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Polyelectrolyte microcapsules for sustained delivery of water-soluble drugs
Materials Science and Engineering C 31 (2011) 342–349
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Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m s e c
Polyelectrolyte microcapsules for sustained delivery of water-soluble drugs S. Anandhakumar a, M. Debapriya a, V. Nagaraja b, Ashok M. Raichur a,⁎ a b
Department of Materials Engineering, Indian Institute of Science, Bangalore, 560012, India Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, India
a r t i c l e
i n f o
Article history: Received 8 April 2010 Received in revised form 1 October 2010 Accepted 11 October 2010 Available online 15 October 2010 Keywords: Weak polyelectrolyte capsules Ciprofloxacin Encapsulation Antibacterial studies
a b s t r a c t Polyelectrolyte capsules composed of weak polyelectrolytes are introduced as a simple and efficient system for spontaneous encapsulation of low molecular weight water-soluble drugs. Polyelectrolyte capsules were prepared by layer-by-layer (LbL) assembling of weak polyelectrolytes, poly(allylamine hydrochloride) (PAH) and poly (methacrylic acid) (PMA) on polystyrene sulfonate (PSS) doped CaCO3 particles followed by core removal with ethylene-diaminetetraacetic acid (EDTA). The loading process was observed by confocal laser scanning microscopy (CLSM) using tetramethylrhodamineisothiocyanate labeled dextran (TRITC-dextran) as a fluorescent probe. The intensity of fluorescent probe inside the capsule decreased with increase in cross-linking time. Ciprofloxacin hydrochloride (a model water-soluble drug) was spontaneously deposited into PAH/PMA capsules and their morphological changes were investigated by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The quantitative study of drug loading was also elucidated which showed that drug loading increased with initial drug concentration, but decreased with increase in pH. The loaded drug was released in a sustained manner for 6 h, which could be further extended by cross-linking the capsule wall. The released drug showed significant antibacterial activity against E. coli. These findings indicate that such capsules can be potential carriers for water-soluble drugs in sustained/controlled drug delivery applications. © 2010 Elsevier B.V. All rights reserved.
1. Introduction In pharmaceutical field, drug delivery systems (DDS) are being developed with the aim of achieving high drug content, controlled release, prolonged circulation in blood and ability to target a specific area, so that they greatly increase the drug efficacy and reduce the drug toxicity and side effects during application. Because of these requirements, variety of DDS (liposomes, microparticles and emulsions) have been developed [1–4]. In spite of some advantages over conventional dosage forms, they have found limited use due to low encapsulation efficiency, burst release and poor storage stability. More efforts are being made to improve the encapsulation efficiency, but only few studies have been reported that focus on both high encapsulation efficiency and sustained/controlled release of encapsulated drug [5,6]. An attractive and flexible DDS that meets all these requirements for drug delivery is the polyelectrolyte microcapsules, which are prepared by alternate deposition of oppositely charged polyelectrolytes onto dissolvable colloidal particles, followed by core removal [7]. These capsules provide numerous ways for drug loading and release. For example, the incorporation of polyanions into microcapsules during fabrication improves the loading of positively charged substances by spontaneous deposition mechanism [8]. The permeability of the capsule wall is the critical parameter for drug delivery, so that loading and subsequent
⁎ Corresponding author. Tel.: + 91 80 22933238; fax: + 91 80 23600472. E-mail address: [email protected] (A.M. Raichur). 0928-4931/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2010.10.005
release can be modified in a desired manner. Many attempts have been made to control the permeability of the capsules by changing pH, ionic strength, temperature, number of layers and polarity [9–14]. The other methods of modulating the permeability are cross-linking the wall components by various mechanisms such as UV irradiation, thermo- or photo-induced cross-linking, water-soluble carbodiimide chemistry and glutaraldehyde cross-linking [15–19]. The capsule surface can also be modified to meet the biological challenges such as shielding drug from human immune system and delivering the drugs at a specific site. Many efforts have been devoted for loading and subsequent release of model macromolecules from the microcapsule interior, but only few studies have used actual drug molecules. The simplest method of encapsulation is to use the drug crystal itself as a template for multilayer assembly [20], but the application is limited to substances having a low solubility under coating conditions. The drug can also be pre-loaded into the template before multilayer assembly [21]; however the template dissolution may affect the properties of the drug. There is another method of “spontaneous deposition” of drugs into microcapsules templated on melamine formaldehyde (MF) particles [22,23]. The negatively charged MF/PSS complex formed after core removal attracts drug molecules into the capsules, so that the deposited drug is in aggregate or complex form rather than in its free state. It should be noted that the concentration of free drugs in the capsule interior is always lower than the bulk [23]. It is also important to consider the biocompatibility of template material when used for drug delivery applications. The recent novel finding of “charge controlled attraction and repulsion” method is practically more attractive because the polyanions are pre-loaded into hollow capsules,
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which increase the interior drug concentration from ten to hundreds of times of the bulk concentration [8]. All the aforementioned encapsulation techniques have been reported on polyelectrolyte capsules prepared from strong polyelectrolytes, which do not respond to changes in pH. Although encapsulation by varying ionic strength seems to be applicable for strong polyelectrolyte capsules, it enables only the entrapment of relatively small amounts [24]. The polyelectrolyte capsules composed of weak polyelectrolytes have attracted considerable interest, because of their distinct and reversible change of properties in response to external stimulus. The pH dependent permeability of these capsules can be used for controlled encapsulation and release of drugs. Further, pH responsive microcapsules can be easily destroyed at extreme pH conditions, when the pH-induced imbalance of charges overcompensates the attractive polymer–polymer interactions [25,26]. Thus it is interesting to study the potential of these capsules for encapsulation and release of water-soluble drugs which have hardly been reported so far. In this publication, we continued our research on polyelectrolyte microcapsules composed of the weak polyelectrolytes PMA and PAH for controlled release. The ability of these capsules to achieve high efficiency encapsulation from low concentration loading solution by charge controlled mechanism was investigated using TRITC-dextran as a fluorescent probe. A water-soluble drug, ciprofloxacin hydrochloride (Fig. 1a), was used as a model drug. Ciprofloxacin hydrochloride is the most active fluoroquinolone and can inhibit 90% of Enterobacteriaceae at concentration below 0.25 μg/ml [27]. Moreover, it can be used in vitro and in vivo especially against gram-negative bacteria irrespective of their growth rate [28]. The possibility of controlling the release rate by cross-linking the wall components with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) has also been investigated. In vitro study was also performed to evaluate the activity of ciprofloxacin after release. Thus, we can combine the features of the pH responsive encapsulation with spontaneous deposition mechanism to obtain high drug encapsulation efficiency, which reduces the need for frequent administration leading to improved patient compliance. 2. Materials and methods 2.1. Materials PAH (Mw = 70 kDa), PMA (Mw = 483 kDa), PSS (Mw = 70 kDa), sodium carbonate, calcium chloride, EDTA, TRITC-dextran (Mw = 65– 76 kDa) and phosphate buffered saline (PBS) were all purchased from
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Sigma-Aldrich and EDC (Spectrochem, India). Ciprofloxacin hydrochloride (CH) was a gift by Dr. Reddy's laboratories Ltd., India. All chemicals were used without any further purification. The water used in all experiment was obtained from Milli-Q system with resistivity greater than 18 MΩ cm. All pH adjustments were done with 0.1 M HCl or 0.1 M NaOH. 2.2. Microcapsule preparation and cross-linking PSS doped CaCO3 particles with diameter of 6 ± 0.5 μm, prepared by colloidal aggregation of sodium carbonate and calcium chloride were used as sacrificial template [8]. Multilayer buildup was performed by LbL technique from 1 mg/ml polyelectrolyte solution of PAH and PMA in 0.5 M NaCl at pH = 6. After each adsorption step, three washing steps were performed. When four layers were deposited, the core was dissolved by 0.2 M EDTA solution, followed by three washing steps with water. As the PSS doped CaCO3 particles possess a negatively charged surface, the multilayer buildup was started with PAH. Cross-linking was performed by incubating the capsule suspension with 0.2 mM EDC at pH = 6 for a desirable time at room temperature, followed by three washings with water. 2.3. Ciprofloxacin loading and release experiments 0.3 ml of microcapsule suspension ((3 ± 0.2) × 108 capsules/ml) in water was centrifuged at 6000 rpm for 10 min and the supernatant was removed. Then the microcapsules were re-dispersed in 1 ml of CH with different loading conditions such as pH and feeding concentration and kept in a shaker at 25 °C. After incubation for 1 h, the mixture was centrifuged and the drug loaded capsules were washed once with water and used in release experiments. The scheme illustrating the loading and release process is depicted in Fig. 1b. The amount of drug loaded inside the microcapsules was estimated from the difference between input amount and amount in the supernatant after loading process. The drug amount in the supernatant was determined directly at 276 nm using a ND-1000 UV–Vis spectrophotometer (Nanodrop Technologies, USA). The number of microcapsules was determined using a hemocytometer with a 40× objective lens. In order to calculate drug concentration in the capsules, hundred microcapsules were selected stochastically and their diameters were measured under CLSM, by which their volumes were calculated respectively. The volume of one capsule (1.13 × 10− 10 ml) was averaged from the total
Fig. 1. (a) Chemical structure of ciprofloxacin hydrochloride. (b) Scheme demonstrating the deposition of ciprofloxacin into the preformed hollow capsules. (A— Prepared hollow capsules pre-loaded with PSS; A → B, spontaneous deposition of ciprofloxacin into preformed hollow capsules; B → C, centrifugation and removal of supernatant drug; C → D, release of the loaded drug in PBS buffer by concentration dependent diffusion mechanism).
