 −1 1 0 1 1
−1 0 1 0 −1 0 1 1 −1
205.5 184.3 256.3 117.1 230.5 250.5 239.3 389.2 270.7
83.37 98.53 81.79 88.77 77.97 98.98 94.32 95.21 83.46
3.4. Structural analysis by FTIR
4.2. Optimization by factorial statistical design
FTIR spectra were recorded on Fourier-transform infrared spectrophotometer (IR Affinity-1, Shimandzu, Japan) using KBr pellet method. Pellets were formed by pressing the mixture of test sample and KBr in a ratio (1:100) and scanned from 4500 to 500 cm−1 .
Nine batches of glycyrrhizin-loaded CSGA-NPs were obtained by using 3-Level factorial design by varying the concentration of CS (A) and GA (B) and the experimental observations of PS (R1 ) and EE (R2 ) are tabulated in Table 2. The preliminary trial revealed that the concentrations of polymers and surfactant influenced the PS and EE of glycyrrhizin-loaded CSGA-NPs. From the observations, it was found that near 0-level, minimum PS and maximum EE were achieved. The regression equation for the model relating R1 and R2 as response are shown in Eqs. (1) and (2), respectively. The positive and negative sign in Eqs. (1) and (2) indicates synergistic and antagonistic effects, respectively. The results of ANOVA analysis of the polynomial equations are portrayed in Table 3 which indicates that quadratic model was best fitted and significant for the development of two responses (PS and EE). The significance of the model was also depicted by the values of R2 which is >0.98, for both the responses. However, other factors are also in reasonable range which depicted the significance of the model.
3.5. In vitro drug release studies Glycyrrhizin-loaded CSGA-NPs (50 mg) were placed in a dialysis sac (cut off between 12 kDA and 14 kDA.) and tied with dialysis clip. The dialysis membrane was then immersed in 150 ml of release medium—PBS (pH 7.4), in a conical flask, kept on a shaking incubator maintained at 100 rpm and 37 ◦ C. Sample aliquots (4 ml) of release medium were withdrawn at regular intervals and replaced by fresh PBS. Drug content was estimated using UV spectrophotometer at 258 nm, taking blank CSGA-NPs in releasing media as blank/reference. This was compared with release of glycyrrhizin from pure glycyrrhizin placed in dialysis sac, taking PBS as blank/reference, under identical conditions. All experiments were performed in triplicate.
PS = +188.86 + 17.73 × A + 13.03 × B + 42.67 × A2 + 31.27 × B2
(1)
3.6. In vitro antibacterial activity The antibacterial activities of glycyrrhizin, blank CSGA-NPs and glycyrrhizin-loaded CSGA-NPs were tested by agar well-diffusion method [25] against B. ceresus, B. polymyxa, P. aeruginosa, E. aerogenes in vitro. Nutrient agar media plates were prepared in laminar air flow, and after their solidification, bacterial cultures were swabbed uniformly by l-shaped spreader. The 4 wells were cut in the agar plate and the solutions of glycyrrhizin-loaded CSGA-NPs (5 mg/ml; CSGA-NPs contains 1.0 mg of the drug), blank CSGA-NPs (5 mg/ml), glycyrrhizin (1 mg/ml) and distilled water were filled in the wells. After 24 h of incubation at 37 ◦ C, zone of inhibition (in mm) obtained around the well was measured. The experiments were performed in triplicate.
EE = +98.34 + 4.85 × A + 4.42 × B + 1.99 × A × B − 4.37 × A2 − 9.40 × B2
(2)
The contour plots, Figs. 1 and 2 render the interaction behavior of concentration of independent factors, CS (A) and GA (B) on the response of dependent factor PS (R1 ) and EE (R2 ), respectively. Significance of the mutual interaction has been predicted form the shape of the contour plots. Figs. 1 and 2 are elliptical contour plots, means the independent factors have significant interaction for PS and EE, respectively. Table 3 ANOVA analysis summary for the PS and EE for glycyrrhizin loaded CSGANPs.
4. Results and discussion 4.1. Protocol for nanoparticles synthesis Polymeric nanoparticles offer valuable delivery system of active ingredient because of high encapsulation efficiency, biodegradability, biocompatibility, less toxicity and high stability [26–28]. The glycyrrhizin-loaded CSGA-NPs were synthesized by ionotropic gelation method. The particle formation attained by successive addition of positively charged chitosan and the negatively charged gum arabic. Positively charged amine groups ( NH2 ) of chitosan interact electrostatically with negatively charged carboxylic groups ( COO ) of gum arabic and form mesh-like polymer structure for drug loading [11].