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volume. Further details concerning the quantification of drug loaded can be found in our previous paper [29]. The loaded capsules ((9 ± 0.1) × 107 capsules) were incubated in 1 ml of PBS at pH = 7.4 in an Eppendorf tube. Then the tube was placed in a shaker at 200 rpm and 37 °C. After 30 min, the capsule suspension was centrifuged and the supernatant was replaced with fresh PBS (pre-warmed to 37 °C). The amount of CH in supernatant solution was determined spectroscopically. After cross-linking the loaded capsules, they were washed thrice with water. The drug present in the supernatant was measured and added to the total drug release in the first half an hour. 2.4. Confocal laser scanning microscopy (CLSM) The size, integrity and degree of filling of individual capsules were determined using a Zeiss LSM 510 META confocal scanning system (Zeiss, Germany) equipped with a 100× oil immersion objective with a numerical aperture of 1.4. The capsules were visualized by electrostatic adsorption of TRITC-dextran at different pH values and cross-linking densities by keeping other parameters such as aperture, gain and laser power constant. The excitation wave length was 543 nm. 2.5. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) The morphology changes of the polyelectrolyte capsules were observed by AFM and SEM. AFM measurements were performed with a Nanosurf Easy Scan2 AFM (Nanoscience Instruments, USA) in air at room temperature by contact mode. AFM samples were prepared by depositing a droplet of the capsule suspension on a silicon wafer and allowing it to dry overnight in a desiccator. SEM images of Au sputtered samples dried on silicon wafer were obtained with a field emission scanning electron microscope (FEI-SIRION, Eindhoven, Netherlands). 2.6. Fourier transform infrared spectroscopy (FTIR) Dried microcapsules were mixed with KBr powder and pelletized. Spectra were acquired in transmission mode using a Nicolet 5700 FTIR spectrometer (Thermo Electron Corporation, USA). 2.7. Differential scanning calorimetry (DSC) Thermograms of pure drug, empty and drug-loaded capsules were performed in order to characterize their physical state after encapsulation, using a computer-interfaced differential scanning calorimeter (DSC 822, Mettler-Toledo Inc., USA) under nitrogen atmosphere. About 5 mg of samples was weighed, crimped into an aluminum pan and heated from 25 °C to 350 °C at the rate of 5 °C/min. 2.8. Sustained antibacterial activity Loaded capsules were suspended in 3 ml of M9 minimal media and incubated in a shaker at 200 rpm and 37 °C. A semi-automatic protocol was used. Briefly, 2.4 ml of the sample was collected at different time intervals by centrifugation and their drug concentration was measured by spectroscopy. Fresh media (2.4 ml) was added to the incubated sample to maintain the initial volume of 3 ml. The collected samples were mixed with 40 μl of E. coli solution grown in M9 minimal media (final concentration of E. coli, 1.2 × 107 cells/ml). After 5 h of incubation period at 37 °C, the bacterial growth of the samples was determined by measuring the optical density (OD) at 600 nm (0.1 OD equals to 108 E. coli/ml). The free growing cell and free drug were maintained as positive and negative controls respectively.
3. Results and discussion Spherical calcium carbonate microparticles are a new category of template, which can be produced in the size range of 4 to 7 μm. This non-toxic template can be easily and gently removed by complexation with EDTA after self assembling of polyelectrolyte multilayer during capsule fabrication. It is known from literature that dissolution of other widely used templates such as MF or PS could either cause capsule wall defects due to osmotic stress produced by core dissolution or modify the capsule properties due to deposition of residual material within the capsule [30,31]. In this study, PSS doped CaCO3 particles were prepared and used for capsule fabrication due to their ability to form defect free capsules without any residues [6,29]. After template dissolution, hollow capsules were obtained. The semipermeability of the polyelectrolyte membrane permits small molecules (EDTA, inorganic ions and dyes) to freely enter and leave the capsule interior whereas large molecules like incorporated PSS are captured inside the capsules. The pre-loaded PSS molecules endow the hollow capsules with the capability to spontaneous load positively charged drug molecules by electrostatic interaction [8]. 3.1. Spontaneous loading of TRITC-dextran into (PAH/PMA)2 capsules The permeability and spontaneous loading of polyelectrolyte capsules composed of weak polyelectrolytes is particularly interesting due to its close relationship with drug loading and release in drug delivery. Water-soluble drugs can be readily incorporated into the capsule interior in high concentration upon simple mixing with capsule suspensions. To understand the loading process and to reveal the distribution of drug molecules inside the capsules, the encapsulation was monitored by CLSM using TRITC-dextran as a fluorescent probe. The confocal images at low and high pH's are shown in Fig. 2. The bulk solution was removed by centrifugation, so that the fluorescence from the capsule interior could be seen clearly. At alkaline conditions, e.g., pH= 8 (Fig. 2b) the interior of the capsules remained dark which implies that there was no loading of TRITC-dextran. In contrast, strong fluorescence was observed from the capsule interior at pH= 3 (Fig. 2a), demonstrating spontaneous deposition of large amount of TRITCdextran in the capsule interior. It has been reported that PSS/PAH microcapsules can be loaded with positively charged substances such as rhodamine, dextran etc., when PSS doped CaCO3 microparticles were used as sacrificial template for assembly. The driving force for spontaneous deposition was ascribed to the electrostatic interaction between the pre-incorporated PSS molecules and the positively charged water-soluble substances [8]. The fluorescence intensity profile gives information about the concentration and distribution of probe molecules in the capsule interior. Therefore, it reflects that a large amount of dextran was encapsulated and homogeneously deposited in the interior of the capsules. The charge density variation of weak polyelectrolytes at pH b4, induced pore formation by loosening the polyelectrolyte networks thus controlling the open and closed states of the capsules [9,29]. These results show the important fact that pH controlled permeability of (PAH/PMA) capsules can be used for encapsulation and subsequent release of drugs in controlled manner, which may find potential application for sustained release. The permeability and release of capsules can also be controlled by cross-linking the layer components, which helps to tune the loading and release at a well-defined rate. Cross-linking density is the key parameter used to tune pH dependent permeability, because higher degree of cross-linking may lead to complete loss of pH responsive properties due to absence of available charged site for electrostatic interaction. It is possible to cross-link the (PAH/PMA) capsules easily by cross-linking the functional groups of PMA and PAH with EDC [18,32]. The main advantage of EDC based zero length cross-linker is that it does not introduce any additional cross-linker molecule or particle that may be toxic during in vivo applications [18,33].