Parameters
Response R1 (PS)
Response R2 (EE)
SS MS DF F-Value P-Value SD CV R2 Adj.-R2 Pred.-R2 Adeq precision
8502.13 2125.53 4 78.93 0.0005 5.19 2.18 0.9875 0.9750 0.9367 27.070
489.07 97.81 5 80.74 0.0021 1.10 1.23 0.9926 0.9803 0.9108 23.959
SS indicates sum of square; MS is mean square; DF is degree of freedom; F is Fischer’s ratio; P is probability; SD is standard deviation; CV is coefficient of variance; R is regression coefficient.
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Fig. 1. Contour plot for PS.
Fig. 2. Contour plot for EE.
Fig. 3. Response surface plot for PS.
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Fig. 4. Response surface plot for EE.
Table 4 Records for the final optimized concentration with minimum % error. Conc. of CS
Conc. of GA
Observations for PS
Observations for EE
Predicted
Experimental
% Error
Predicted
Experimental
% Error
0.05
0.06
185.235
181.4
2.072
98.81
99.84
−0.0104
The response surface plots, Figs. 3 and 4 reveal the concentration effect of CS (A) and GA (B) on the PS (R1 ) and %EE (R2 ), respectively, in the glycyrrhizin-loaded CSGA-NPs. Figs. 3 and 4 have upward and downward curve-like relationship with PS and EE, respectively. It means an increase in concentration of polymers first leads to a decrease in PS and increase in EE upto a certain level while further increase in concentrations resulted in increase in PS and decrease in EE. After ANOVA analysis, numerical optimization was employed for obtaining the final optimized concentration of the formulation with desired responses like minimum PS (R1 ) and maximum EE (R2 ). The results for final optimized concentration are tabulated in Table 4. 4.3. Zeta potential Zeta potential is a good tool that represents the interaction behavior between the polymers to make the formulation more stable. Chitosan was found to show high positive zeta potential, whereas gum arabic reflected negative zeta potential [29]. Zeta potential confirms the stability of the nanoformulation and the
potential depends upon the concentration of CS and GA. In final optimized batch, GA concentration is about 10 times lesser than the CS. Therefore CS charge dominate over the GA charge and attributes positive value for the zeta potential. Fig. 5 reflects zeta potential curve with +31.2 mV value of the final optimized batch, which shows stability. 4.4. Transmission electron microscopy of nanoformulation Fig. 6 shows TEM images of glycyrrhizin-loaded CSGA NPs with a spherical shape having diameters ranging from 140 to 200 nm. The results of DLS based particle size analyzer (Fig. 7) and TEM are consistent and little variation is due to the fact that particle size analyzer measures average size of the particles taking into account possible agglomeration of different size classes. 4.5. Structural analysis by FTIR The FTIR spectroscopy is not only used for the interaction studies [30], but also used to analyze the encapsulation of bioactive molecules on the NPs [31]. Fig. 8 shows the overlay spectra of
Fig. 5. Zeta potential curve of final optimized glycyrrhizin loaded CSGANPs.
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Fig. 6. TEM micrograph of glycyrrhizin loaded CSGANPs.
glycyrrhizin and glycyrrhizin-loaded CSGA-NPs. Spectra of glycyrrhizin gives broad band at 3454.50 cm−1 due to OH stretching in the COOH group, peak at 2929.87 cm−1 and 2875.87 cm−1 which can be attributed to C H stretching of alkane, peak at 1726.29 cm−1 due to C O stretching of ketone, peak at 1651.06 cm−1 due to C C stretching vibration, peak at 1452.39 cm−1 due to C H deformation, 1080.14 cm−1 due to C OH stretching and peak at 1041.56 cm−1 can be attributed to C O stretching of primary alcohol [32,33]. The IR spectrum documented the loading of glycyrrhizin in glycyrrhizin-loaded CSGA-NPs without any chemical interaction. Peaks similarity between glycyrrhizin and glycyrrhizin-loaded CSGA-NPs like appearance of strong band at 3421.72 cm−1 ( OH stretching), strong intensity bands at 2924.09 cm−1 , 2856.58 cm−1 (C H stretching) and peak at 1737.86 cm−1 ( C O stretching), 1641.42 cm−1 (C C stretching vibration), 1462.04 cm−1 (C H deformation) and 1107.14 cm−1 (C OH stretching) revealed the presence of glycyrrhizin. Therefore this could depict the presence of drug molecules glycyrrhizin in the glycyrrhizin-loaded CSGA-NPs without any chemical interaction.