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Fig. 2. CLSM images of microcapsules incubated with TRITC-dextran at pH = 3 (a) and pH = 8 (b). Scale bar = 10 μm.
The cross-linked capsules, regardless of additional cross-linking showed very good dispersion without any shape change and aggregation. The encapsulation process of cross-linked hollow capsules was monitored by CLSM at pH =3 using TRITC-dextran. Representative CLSM images at different cross-linking times are presented in Fig. 3. All the capsules showed strong fluorescence from the capsule interior than that from the bulk demonstrating spontaneous deposition of dextran. The degree of
filling decreased when the cross-linking time was increased as shown in the fluorescence cross section profile (Fig. 3a and b) which can be attributed to decrease in wall permeability. It is important to note that the process of cross-linking did not affect the spontaneous deposition properties of the capsules. To better understand the mechanism, the cross-linked capsules were investigated in SEM to reveal their morphological changes (Fig. 4). SEM measurements were performed on both
Fig. 3. CLSM images of microcapsules to show spontaneous deposition of TRITC-dextran into cross-linked microcapsules. (a) 0.5 h and (b) 7 h. Scale bar = 10 μm.
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Fig. 5. FTIR spectra of hollow microcapsules as a function of cross-linking time.
cross-linked capsules. The spectrum of control capsules exhibits two sharp absorption bands at 1405 and 1632 cm− 1, which are attributed to the symmetric stretches of carboxylate (–COO−) group and N–H bending (scissoring) vibration of PAH [17,33]. After cross-linking, these two peaks are significantly decreased. At the same time, new broad peaks centered at 1635 cm− 1 and 1541 cm− 1 are evident in the spectrum, which are attributed to the formation of amide I and amide II bands respectively [17]. The amide I band formed as a result of cross-linking is not discernible because of the overlap with band associated with N–H bending vibration of PAH (1632 cm− 1) in the region. When the cross-linking time is increased, the spectrum remained the same, but the amide to carboxylate peak height ratio is larger suggesting increased cross-linking. 3.2. Spontaneous deposition of ciprofloxacin hydrochloride
Fig. 4. SEM images of microcapsules to investigate the morphological changes induced by cross-linking. (a) Control; (b) and (c) cross-linked capsules for 0.5 and 7 h respectively. Scale bar = 2 μm.
cross-linked and uncross-linked capsules. All the capsules showed collapsed structure as a result of water evaporation during drying and core removal as shown in Fig. 4(a–c). Cross-linked capsules showed more folds when compared to control. When the cross-linking time was increased, more folds had formed and the wall thickness increased from 110±10 nm (for uncross-linked capsules) to 150±20 nm (for 7 h crosslinked capsules). Tong et al. also observed similar results for cross-linking of PSS/PAH capsules by glutaraldehyde [19]. They proposed that crosslinking the capsules stiffen the capsule walls, so that capsule walls cannot closely approach as that of control, which could increase thickness and number of folds in cross-linked capsules. Therefore, it can be concluded that cross-linking not only enhances the stability of the microcapsules at extreme pH conditions [26], but also controls the permeability of capsules without affecting their ability for spontaneous deposition of watersoluble substances. The cross-linking between ammonium groups of PAH and carboxylate groups of PMA in the presence of EDC was investigated by FTIR spectroscopy. Fig. 5 shows the FTIR spectrum of control and
Water-soluble drugs have been shown to accumulate spontaneously in PSS/PAH multilayer polyelectrolyte capsules using MF or CaCO3 templates by the LbL technique [22,34]. Recently, encapsulation of procainamide hydrochloride in thermally responsive PDADMAC/PSS capsule by heat treatment method has also been reported [35]. The major advantage of the present method is that encapsulation proceeds easily and effectively by simple mixing of capsule suspension with drug solution at room temperature. Further, the drug concentration attained in the capsule interior can be ten to hundred times higher than the bulk concentration without any other modification of the physical or chemical structure of the capsules. The electrostatic interaction between pre-loaded PSS in the capsules and drug is the primary reason for efficient drug encapsulation. Here ciprofloxacin hydrochloride was chosen as low molecular weight water-soluble drug (Mw = 385.8 g/ mol) to study the encapsulation and release process. The morphological changes of capsules prior and after encapsulation were monitored by AFM and SEM investigations. The AFM images (Fig. 6) of empty and loaded capsules showed different collapse behaviors and morphologies. The control capsules were completely collapsed and flat (Fig. 6a) whereas the loaded capsules exhibited much higher average vertical height as shown in Fig. 6b. The typical double wall thickness of a control capsule was 110 ± 10 nm which increased to 600 ± 50 nm after encapsulation. This increase in thickness is attributed to the ciprofloxacin loading. Fig. 7 shows SEM the images of the loaded capsules. The collapsed structure of the control capsules was similar to the AFM images shown in Fig. 6a, but the loaded capsules demonstrated a swollen structure without any folding (only folding marks were visible) on the surface of the capsules (Fig. 7a). Moreover, the loaded capsules were well dispersed without any aggregation. The SEM picture (Fig. 7b) shows the loaded drug that is expelled from the broken capsule (during drying). The drug precipitates are clearly seen from where the capsule
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Fig. 6. AFM images of dried capsules and their corresponding height profiles. (a) Empty capsules; (b) CH loaded capsules at pH = 3. After drug loading, the average height of capsules increased from 110 ± 10 nm to 600 ± 50 nm.
has broken. These results confirmed that the drug was mostly encapsulated in the capsule interior and not adsorbed on the surface of the capsule wall. DSC was used to analyze the physical state of drug in pure and loaded form as shown in Fig. 8. The drug might have been in crystalline, amorphous or dissolved in polymeric matrix after encapsulation process. Thermograms of drug in pure and loaded form showed that
there were two significant endothermic peaks at 100–150 °C and 310 °C attributed to sample dehydration and melting of CH [36]. The endothermic peak in loaded capsules at 305 °C confirmed the loading of CH, but the sharpness of peak slightly lost due to weaker electrostatic interaction between the pre-loaded PSS and drug. But it should be noted that significant amount of drug appeared as crystalline precipitate as shown in SEM image of broken capsule (Fig. 7b).
Fig. 7. SEM images of loaded capsules. (a) Intact loaded capsule and (b) broken capsule.
Fig. 8. DSC thermograms of empty (control), pure drug and drug-loaded capsules.