Fig. 8. FTIR overlay spectra for glycyrrhizin and glycyrrhizin loaded CSGANPs.
4.6. In vitro drug release studies Fig. 9 compares the release of encapsulated glycyrrhizin from the glycyrrhizin-loaded CSGA-NPs. The optimized nanoformulation showed a sustained release of glycyrrhizin. A slow but incomplete glycyrrhizin release into the media from the glycyrrhizin-loaded CSGA NPs was observed throughout the assay. During the first 4 h, 25.06% of the drug was released followed by a sustained release of glycyrrhizin. The initial release may be due to presence of glycyrrhizin at or near the surface of nanoparticles [34].
Fig. 9. In vitro release profile for glycyrrhizin and glycyrrhizin loaded CSGANPs.
Glycyrrhizin-loaded nanoparticles resulted in nearly 52.12% release of glycyrrhizin in 24 h as compared to 86.30% release of pure glycyrrhizin. This in vitro release behavior indicates that glycyrrhizin availability is prolonged.
Fig. 7. PSA image of final optimized concentration.
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Acknowledgements The authors are thankful to Nano Mission, Department of Science & Technology, Govt. of India for providing financial assistance to establish research facilities. Ruma Rani thanks DST for providing INSPIRE fellowship. References
Fig. 10. Antibacterial activity of glycyrrhizin loaded CSGANPs, glycyrrhizin and blank CSGANPs against different bacterial strains.
4.7. In vitro antibacterial activity Well puncture method was used to study the antibacterial effect of nanoparticles in comparison to polymer and glycyrrhizin on two species of Gram-negative bacteria (P. aerugenosa and E. aerogenes) and two Gram-positive bacteria (B. ceresus and K. pnemonae). The experiments were done in triplicate and zone of inhibition were measured which are illustrated in Fig. 10. Results revealed prolonged antibacterial activity in the case of glycyrrhizin-loaded CSGA-NPs due to encapsulation of glycyrrhizin in comparison to aqueous solution of pure glycyrrhizin and blank CSGA-NPs [35]. The largest zone of inhibition of glycyrrhizin-loaded CSGA-NPs was observed in B. ceresus. Some antibacterial activity was observed in case of blank CSGA-NPs with smaller zone of inhibition. 5. Conclusion Ionotropic gelation method was used for preparation of glycyrrhizin-loaded CSGA-NPs and further optimized using 3level factorial design. From the observations, it is concluded that minimum PS and maximum EE has been achieved near the 0-level of chitosan and gum arabic. Further the optimized glycyrrhizin-loaded CSGA-NPs were found to be in the size range of 140–200 nm as confirmed by TEM. In addition, the nanoparticulate formulation has high encapsulation efficiency; showing sustained release of glycyrrhizin and also possessing antibacterial activity, with maximum zone of inhibition against B. ceresus which demonstrated that nano-formulation provides higher efficacy for a long time. However, further suitable animal model (in vivo) studies are still required to validate the results of in vitro evaluation and use of glycyrrhizin-loaded CSGA-NPs various applications.