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Owing to the electrostatic attraction based on spontaneous deposition, the concentration of loaded drug inside the microcapsules is possibly higher than that in the bulk solution. Thus we evaluated some factors that affect the CH encapsulation especially for weak polyelectrolytes, which in turn may influence charge based loading behavior. The loading time was fixed at 1 h as it was enough to attain equilibrium loading (data not shown). The influence of pH value on the amount of drug encapsulation was studied from pH 6 to 3. As the pH decreased, both the loading capacity and capsule interior concentration increased to 4.16 × 1010 molecules/capsule and 236 mg/ml respectively at pH = 3 as shown in Fig. 9a and b. These values were twice than that at pH= 6. This fact can be explained by two primary reasons, (a) higher protonation degree of ciprofloxacin at pH= 3 (pKa 1, 6.09; pKa 2, 8.8) [37]; and (b) pH-induced permeability, which favored the diffusion of drug molecules from the bulk into the capsule interior [29]. The driving force for spontaneous deposition of CH is electrostatic interaction between drug and pre-loaded PSS, which is further increased by the protonation degree of drug molecules. This is reflected in increased loading efficiency from 13 to 32% when pH is decreased from 6 to 3. The relationship between initial drug concentration and the loading capacity of the (PAH/PMA) capsules is shown in Fig. 10. When the bulk drug concentration was increased, the loading capacity increased first and reached a saturation value of 4.21× 1010 molecules/capsule, but the ratio of interior to bulk concentration decreased continuously from 221 at 0.5 mg/ml to 28.5 at 10 mg/ml. These results demonstrated that the drug loading was controlled by both spontaneous deposition mechanism and ordinary diffusion (driven by concentration difference). In brief, spontaneous loading mechanism dominates the loading process at lower drug concentration resulting in high loading efficiency. But at higher concentration, the diffusion mechanism controls the loading process which is reflected in decreased loading efficiency though the loading capacity is high (Fig. 10b).
Fig. 10. (a) Effect of CH concentration on loading capacity and (b) interior concentration and loading efficiency. The results represent the mean ± SD (n = 3).
3.3. In vitro release Release of ciprofloxacin hydrochloride from loaded capsules was investigated in PBS using centrifuge method. The release profile has two regions; an initial burst release followed by a sustained release. Fig. 11a shows the release profile of the encapsulated ciprofloxacin when the bulk solution was exchanged with fresh PBS semiautomatically. The initial burst release in the first 1 h is attributed to the large imbalance of drug concentration between capsule interior and bulk. This phase was followed by sustained delivery of drug at an almost constant rate with nearly 70% of drug released in 6 h. No further release was observed after 6 h. Previously, it has been reported that the release rate of loaded drug can be significantly reduced by cross-linking the wall components of polyelectrolyte capsules [38]. Here, we investigated ciprofloxacin release by cross-linking the capsule wall after loading. Fig. 11b shows the release rate of ciprofloxacin as a function of cross-linking time. As the cross-linking time is increased, the release rate as well as cumulative release decreased due to the increase in cross-linking density, which provides resistance to the diffusion of drug from capsule interior to the bulk. The ability of sustained release from these capsules offers a promising drug delivery system for low molecular weight water-soluble drugs. 3.4. Sustained antibacterial activity
Fig. 9. (a) Effect of pH on CH loading capacity and (b) capsule interior concentration and loading efficiency. The results represent the mean ± SD (n = 3).
We investigated sustained bactericidal activity of CH released from the capsules against tested bacterial pathogen E. coli. Table 1 shows the proliferation behavior of the E. coli cultured in CH samples at various time intervals. When E. coli was added at infecting dose of 1.2 × 107 CFU/ml to CH samples, no growth was observed up to 7 h. In the control samples (empty capsules), robust bacterial growth was observed. Because of continuous release of CH from the capsules, antibacterial activity continued for at least 7 h. When the concentration of drug present in
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increased, the capsule interior concentration increased initially and saturated at 239 mg/ml. The loaded drug was released in a controlled manner, which could be further extended by cross-linking the capsule wall. The released CH showed significant antibacterial activity against bacterial pathogen E. coli. Hence this kind of weak polyelectrolyte capsules provides a promising drug delivery vehicle for controlled drug delivery applications.
Acknowledgements The authors wish to thank the Department of Biotechnology & Department of Science and Technology, Government of India for financial assistance.
References [1] K.E. Uhrich, S.M. Cannizzaro, R.S. Langer, K.M. Shakesheff, Chem. Rev. 99 (1999) 3181. [2] C.P. Torchlin, Adv. Drug Delivery Rev. 58 (2006) 1532. [3] L. Yang, P. Alexandridis, Curr. Opin. Colloid Interface Sci. 5 (2000) 132. [4] M.L. Hans, A.M. Lowman, Curr. Opin. Solid State Mater. Sci. 6 (2002) 319. [5] P. Tardi, E. Choice, D. Masin, T. Redelmeier, M. Bally, T.D. Madden, Cancer Res. 60 (2000) 3389. [6] R. Pandey, G.K. Khuller, J. Antimicrob. Chemother. 53 (2004) 635. [7] E. Donath, G.B. Sukhorukov, F. Caruso, S.A. Davis, H. Möhwald, Angew. Chem. Int. Ed. 37 (16) (1998) 2201. [8] W. Tong, W. Dong, C. Gao, H. Möhwald, J. Phys. Chem. B 109 (2005) 13159. [9] G.B. Sukhorukov, A.A. Antipov, A. Voigt, E. Donath, H. Möhwald, Macromol. Rapid Commun. 22 (2001) 44. [10] O.P. Tiourina, A.A. Antipov, G.B. Sukhorukov, N.I. Larionova, Y. Lvov, H. Möhwald, Macromol. Biosci. 1 (2001) 209. [11] G. Ibarz, L. Dähne, E. Donath, H. Möhwald, Adv. Mater. 13 (17) (2001) 1324. [12] K. Köhler, G.B. Sukhorukov, Adv. Funct. Mater. 17 (2007) 2053. [13] A.A. Antipov, G.B. Sukhorukov, E. Donath, H. Möhwald, J. Phys. Chem. B 105 (2001) 2281. [14] Y. Lvov, A.A. Antipov, A. Mamedov, H. Möhwald, G.B. Sukhorukov, Nano Lett. 1 (3) (2001) 125. [15] M.K. Park, S. Deng, R.C. Advincula, Langmuir 21 (2005) 5272. [16] S.Y. Yang, M.F. Rubner, J. Am. Chem. Soc. 124 (2002) 2100. [17] J.J. Harris, P.M. DeRose, M.L. Bruening, J. Am. Chem. Soc. 121 (1999) 1978. [18] L. Richert, F. Boulmedais, P. Lavelle, J. Mutterer, E. Ferreux, G. Decher, P. Schaaf, J.C. Voegel, C. Picart, Biomacromolecules 5 (2004) 284. [19] W. Tong, C. Gao, H. Möhwald, Chem. Mater. 17 (2005) 4610. [20] H. Ai, S.A. Jones, M.M. de Villiers, Y.M. Lvov, J. Controlled Release 86 (2003) 59. [21] S.M. Marinakos, M.F. Anderson, J.A. Ryan, L.D. Martin, D.L. Feldheim, J. Phys. Chem. B 105 (2001) 8872. [22] Z. Mao, L. Ma, C. Gao, J. Shen, J. Controlled Release 104 (2005) 193. [23] C. Gao, E. Donath, H. Möhwald, J. Shen, Angew. Chem. Int. Ed. 41 (20) (2002) 3789. [24] C. Gao, H. Möhwald, J.C. Shen, Chemphyschem 5 (2004) 116. [25] C. Gao, H. Möhwald, J. Shen, Adv. Mater. 15 (2003) 930. [26] T. Mauser, C. Dejugnat, G.B. Sukhorukov, Macromol. Rapid Commun. 25 (2004) 1781. [27] H.C. Neu, Am. J. Ophthalmol. 112 (1991) 15S. [28] H.J. Zeiler, Antimicrob. Agents Chemother. 28 (1985) 524. [29] S. Anandhakumar, V. Nagaraja, A.M. Raichur, Colloids Surf., B 78 (2010) 266. [30] C. Gao, S. Moya, H. Lichtenfeld, A. Casoli, H. Fiedler, E. Donath, H. Möhwald, Macromol. Mater. Eng. 286 (2001) 355. [31] C. Dejugnat, G.B. Sukhorukov, Langmuir 20 (2004) 7265. [32] K. Tomihata, Y. Ikada, J. Biomed. Mater. Res. A 37 (1997) 243. [33] Y. Wang, A. Yu, F. Caruso, Angew. Chem. Int. Ed. 44 (2005) 2888. [34] Q. Zhao, S. Zhang, W. Tong, C. Gao, J. Shen, Eur. Polym. J. 42 (2006) 3341. [35] W. Song, Q. He, H. Möhwald, Y. Yang, J. Li, J. Controlled Release 139 (2009) 160. [36] A.A. Silva-Junior, M.V. Scarpa, K.C. Pestana, L.P. Mercuri, J.R. Matos, A.G. Oliveira, Thermochim. Acta 467 (2008) 91. [37] J. Barbosa, D. Barrón, E. Jiménez-Lozano, V. Sanz-Nebot, Anal. Chim. Acta 437 (2001) 309. [38] S. Ye, C. Wang, X. Liu, Z. Tong, J. Biomater. Sci., Polym. Ed. 16 (2005) 909.
Fig. 11. Release profile of (a) control and (b) cross-linked capsules. The results represent the mean± SD (n= 3).
the capsules decreased below the minimal inhibition concentration (concentration required to completely sterilize the bacterial pathogen), gradual growth of E. coli was observed. But compared to the control capsules, in which the E. coli concentration increased nearly 900 times, one can conclude that the concentration of CH released even after 25 h can still effectively restrain the proliferation of the E. coli. The loaded capsules were cross-linked to reduce the drug release rate. It can be expected that cross-linking will increase the release time which could extend the antibacterial activity further (Fig. 11b). The antibacterial activity was increased from 7 to 10 h when the capsules cross-linked for 7 h as shown in Table 1. The cross-linking gives an extra barrier to the drug movement from capsule interior to bulk, thus slowing the release rate. 4. Conclusion We have demonstrated a drug delivery system based on polyelectrolyte capsules that can efficiently encapsulate water-soluble drugs. Hollow capsules composed of weak polyelectrolytes PAH and PMA prepared by LbL assembly which showed excellent loading capacity to ciprofloxacin. The main feature of PAH/PMA capsules is the pH-induced permeability changes which enables fine control over loading and subsequent release of drug in controlled manner. Loading capacity and interior drug concentration were increased when pH of the feeding solution decreased. When the concentration of feeding solution was
Table 1 Proliferation of E. coli cultured in medium containing the released CH drug at different time intervals. Time, h
1
Infecting dose (107 CFU/ml)
1.2
Final E. coli concentration after being cultured for 5 h (107 CFU/ml)
Uncross-linked capsules 0.5 h cross-linked 7 h cross-linked
0 0 0
2
3
4
5
6
7
8
9
10
25
30
Control
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
127 90 0
331 288 0
429 377 0
639 528 139
– – 310
904
Contents lists available at ScienceDirect
Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m s e c
Polyelectrolyte microcapsules for sustained delivery of water-soluble drugs S. Anandhakumar a, M. Debapriya a, V. Nagaraja b, Ashok M. Raichur a,⁎ a b
Department of Materials Engineering, Indian Institute of Science, Bangalore, 560012, India Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, India
a r t i c l e
i n f o
Article history: Received 8 April 2010 Received in revised form 1 October 2010 Accepted 11 October 2010 Available online 15 October 2010 Keywords: Weak polyelectrolyte capsules Ciprofloxacin Encapsulation Antibacterial studies
a b s t r a c t Polyelectrolyte capsules composed of weak polyelectrolytes are introduced as a simple and efficient system for spontaneous encapsulation of low molecular weight water-soluble drugs. Polyelectrolyte capsules were prepared by layer-by-layer (LbL) assembling of weak polyelectrolytes, poly(allylamine hydrochloride) (PAH) and poly (methacrylic acid) (PMA) on polystyrene sulfonate (PSS) doped CaCO3 particles followed by core removal with ethylene-diaminetetraacetic acid (EDTA). The loading process was observed by confocal laser scanning microscopy (CLSM) using tetramethylrhodamineisothiocyanate labeled dextran (TRITC-dextran) as a fluorescent probe. The intensity of fluorescent probe inside the capsule decreased with increase in cross-linking time. Ciprofloxacin hydrochloride (a model water-soluble drug) was spontaneously deposited into PAH/PMA capsules and their morphological changes were investigated by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The quantitative study of drug loading was also elucidated which showed that drug loading increased with initial drug concentration, but decreased with increase in pH. The loaded drug was released in a sustained manner for 6 h, which could be further extended by cross-linking the capsule wall. The released drug showed significant antibacterial activity against E. coli. These findings indicate that such capsules can be potential carriers for water-soluble drugs in sustained/controlled drug delivery applications. © 2010 Elsevier B.V. All rights reserved.
1. Introduction In pharmaceutical field, drug delivery systems (DDS) are being developed with the aim of achieving high drug content, controlled release, prolonged circulation in blood and ability to target a specific area, so that they greatly increase the drug efficacy and reduce the drug toxicity and side effects during application. Because of these requirements, variety of DDS (liposomes, microparticles and emulsions) have been developed [1–4]. In spite of some advantages over conventional dosage forms, they have found limited use due to low encapsulation efficiency, burst release and poor storage stability. More efforts are being made to improve the encapsulation efficiency, but only few studies have been reported that focus on both high encapsulation efficiency and sustained/controlled release of encapsulated drug [5,6]. An attractive and flexible DDS that meets all these requirements for drug delivery is the polyelectrolyte microcapsules, which are prepared by alternate deposition of oppositely charged polyelectrolytes onto dissolvable colloidal particles, followed by core removal [7]. These capsules provide numerous ways for drug loading and release. For example, the incorporation of polyanions into microcapsules during fabrication improves the loading of positively charged substances by spontaneous deposition mechanism [8]. The permeability of the capsule wall is the critical parameter for drug delivery, so that loading and subsequent
⁎ Corresponding author. Tel.: + 91 80 22933238; fax: + 91 80 23600472. E-mail address: [email protected] (A.M. Raichur). 0928-4931/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2010.10.005
release can be modified in a desired manner. Many attempts have been made to control the permeability of the capsules by changing pH, ionic strength, temperature, number of layers and polarity [9–14]. The other methods of modulating the permeability are cross-linking the wall components by various mechanisms such as UV irradiation, thermo- or photo-induced cross-linking, water-soluble carbodiimide chemistry and glutaraldehyde cross-linking [15–19]. The capsule surface can also be modified to meet the biological challenges such as shielding drug from human immune system and delivering the drugs at a specific site. Many efforts have been devoted for loading and subsequent release of model macromolecules from the microcapsule interior, but only few studies have used actual drug molecules. The simplest method of encapsulation is to use the drug crystal itself as a template for multilayer assembly [20], but the application is limited to substances having a low solubility under coating conditions. The drug can also be pre-loaded into the template before multilayer assembly [21]; however the template dissolution may affect the properties of the drug. There is another method of “spontaneous deposition” of drugs into microcapsules templated on melamine formaldehyde (MF) particles [22,23]. The negatively charged MF/PSS complex formed after core removal attracts drug molecules into the capsules, so that the deposited drug is in aggregate or complex form rather than in its free state. It should be noted that the concentration of free drugs in the capsule interior is always lower than the bulk [23]. It is also important to consider the biocompatibility of template material when used for drug delivery applications. The recent novel finding of “charge controlled attraction and repulsion” method is practically more attractive because the polyanions are pre-loaded into hollow capsules,