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Optimization and evaluation of bioactive drug-loaded polymeric nanoparticles for drug delivery Ruma Rani a , Neeraj Dilbaghi a , Dinesh Dhingra b , Sandeep Kumar a,∗ a b
Department of Bio & Nanotechnology, Guru Jambheshwar University of Science & Technology, Hisar 125001, India Department of Pharmaceutical Science, Guru Jambheshwar University of Science & Technology, Hisar 125001, India
a r t i c l e
i n f o
Article history: Received 16 December 2014 Received in revised form 24 March 2015 Accepted 30 March 2015 Available online 13 April 2015 Keywords: Glycyrrhizin Antibacterial Nanoformulation Sustained release
a b s t r a c t The premise of the present study was to suitably select or modify the constitution of the polymer matrix to achieve significantly high entrapment of hydrophilic drugs within polymeric nanoparticles (NPs). Glycyrrhizin (GL), the bioactive drug was selected as a representative hydrophilic drug. Ionotropic gelation technique was used for the preparation of glycyrrhizin-loaded NPs. Concentration of polymers were optimized by 3-level factorial design which affected the particle size and encapsulation efficiency. The formulation was subjected to morphological, physiochemical and in vitro drug release studies. Mean particle size of nanoparticles was around 181 nm as estimated with particle size analyzer. TEM observations revealed spherical shape and size in the range of 140–200 nm. Fourier transform-infrared analysis did not reveal any chemical interaction among the drug and polymers used for the nano-formulation. A release study conducted in vitro over a period of 24 h indicated primarily burst release after that controlled release of glycyrrhizin from the formulation. Antibacterial activities of glycyrrhizin, blank chitosan–gum arabic NPs and glycyrrhizin-loaded chitosan–gum arabic NPs were tested against two Gram negative and two Gram positive bacteria. The study demonstrates the benefit of excipient screening techniques in improving entrapment efficiency of a hydrophilic drug. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Transport and delivery of the drug molecules specifically, gradually or securely to the site of action with the help of effective drug delivery systems (DDS) is becoming a highly important research area for the pharmaceutical researchers. Nanotechnology provides an effective DDS for delivering at the targeted sites and release of therapeutic compound in a controlled manner. Nanoencapsulation is the formation of drug-loaded NPs that increase the efficiency, efficacy, protect drug from degradation and maintains functional activity of encapsulated drugs [1]. Unique features of nanosized particles expand the scope of nanotechnology to many fields, especially in nanomedicine which namely include diagnosis, prevention and treatment of disease. Several nanoscale DDS have been used like liposomes, solid-lipid nanoparticles, micelles, hydrogels and dendrimers. Therefore NPs are fabricated from natural and synthetic macro-molecules for the fulfillments of drug delivery purpose [2,3]. Polysaccharides have gained attention due to mucoadhesive properties, biodegradability, sustainability, lack
∗ Corresponding author. Tel.: +91 1662-263378. E-mail address: [email protected] (S. Kumar). http://dx.doi.org/10.1016/j.ijbiomac.2015.03.070 0141-8130/© 2015 Elsevier B.V. All rights reserved.
of toxicity and non-antigenicity, therefore they are the most abundant industrial raw material for pharmaceutical applications [4]. For polymeric nanoparticulate system, several natural polymers like chitosan, starch, dextran, albumin, gelatin, alginate, gums and synthetic polymers like polylactic acid (PLA), poly-(lactide-coglycolide) (PLGA), polyanhydrides, poly--caprolactone (PCL) have been used [5]. The polymeric materials that are to be engulfed, injected or implanted in the body should have the properties of biodegradability and biocompatibility. Several reports exist in the literature wherein polymeric NPs have been used for delivery of formulation to the oral [6], parental [7], and ocular purpose [8]. The polymeric material can protect the labile molecule from enzymatic and hydrolytic degradation in the gastrointestinal tract and enhances the transport across the systemic circulation [5,9]. Some researchers have used polymer in conjugation with other polymers [10–12] so as to enhance the solubility, encapsulation efficiency, reducing the degradation of drug and prolonging the residence time by increasing the contact time between therapeutic molecule and biomembrane at the target or absorption site. Ionotropic-gelation is a method for the encapsulation of macromolecules such as insulin [11], using natural polysaccharides; chitosan (CS) and gum arabic (GA). Chitosan and gum arabic are naturally occurring polysaccharides, basically polycationic and