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which increase the interior drug concentration from ten to hundreds of times of the bulk concentration [8]. All the aforementioned encapsulation techniques have been reported on polyelectrolyte capsules prepared from strong polyelectrolytes, which do not respond to changes in pH. Although encapsulation by varying ionic strength seems to be applicable for strong polyelectrolyte capsules, it enables only the entrapment of relatively small amounts [24]. The polyelectrolyte capsules composed of weak polyelectrolytes have attracted considerable interest, because of their distinct and reversible change of properties in response to external stimulus. The pH dependent permeability of these capsules can be used for controlled encapsulation and release of drugs. Further, pH responsive microcapsules can be easily destroyed at extreme pH conditions, when the pH-induced imbalance of charges overcompensates the attractive polymer–polymer interactions [25,26]. Thus it is interesting to study the potential of these capsules for encapsulation and release of water-soluble drugs which have hardly been reported so far. In this publication, we continued our research on polyelectrolyte microcapsules composed of the weak polyelectrolytes PMA and PAH for controlled release. The ability of these capsules to achieve high efficiency encapsulation from low concentration loading solution by charge controlled mechanism was investigated using TRITC-dextran as a fluorescent probe. A water-soluble drug, ciprofloxacin hydrochloride (Fig. 1a), was used as a model drug. Ciprofloxacin hydrochloride is the most active fluoroquinolone and can inhibit 90% of Enterobacteriaceae at concentration below 0.25 μg/ml [27]. Moreover, it can be used in vitro and in vivo especially against gram-negative bacteria irrespective of their growth rate [28]. The possibility of controlling the release rate by cross-linking the wall components with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) has also been investigated. In vitro study was also performed to evaluate the activity of ciprofloxacin after release. Thus, we can combine the features of the pH responsive encapsulation with spontaneous deposition mechanism to obtain high drug encapsulation efficiency, which reduces the need for frequent administration leading to improved patient compliance. 2. Materials and methods 2.1. Materials PAH (Mw = 70 kDa), PMA (Mw = 483 kDa), PSS (Mw = 70 kDa), sodium carbonate, calcium chloride, EDTA, TRITC-dextran (Mw = 65– 76 kDa) and phosphate buffered saline (PBS) were all purchased from
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Sigma-Aldrich and EDC (Spectrochem, India). Ciprofloxacin hydrochloride (CH) was a gift by Dr. Reddy's laboratories Ltd., India. All chemicals were used without any further purification. The water used in all experiment was obtained from Milli-Q system with resistivity greater than 18 MΩ cm. All pH adjustments were done with 0.1 M HCl or 0.1 M NaOH. 2.2. Microcapsule preparation and cross-linking PSS doped CaCO3 particles with diameter of 6 ± 0.5 μm, prepared by colloidal aggregation of sodium carbonate and calcium chloride were used as sacrificial template [8]. Multilayer buildup was performed by LbL technique from 1 mg/ml polyelectrolyte solution of PAH and PMA in 0.5 M NaCl at pH = 6. After each adsorption step, three washing steps were performed. When four layers were deposited, the core was dissolved by 0.2 M EDTA solution, followed by three washing steps with water. As the PSS doped CaCO3 particles possess a negatively charged surface, the multilayer buildup was started with PAH. Cross-linking was performed by incubating the capsule suspension with 0.2 mM EDC at pH = 6 for a desirable time at room temperature, followed by three washings with water. 2.3. Ciprofloxacin loading and release experiments 0.3 ml of microcapsule suspension ((3 ± 0.2) × 108 capsules/ml) in water was centrifuged at 6000 rpm for 10 min and the supernatant was removed. Then the microcapsules were re-dispersed in 1 ml of CH with different loading conditions such as pH and feeding concentration and kept in a shaker at 25 °C. After incubation for 1 h, the mixture was centrifuged and the drug loaded capsules were washed once with water and used in release experiments. The scheme illustrating the loading and release process is depicted in Fig. 1b. The amount of drug loaded inside the microcapsules was estimated from the difference between input amount and amount in the supernatant after loading process. The drug amount in the supernatant was determined directly at 276 nm using a ND-1000 UV–Vis spectrophotometer (Nanodrop Technologies, USA). The number of microcapsules was determined using a hemocytometer with a 40× objective lens. In order to calculate drug concentration in the capsules, hundred microcapsules were selected stochastically and their diameters were measured under CLSM, by which their volumes were calculated respectively. The volume of one capsule (1.13 × 10− 10 ml) was averaged from the total
Fig. 1. (a) Chemical structure of ciprofloxacin hydrochloride. (b) Scheme demonstrating the deposition of ciprofloxacin into the preformed hollow capsules. (A— Prepared hollow capsules pre-loaded with PSS; A → B, spontaneous deposition of ciprofloxacin into preformed hollow capsules; B → C, centrifugation and removal of supernatant drug; C → D, release of the loaded drug in PBS buffer by concentration dependent diffusion mechanism).
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volume. Further details concerning the quantification of drug loaded can be found in our previous paper [29]. The loaded capsules ((9 ± 0.1) × 107 capsules) were incubated in 1 ml of PBS at pH = 7.4 in an Eppendorf tube. Then the tube was placed in a shaker at 200 rpm and 37 °C. After 30 min, the capsule suspension was centrifuged and the supernatant was replaced with fresh PBS (pre-warmed to 37 °C). The amount of CH in supernatant solution was determined spectroscopically. After cross-linking the loaded capsules, they were washed thrice with water. The drug present in the supernatant was measured and added to the total drug release in the first half an hour. 2.4. Confocal laser scanning microscopy (CLSM) The size, integrity and degree of filling of individual capsules were determined using a Zeiss LSM 510 META confocal scanning system (Zeiss, Germany) equipped with a 100× oil immersion objective with a numerical aperture of 1.4. The capsules were visualized by electrostatic adsorption of TRITC-dextran at different pH values and cross-linking densities by keeping other parameters such as aperture, gain and laser power constant. The excitation wave length was 543 nm. 2.5. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) The morphology changes of the polyelectrolyte capsules were observed by AFM and SEM. AFM measurements were performed with a Nanosurf Easy Scan2 AFM (Nanoscience Instruments, USA) in air at room temperature by contact mode. AFM samples were prepared by depositing a droplet of the capsule suspension on a silicon wafer and allowing it to dry overnight in a desiccator. SEM images of Au sputtered samples dried on silicon wafer were obtained with a field emission scanning electron microscope (FEI-SIRION, Eindhoven, Netherlands). 2.6. Fourier transform infrared spectroscopy (FTIR) Dried microcapsules were mixed with KBr powder and pelletized. Spectra were acquired in transmission mode using a Nicolet 5700 FTIR spectrometer (Thermo Electron Corporation, USA). 2.7. Differential scanning calorimetry (DSC) Thermograms of pure drug, empty and drug-loaded capsules were performed in order to characterize their physical state after encapsulation, using a computer-interfaced differential scanning calorimeter (DSC 822, Mettler-Toledo Inc., USA) under nitrogen atmosphere. About 5 mg of samples was weighed, crimped into an aluminum pan and heated from 25 °C to 350 °C at the rate of 5 °C/min. 2.8. Sustained antibacterial activity Loaded capsules were suspended in 3 ml of M9 minimal media and incubated in a shaker at 200 rpm and 37 °C. A semi-automatic protocol was used. Briefly, 2.4 ml of the sample was collected at different time intervals by centrifugation and their drug concentration was measured by spectroscopy. Fresh media (2.4 ml) was added to the incubated sample to maintain the initial volume of 3 ml. The collected samples were mixed with 40 μl of E. coli solution grown in M9 minimal media (final concentration of E. coli, 1.2 × 107 cells/ml). After 5 h of incubation period at 37 °C, the bacterial growth of the samples was determined by measuring the optical density (OD) at 600 nm (0.1 OD equals to 108 E. coli/ml). The free growing cell and free drug were maintained as positive and negative controls respectively.
3. Results and discussion Spherical calcium carbonate microparticles are a new category of template, which can be produced in the size range of 4 to 7 μm. This non-toxic template can be easily and gently removed by complexation with EDTA after self assembling of polyelectrolyte multilayer during capsule fabrication. It is known from literature that dissolution of other widely used templates such as MF or PS could either cause capsule wall defects due to osmotic stress produced by core dissolution or modify the capsule properties due to deposition of residual material within the capsule [30,31]. In this study, PSS doped CaCO3 particles were prepared and used for capsule fabrication due to their ability to form defect free capsules without any residues [6,29]. After template dissolution, hollow capsules were obtained. The semipermeability of the polyelectrolyte membrane permits small molecules (EDTA, inorganic ions and dyes) to freely enter and leave the capsule interior whereas large molecules like incorporated PSS are captured inside the capsules. The pre-loaded PSS molecules endow the hollow capsules with the capability to spontaneous load positively charged drug molecules by electrostatic interaction [8]. 3.1. Spontaneous loading of TRITC-dextran into (PAH/PMA)2 capsules The permeability and spontaneous loading of polyelectrolyte capsules composed of weak polyelectrolytes is particularly interesting due to its close relationship with drug loading and release in drug delivery. Water-soluble drugs can be readily incorporated into the capsule interior in high concentration upon simple mixing with capsule suspensions. To understand the loading process and to reveal the distribution of drug molecules inside the capsules, the encapsulation was monitored by CLSM using TRITC-dextran as a fluorescent probe. The confocal images at low and high pH's are shown in Fig. 2. The bulk solution was removed by centrifugation, so that the fluorescence from the capsule interior could be seen clearly. At alkaline conditions, e.g., pH= 8 (Fig. 2b) the interior of the capsules remained dark which implies that there was no loading of TRITC-dextran. In contrast, strong fluorescence was observed from the capsule interior at pH= 3 (Fig. 2a), demonstrating spontaneous deposition of large amount of TRITCdextran in the capsule interior. It has been reported that PSS/PAH microcapsules can be loaded with positively charged substances such as rhodamine, dextran etc., when PSS doped CaCO3 microparticles were used as sacrificial template for assembly. The driving force for spontaneous deposition was ascribed to the electrostatic interaction between the pre-incorporated PSS molecules and the positively charged water-soluble substances [8]. The fluorescence intensity profile gives information about the concentration and distribution of probe molecules in the capsule interior. Therefore, it reflects that a large amount of dextran was encapsulated and homogeneously deposited in the interior of the capsules. The charge density variation of weak polyelectrolytes at pH b4, induced pore formation by loosening the polyelectrolyte networks thus controlling the open and closed states of the capsules [9,29]. These results show the important fact that pH controlled permeability of (PAH/PMA) capsules can be used for encapsulation and subsequent release of drugs in controlled manner, which may find potential application for sustained release. The permeability and release of capsules can also be controlled by cross-linking the layer components, which helps to tune the loading and release at a well-defined rate. Cross-linking density is the key parameter used to tune pH dependent permeability, because higher degree of cross-linking may lead to complete loss of pH responsive properties due to absence of available charged site for electrostatic interaction. It is possible to cross-link the (PAH/PMA) capsules easily by cross-linking the functional groups of PMA and PAH with EDC [18,32]. The main advantage of EDC based zero length cross-linker is that it does not introduce any additional cross-linker molecule or particle that may be toxic during in vivo applications [18,33].
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Fig. 2. CLSM images of microcapsules incubated with TRITC-dextran at pH = 3 (a) and pH = 8 (b). Scale bar = 10 μm.
The cross-linked capsules, regardless of additional cross-linking showed very good dispersion without any shape change and aggregation. The encapsulation process of cross-linked hollow capsules was monitored by CLSM at pH =3 using TRITC-dextran. Representative CLSM images at different cross-linking times are presented in Fig. 3. All the capsules showed strong fluorescence from the capsule interior than that from the bulk demonstrating spontaneous deposition of dextran. The degree of
filling decreased when the cross-linking time was increased as shown in the fluorescence cross section profile (Fig. 3a and b) which can be attributed to decrease in wall permeability. It is important to note that the process of cross-linking did not affect the spontaneous deposition properties of the capsules. To better understand the mechanism, the cross-linked capsules were investigated in SEM to reveal their morphological changes (Fig. 4). SEM measurements were performed on both
Fig. 3. CLSM images of microcapsules to show spontaneous deposition of TRITC-dextran into cross-linked microcapsules. (a) 0.5 h and (b) 7 h. Scale bar = 10 μm.
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Fig. 5. FTIR spectra of hollow microcapsules as a function of cross-linking time.
cross-linked capsules. The spectrum of control capsules exhibits two sharp absorption bands at 1405 and 1632 cm− 1, which are attributed to the symmetric stretches of carboxylate (–COO−) group and N–H bending (scissoring) vibration of PAH [17,33]. After cross-linking, these two peaks are significantly decreased. At the same time, new broad peaks centered at 1635 cm− 1 and 1541 cm− 1 are evident in the spectrum, which are attributed to the formation of amide I and amide II bands respectively [17]. The amide I band formed as a result of cross-linking is not discernible because of the overlap with band associated with N–H bending vibration of PAH (1632 cm− 1) in the region. When the cross-linking time is increased, the spectrum remained the same, but the amide to carboxylate peak height ratio is larger suggesting increased cross-linking. 3.2. Spontaneous deposition of ciprofloxacin hydrochloride
Fig. 4. SEM images of microcapsules to investigate the morphological changes induced by cross-linking. (a) Control; (b) and (c) cross-linked capsules for 0.5 and 7 h respectively. Scale bar = 2 μm.
cross-linked and uncross-linked capsules. All the capsules showed collapsed structure as a result of water evaporation during drying and core removal as shown in Fig. 4(a–c). Cross-linked capsules showed more folds when compared to control. When the cross-linking time was increased, more folds had formed and the wall thickness increased from 110±10 nm (for uncross-linked capsules) to 150±20 nm (for 7 h crosslinked capsules). Tong et al. also observed similar results for cross-linking of PSS/PAH capsules by glutaraldehyde [19]. They proposed that crosslinking the capsules stiffen the capsule walls, so that capsule walls cannot closely approach as that of control, which could increase thickness and number of folds in cross-linked capsules. Therefore, it can be concluded that cross-linking not only enhances the stability of the microcapsules at extreme pH conditions [26], but also controls the permeability of capsules without affecting their ability for spontaneous deposition of watersoluble substances. The cross-linking between ammonium groups of PAH and carboxylate groups of PMA in the presence of EDC was investigated by FTIR spectroscopy. Fig. 5 shows the FTIR spectrum of control and
Water-soluble drugs have been shown to accumulate spontaneously in PSS/PAH multilayer polyelectrolyte capsules using MF or CaCO3 templates by the LbL technique [22,34]. Recently, encapsulation of procainamide hydrochloride in thermally responsive PDADMAC/PSS capsule by heat treatment method has also been reported [35]. The major advantage of the present method is that encapsulation proceeds easily and effectively by simple mixing of capsule suspension with drug solution at room temperature. Further, the drug concentration attained in the capsule interior can be ten to hundred times higher than the bulk concentration without any other modification of the physical or chemical structure of the capsules. The electrostatic interaction between pre-loaded PSS in the capsules and drug is the primary reason for efficient drug encapsulation. Here ciprofloxacin hydrochloride was chosen as low molecular weight water-soluble drug (Mw = 385.8 g/ mol) to study the encapsulation and release process. The morphological changes of capsules prior and after encapsulation were monitored by AFM and SEM investigations. The AFM images (Fig. 6) of empty and loaded capsules showed different collapse behaviors and morphologies. The control capsules were completely collapsed and flat (Fig. 6a) whereas the loaded capsules exhibited much higher average vertical height as shown in Fig. 6b. The typical double wall thickness of a control capsule was 110 ± 10 nm which increased to 600 ± 50 nm after encapsulation. This increase in thickness is attributed to the ciprofloxacin loading. Fig. 7 shows SEM the images of the loaded capsules. The collapsed structure of the control capsules was similar to the AFM images shown in Fig. 6a, but the loaded capsules demonstrated a swollen structure without any folding (only folding marks were visible) on the surface of the capsules (Fig. 7a). Moreover, the loaded capsules were well dispersed without any aggregation. The SEM picture (Fig. 7b) shows the loaded drug that is expelled from the broken capsule (during drying). The drug precipitates are clearly seen from where the capsule
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Fig. 6. AFM images of dried capsules and their corresponding height profiles. (a) Empty capsules; (b) CH loaded capsules at pH = 3. After drug loading, the average height of capsules increased from 110 ± 10 nm to 600 ± 50 nm.