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polyanionic, respectively, and are biodegradable, biocompatible and less toxic [13–15]. Cationic nature of chitosan shows good mucoadhesive and membrane permeability properties [16]. The mechanisms of mucoadhesive and membrane permeation is based on interaction of positive charges with negatively charged cell membrane and prevent structural recognition by membrane associated gate proteins, respectively [16]. Negative charge of GA helps in the interaction with positively charged chitosan polymer leading to the formation of polymer matrix for the purpose of encapsulation. Along with these polymers, surfactant is also used for enhancing the interaction and for reducing the particle size. Glycyrrhizin is bioactive compound extracted from the roots of liquorice (Glycyrrhiza glabra). After hydrolysis glycyrrhizin changed into two molecules of d-glucuronic acid and one molecule of aglycone named 18 -glycyrrhetinic acid. Pharmacological actions, including antiviral activity [17,18], anti-hepatotoxic activity [19], anti-allergic activity [20], anti-inflammatory activity [21], protection against autoimmune disorders [22] and anti-hyperglycaemic effects [23,24] have been reported for glycyrrhizin and 18 glycyrrhetinic acid. Commercial products of glycyrrhizin are available in oral (25 mg/tablet) and intravenous (2 mg/ml glycyrrhizin solution containing glycine and l-cysteine) formulations. The intravenous formulation is administered 2 or 3 times per week over a longer time. Frequent intravenous injections increase patient non-compliances. Therefore, the formulations which are compatible for the patient will be more suitable. The present work is undertaken with an aim to form glycyrrhizin-loaded nanoformulation for drug delivery with increased efficacy and sustained release, using biocompatible polymers. Ionotropic gelation method was used for the preparation of nanoformulations and optimization of concentration of polymers (CS and GA) was done with the help of factorial design with aim of minimum particle size (PS) and maximum encapsulation efficiency (EE). Synthesized glycyrrhizin-loaded CSGA-NPs were further characterized by FTIR ((Fourier Transform Infrared Spectroscopy) for interactive studies and TEM (Transmission Electron Microscope) for microscopic evaluation. Furthermore, in vitro evaluation of antibacterial activity has been conducted against various bacterial strains and in vitro release of the nanoformulation was evaluated in phosphate buffer saline (PBS) at physiological condition (pH 7.4) using dialysis sac method. 2. Experimental 2.1. Materials Chitosan (CS), gum arabic (GA) and polysorbate-60 were obtained from Hi-Media Laboratories Pvt. Ltd. (Mumbia, India). Bioactive compound, glycyrrhizin was procured from SigmaAldrich. The other materials used in experiment were of pharmaceutical and analytical grade. Bacterial cultures i.e. Bacillus ceresus NCDC no. 240, Bacillus polymyxa NCDC no. 068, Pseudomonas aeruginosa NCDC no. 105, Enterobacter aerogenes NCDC no. 106 were procured from National Collection of Dairy Cultures (NCDC), NDRI, Karnal.
Table 1 Actual values of parameters for glycyrrhizin loaded CSGANPs. Coded values
−1 0 1
Actual values Concentration of CS (%)
Concentration of GA (%)
1 1.3 1.5
0.1 0.15 0.2
(1% of the total solution) was added to the mixture and stirring was continued to achieve reduced size of nanoparticles. Glycyrrhizinloaded CSGA-NPs were prepared by gradual addition of aqueous solution of glycyrrhizin in CS solution initially and then same procedure was followed. Polymer–drug ratio was taken 7:1. The obtained nanosuspension was analyzed for PS and % EE. PS was determined by particle size analyzer and % EE was determined by estimating amount of free drug by UV–vis spectrophotometer. The sediment having NPs freeze dried at −80 ◦ C for 4 h followed by lyophilization using a lyopholizer (Alpha 2–4 LD plus, Martin Christ, Germany) for 24 h at −90 ◦ C and 0.0010 mbar, using mannitol (1% w/v) as cryoprotectant. 2.3. Optimization by factorial statistical design The factorial design was applied for the optimization of formulation ingredient to develop a nanoformulation. Based on the preliminary experimental observations, concentrations of polymers were found to be more affective on PS and EE. A 3-factorial design was chosen for optimization of nanoformulation to achieve minimum PS and maximum EE. The concentration parameters for factorial design of glycyrrhizin-loaded CSGA-NPs are depicted in Table 1. Design Expert Software Version 8.0.7.1. was used for the data analysis and for plotting of contour plots and 3-D response surface plots. 3. Characterization 3.1. Particle size (PS) and encapsulation efficiency (EE) The particle size of glycyrrhizin-loaded CSGA-NPs samples were determined by the Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). The % EE was determined by UV–vis spectrophotometer (Shimardzu) by estimating the free amount of drug in the clear supernatant by using supernatant of blank-CSGA-NPs as basic correction. The absorbance was recorded at 258 nm for GL in supernatant. % EE =
(Total glycyrrhizin − Unbound glycyrrhizin) × 100 Total glycyrrhizin
3.2. Zeta potential Zeta potential of the optimized final batch was measured by Zetasizer Nano ZS (Malvern Instruments, Malvern, UK).
2.2. Protocol for nanoparticles synthesis 3.3. Transmission electron microscopy of nanoformulation Glycyrrhizin-loaded chitosan–gum arabic nanoparticles (GLloaded CSGA-NPs) were formulated by ionotropic gelation method using chitosan as primary polymer, gum arabic as secondary polymer i.e. co-polymer and polysorbate-60 as surfactant. CS solution (1–1.5%) was prepared in 2% (v/v) acetic acid solution and GA solution was prepared in distilled water by stirring. Thereafter, GA solution was added to CS solution during stirring. Finally surfactant
TEM (TEM Morgagni 268D, Fei Electron Optics) analysis was used to study the morphology of prepared nanoparticles. The lypholized sample was diluted with distilled water and homogenized using an ultrasonicator. A drop was casted on a copper grid, air dried for 5 min and loaded in the goniometer. The processed sample was observed under microscope and TEM images were recorded.
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Table 2 Factorial design layout for formulation with experimental observations. Exp. no.
Coded values of CS
Coded values of GA
PS (nm) (R1 )
EE (%) (R2 )
1. 2. 3. 4. 5. 6. 7. 8. 9.
0 0 −1 −1 −1 1 0 1 1
−1 0 1 0 −1 0 1 1 −1
205.5 184.3 256.3 117.1 230.5 250.5 239.3 389.2 270.7
83.37 98.53 81.79 88.77 77.97 98.98 94.32 95.21 83.46
3.4. Structural analysis by FTIR
4.2. Optimization by factorial statistical design
FTIR spectra were recorded on Fourier-transform infrared spectrophotometer (IR Affinity-1, Shimandzu, Japan) using KBr pellet method. Pellets were formed by pressing the mixture of test sample and KBr in a ratio (1:100) and scanned from 4500 to 500 cm−1 .
Nine batches of glycyrrhizin-loaded CSGA-NPs were obtained by using 3-Level factorial design by varying the concentration of CS (A) and GA (B) and the experimental observations of PS (R1 ) and EE (R2 ) are tabulated in Table 2. The preliminary trial revealed that the concentrations of polymers and surfactant influenced the PS and EE of glycyrrhizin-loaded CSGA-NPs. From the observations, it was found that near 0-level, minimum PS and maximum EE were achieved. The regression equation for the model relating R1 and R2 as response are shown in Eqs. (1) and (2), respectively. The positive and negative sign in Eqs. (1) and (2) indicates synergistic and antagonistic effects, respectively. The results of ANOVA analysis of the polynomial equations are portrayed in Table 3 which indicates that quadratic model was best fitted and significant for the development of two responses (PS and EE). The significance of the model was also depicted by the values of R2 which is >0.98, for both the responses. However, other factors are also in reasonable range which depicted the significance of the model.
3.5. In vitro drug release studies Glycyrrhizin-loaded CSGA-NPs (50 mg) were placed in a dialysis sac (cut off between 12 kDA and 14 kDA.) and tied with dialysis clip. The dialysis membrane was then immersed in 150 ml of release medium—PBS (pH 7.4), in a conical flask, kept on a shaking incubator maintained at 100 rpm and 37 ◦ C. Sample aliquots (4 ml) of release medium were withdrawn at regular intervals and replaced by fresh PBS. Drug content was estimated using UV spectrophotometer at 258 nm, taking blank CSGA-NPs in releasing media as blank/reference. This was compared with release of glycyrrhizin from pure glycyrrhizin placed in dialysis sac, taking PBS as blank/reference, under identical conditions. All experiments were performed in triplicate.
PS = +188.86 + 17.73 × A + 13.03 × B + 42.67 × A2 + 31.27 × B2
(1)
3.6. In vitro antibacterial activity The antibacterial activities of glycyrrhizin, blank CSGA-NPs and glycyrrhizin-loaded CSGA-NPs were tested by agar well-diffusion method [25] against B. ceresus, B. polymyxa, P. aeruginosa, E. aerogenes in vitro. Nutrient agar media plates were prepared in laminar air flow, and after their solidification, bacterial cultures were swabbed uniformly by l-shaped spreader. The 4 wells were cut in the agar plate and the solutions of glycyrrhizin-loaded CSGA-NPs (5 mg/ml; CSGA-NPs contains 1.0 mg of the drug), blank CSGA-NPs (5 mg/ml), glycyrrhizin (1 mg/ml) and distilled water were filled in the wells. After 24 h of incubation at 37 ◦ C, zone of inhibition (in mm) obtained around the well was measured. The experiments were performed in triplicate.