has broken. These results confirmed that the drug was mostly encapsulated in the capsule interior and not adsorbed on the surface of the capsule wall. DSC was used to analyze the physical state of drug in pure and loaded form as shown in Fig. 8. The drug might have been in crystalline, amorphous or dissolved in polymeric matrix after encapsulation process. Thermograms of drug in pure and loaded form showed that
there were two significant endothermic peaks at 100–150 °C and 310 °C attributed to sample dehydration and melting of CH [36]. The endothermic peak in loaded capsules at 305 °C confirmed the loading of CH, but the sharpness of peak slightly lost due to weaker electrostatic interaction between the pre-loaded PSS and drug. But it should be noted that significant amount of drug appeared as crystalline precipitate as shown in SEM image of broken capsule (Fig. 7b).
Fig. 7. SEM images of loaded capsules. (a) Intact loaded capsule and (b) broken capsule.
Fig. 8. DSC thermograms of empty (control), pure drug and drug-loaded capsules.
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Owing to the electrostatic attraction based on spontaneous deposition, the concentration of loaded drug inside the microcapsules is possibly higher than that in the bulk solution. Thus we evaluated some factors that affect the CH encapsulation especially for weak polyelectrolytes, which in turn may influence charge based loading behavior. The loading time was fixed at 1 h as it was enough to attain equilibrium loading (data not shown). The influence of pH value on the amount of drug encapsulation was studied from pH 6 to 3. As the pH decreased, both the loading capacity and capsule interior concentration increased to 4.16 × 1010 molecules/capsule and 236 mg/ml respectively at pH = 3 as shown in Fig. 9a and b. These values were twice than that at pH= 6. This fact can be explained by two primary reasons, (a) higher protonation degree of ciprofloxacin at pH= 3 (pKa 1, 6.09; pKa 2, 8.8) [37]; and (b) pH-induced permeability, which favored the diffusion of drug molecules from the bulk into the capsule interior [29]. The driving force for spontaneous deposition of CH is electrostatic interaction between drug and pre-loaded PSS, which is further increased by the protonation degree of drug molecules. This is reflected in increased loading efficiency from 13 to 32% when pH is decreased from 6 to 3. The relationship between initial drug concentration and the loading capacity of the (PAH/PMA) capsules is shown in Fig. 10. When the bulk drug concentration was increased, the loading capacity increased first and reached a saturation value of 4.21× 1010 molecules/capsule, but the ratio of interior to bulk concentration decreased continuously from 221 at 0.5 mg/ml to 28.5 at 10 mg/ml. These results demonstrated that the drug loading was controlled by both spontaneous deposition mechanism and ordinary diffusion (driven by concentration difference). In brief, spontaneous loading mechanism dominates the loading process at lower drug concentration resulting in high loading efficiency. But at higher concentration, the diffusion mechanism controls the loading process which is reflected in decreased loading efficiency though the loading capacity is high (Fig. 10b).
Fig. 10. (a) Effect of CH concentration on loading capacity and (b) interior concentration and loading efficiency. The results represent the mean ± SD (n = 3).
3.3. In vitro release Release of ciprofloxacin hydrochloride from loaded capsules was investigated in PBS using centrifuge method. The release profile has two regions; an initial burst release followed by a sustained release. Fig. 11a shows the release profile of the encapsulated ciprofloxacin when the bulk solution was exchanged with fresh PBS semiautomatically. The initial burst release in the first 1 h is attributed to the large imbalance of drug concentration between capsule interior and bulk. This phase was followed by sustained delivery of drug at an almost constant rate with nearly 70% of drug released in 6 h. No further release was observed after 6 h. Previously, it has been reported that the release rate of loaded drug can be significantly reduced by cross-linking the wall components of polyelectrolyte capsules [38]. Here, we investigated ciprofloxacin release by cross-linking the capsule wall after loading. Fig. 11b shows the release rate of ciprofloxacin as a function of cross-linking time. As the cross-linking time is increased, the release rate as well as cumulative release decreased due to the increase in cross-linking density, which provides resistance to the diffusion of drug from capsule interior to the bulk. The ability of sustained release from these capsules offers a promising drug delivery system for low molecular weight water-soluble drugs. 3.4. Sustained antibacterial activity
Fig. 9. (a) Effect of pH on CH loading capacity and (b) capsule interior concentration and loading efficiency. The results represent the mean ± SD (n = 3).
We investigated sustained bactericidal activity of CH released from the capsules against tested bacterial pathogen E. coli. Table 1 shows the proliferation behavior of the E. coli cultured in CH samples at various time intervals. When E. coli was added at infecting dose of 1.2 × 107 CFU/ml to CH samples, no growth was observed up to 7 h. In the control samples (empty capsules), robust bacterial growth was observed. Because of continuous release of CH from the capsules, antibacterial activity continued for at least 7 h. When the concentration of drug present in
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increased, the capsule interior concentration increased initially and saturated at 239 mg/ml. The loaded drug was released in a controlled manner, which could be further extended by cross-linking the capsule wall. The released CH showed significant antibacterial activity against bacterial pathogen E. coli. Hence this kind of weak polyelectrolyte capsules provides a promising drug delivery vehicle for controlled drug delivery applications.
Acknowledgements The authors wish to thank the Department of Biotechnology & Department of Science and Technology, Government of India for financial assistance.
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Fig. 11. Release profile of (a) control and (b) cross-linked capsules. The results represent the mean± SD (n= 3).
the capsules decreased below the minimal inhibition concentration (concentration required to completely sterilize the bacterial pathogen), gradual growth of E. coli was observed. But compared to the control capsules, in which the E. coli concentration increased nearly 900 times, one can conclude that the concentration of CH released even after 25 h can still effectively restrain the proliferation of the E. coli. The loaded capsules were cross-linked to reduce the drug release rate. It can be expected that cross-linking will increase the release time which could extend the antibacterial activity further (Fig. 11b). The antibacterial activity was increased from 7 to 10 h when the capsules cross-linked for 7 h as shown in Table 1. The cross-linking gives an extra barrier to the drug movement from capsule interior to bulk, thus slowing the release rate. 4. Conclusion We have demonstrated a drug delivery system based on polyelectrolyte capsules that can efficiently encapsulate water-soluble drugs. Hollow capsules composed of weak polyelectrolytes PAH and PMA prepared by LbL assembly which showed excellent loading capacity to ciprofloxacin. The main feature of PAH/PMA capsules is the pH-induced permeability changes which enables fine control over loading and subsequent release of drug in controlled manner. Loading capacity and interior drug concentration were increased when pH of the feeding solution decreased. When the concentration of feeding solution was
Table 1 Proliferation of E. coli cultured in medium containing the released CH drug at different time intervals. Time, h
1
Infecting dose (107 CFU/ml)
1.2
Final E. coli concentration after being cultured for 5 h (107 CFU/ml)
Uncross-linked capsules 0.5 h cross-linked 7 h cross-linked
0 0 0
2
3
4
5
6
7
8
9
10
25
30
Control
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
127 90 0
331 288 0
429 377 0
639 528 139
– – 310
904