EE = +98.34 + 4.85 × A + 4.42 × B + 1.99 × A × B − 4.37 × A2 − 9.40 × B2
(2)
The contour plots, Figs. 1 and 2 render the interaction behavior of concentration of independent factors, CS (A) and GA (B) on the response of dependent factor PS (R1 ) and EE (R2 ), respectively. Significance of the mutual interaction has been predicted form the shape of the contour plots. Figs. 1 and 2 are elliptical contour plots, means the independent factors have significant interaction for PS and EE, respectively. Table 3 ANOVA analysis summary for the PS and EE for glycyrrhizin loaded CSGANPs.
4. Results and discussion 4.1. Protocol for nanoparticles synthesis Polymeric nanoparticles offer valuable delivery system of active ingredient because of high encapsulation efficiency, biodegradability, biocompatibility, less toxicity and high stability [26–28]. The glycyrrhizin-loaded CSGA-NPs were synthesized by ionotropic gelation method. The particle formation attained by successive addition of positively charged chitosan and the negatively charged gum arabic. Positively charged amine groups ( NH2 ) of chitosan interact electrostatically with negatively charged carboxylic groups ( COO ) of gum arabic and form mesh-like polymer structure for drug loading [11].
Parameters
Response R1 (PS)
Response R2 (EE)
SS MS DF F-Value P-Value SD CV R2 Adj.-R2 Pred.-R2 Adeq precision
8502.13 2125.53 4 78.93 0.0005 5.19 2.18 0.9875 0.9750 0.9367 27.070
489.07 97.81 5 80.74 0.0021 1.10 1.23 0.9926 0.9803 0.9108 23.959
SS indicates sum of square; MS is mean square; DF is degree of freedom; F is Fischer’s ratio; P is probability; SD is standard deviation; CV is coefficient of variance; R is regression coefficient.
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Fig. 1. Contour plot for PS.
Fig. 2. Contour plot for EE.
Fig. 3. Response surface plot for PS.
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Fig. 4. Response surface plot for EE.
Table 4 Records for the final optimized concentration with minimum % error. Conc. of CS
Conc. of GA
Observations for PS
Observations for EE
Predicted
Experimental
% Error
Predicted
Experimental
% Error
0.05
0.06
185.235
181.4
2.072
98.81
99.84
−0.0104
The response surface plots, Figs. 3 and 4 reveal the concentration effect of CS (A) and GA (B) on the PS (R1 ) and %EE (R2 ), respectively, in the glycyrrhizin-loaded CSGA-NPs. Figs. 3 and 4 have upward and downward curve-like relationship with PS and EE, respectively. It means an increase in concentration of polymers first leads to a decrease in PS and increase in EE upto a certain level while further increase in concentrations resulted in increase in PS and decrease in EE. After ANOVA analysis, numerical optimization was employed for obtaining the final optimized concentration of the formulation with desired responses like minimum PS (R1 ) and maximum EE (R2 ). The results for final optimized concentration are tabulated in Table 4. 4.3. Zeta potential Zeta potential is a good tool that represents the interaction behavior between the polymers to make the formulation more stable. Chitosan was found to show high positive zeta potential, whereas gum arabic reflected negative zeta potential [29]. Zeta potential confirms the stability of the nanoformulation and the
potential depends upon the concentration of CS and GA. In final optimized batch, GA concentration is about 10 times lesser than the CS. Therefore CS charge dominate over the GA charge and attributes positive value for the zeta potential. Fig. 5 reflects zeta potential curve with +31.2 mV value of the final optimized batch, which shows stability. 4.4. Transmission electron microscopy of nanoformulation Fig. 6 shows TEM images of glycyrrhizin-loaded CSGA NPs with a spherical shape having diameters ranging from 140 to 200 nm. The results of DLS based particle size analyzer (Fig. 7) and TEM are consistent and little variation is due to the fact that particle size analyzer measures average size of the particles taking into account possible agglomeration of different size classes. 4.5. Structural analysis by FTIR The FTIR spectroscopy is not only used for the interaction studies [30], but also used to analyze the encapsulation of bioactive molecules on the NPs [31]. Fig. 8 shows the overlay spectra of
Fig. 5. Zeta potential curve of final optimized glycyrrhizin loaded CSGANPs.
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Fig. 6. TEM micrograph of glycyrrhizin loaded CSGANPs.
glycyrrhizin and glycyrrhizin-loaded CSGA-NPs. Spectra of glycyrrhizin gives broad band at 3454.50 cm−1 due to OH stretching in the COOH group, peak at 2929.87 cm−1 and 2875.87 cm−1 which can be attributed to C H stretching of alkane, peak at 1726.29 cm−1 due to C O stretching of ketone, peak at 1651.06 cm−1 due to C C stretching vibration, peak at 1452.39 cm−1 due to C H deformation, 1080.14 cm−1 due to C OH stretching and peak at 1041.56 cm−1 can be attributed to C O stretching of primary alcohol [32,33]. The IR spectrum documented the loading of glycyrrhizin in glycyrrhizin-loaded CSGA-NPs without any chemical interaction. Peaks similarity between glycyrrhizin and glycyrrhizin-loaded CSGA-NPs like appearance of strong band at 3421.72 cm−1 ( OH stretching), strong intensity bands at 2924.09 cm−1 , 2856.58 cm−1 (C H stretching) and peak at 1737.86 cm−1 ( C O stretching), 1641.42 cm−1 (C C stretching vibration), 1462.04 cm−1 (C H deformation) and 1107.14 cm−1 (C OH stretching) revealed the presence of glycyrrhizin. Therefore this could depict the presence of drug molecules glycyrrhizin in the glycyrrhizin-loaded CSGA-NPs without any chemical interaction.
Fig. 8. FTIR overlay spectra for glycyrrhizin and glycyrrhizin loaded CSGANPs.
4.6. In vitro drug release studies Fig. 9 compares the release of encapsulated glycyrrhizin from the glycyrrhizin-loaded CSGA-NPs. The optimized nanoformulation showed a sustained release of glycyrrhizin. A slow but incomplete glycyrrhizin release into the media from the glycyrrhizin-loaded CSGA NPs was observed throughout the assay. During the first 4 h, 25.06% of the drug was released followed by a sustained release of glycyrrhizin. The initial release may be due to presence of glycyrrhizin at or near the surface of nanoparticles [34].
Fig. 9. In vitro release profile for glycyrrhizin and glycyrrhizin loaded CSGANPs.
Glycyrrhizin-loaded nanoparticles resulted in nearly 52.12% release of glycyrrhizin in 24 h as compared to 86.30% release of pure glycyrrhizin. This in vitro release behavior indicates that glycyrrhizin availability is prolonged.
Fig. 7. PSA image of final optimized concentration.
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Acknowledgements The authors are thankful to Nano Mission, Department of Science & Technology, Govt. of India for providing financial assistance to establish research facilities. Ruma Rani thanks DST for providing INSPIRE fellowship. References
Fig. 10. Antibacterial activity of glycyrrhizin loaded CSGANPs, glycyrrhizin and blank CSGANPs against different bacterial strains.
4.7. In vitro antibacterial activity Well puncture method was used to study the antibacterial effect of nanoparticles in comparison to polymer and glycyrrhizin on two species of Gram-negative bacteria (P. aerugenosa and E. aerogenes) and two Gram-positive bacteria (B. ceresus and K. pnemonae). The experiments were done in triplicate and zone of inhibition were measured which are illustrated in Fig. 10. Results revealed prolonged antibacterial activity in the case of glycyrrhizin-loaded CSGA-NPs due to encapsulation of glycyrrhizin in comparison to aqueous solution of pure glycyrrhizin and blank CSGA-NPs [35]. The largest zone of inhibition of glycyrrhizin-loaded CSGA-NPs was observed in B. ceresus. Some antibacterial activity was observed in case of blank CSGA-NPs with smaller zone of inhibition. 5. Conclusion Ionotropic gelation method was used for preparation of glycyrrhizin-loaded CSGA-NPs and further optimized using 3level factorial design. From the observations, it is concluded that minimum PS and maximum EE has been achieved near the 0-level of chitosan and gum arabic. Further the optimized glycyrrhizin-loaded CSGA-NPs were found to be in the size range of 140–200 nm as confirmed by TEM. In addition, the nanoparticulate formulation has high encapsulation efficiency; showing sustained release of glycyrrhizin and also possessing antibacterial activity, with maximum zone of inhibition against B. ceresus which demonstrated that nano-formulation provides higher efficacy for a long time. However, further suitable animal model (in vivo) studies are still required to validate the results of in vitro evaluation and use of glycyrrhizin-loaded CSGA-NPs various applications.
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