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Acrylated chitosan for mucoadhesive drug delivery systems
Accepted Manuscript Title: Acrylated Chitosan for Mucoadhesive Drug Delivery Systems Author: Yulia Shitrit Havazelet Bianco-Peled PII: DOI: Reference:
S0378-5173(16)31165-6 http://dx.doi.org/doi:10.1016/j.ijpharm.2016.12.023 IJP 16294
To appear in:
International Journal of Pharmaceutics
Received date: Revised date: Accepted date:
25-7-2016 13-11-2016 10-12-2016
Please cite this article as: Shitrit, Yulia, Bianco-Peled, Havazelet, Acrylated Chitosan for Mucoadhesive Drug Delivery Systems.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2016.12.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Acrylated Chitosan for Mucoadhesive Drug
Delivery Systems
Yulia Shitrit and Havazelet Bianco-Peled*
Department of Chemical Engineering, Technion – Israel Institute of Technology,
Haifa 32000, Israel
*Corresponding author: Professor Havazelet Bianco-Peled, Department of Chemical Engineering, Technion – Israel Institute of Technology, Haifa 32000, Israel. Tel.
+972-4-829-3588, Fax. +972-4-829-5672, email [email protected]
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Graphical abstract
Abstract A new mucoadhesive polymer was synthesized by conjugating chitosan to poly(ethylene glycol)diacrylate (PEGDA) via the Michael type reaction. The product was characterized using NMR. Higher PEGDA grafting efficacy was observed with low molecular weight PEGDA (0.7 kDa), compared to long 10 kDa PEGDA. The acrylation percentage was calculated based on the reaction of ninhydrin with chitosan, and supported the qualitative NMR findings. The adhesive properties were studied by tensile test and rotating system involving detachment of polymer tablets from a fresh intestine sample. Chitosan modified with high molecular weight PEGDA presented improvement in mucoadhesive properties compared to both non-modified and thiolated chitosan. On the molecular level, rheology measurements of polymer/mucin mixtures provided additional evidence of strong interaction between modified chitosan and mucin glycoproteins. This new polymer shows promise as a useful polymeric carrier matrix for delivery systems, which could provide prolonged residence time of the vehicle on the mucosa surface. 2
Keywords Chitosan; PEGDA (poly(ethylene glycol) diacrylate); mucoadhesion 1.
2. Introduction The mucous surface is the first barrier that interacts with nutrients and drugs before they can be absorbed by diffusion and reach their target organs (Bansil and Turner, 2006; Cone, 2009). Over the last three decades, researchers have been studying mucoadhesion due to its potential to optimize localized drug delivery. This delivery method utilizes vehicles capable of attaching to mucosal surfaces, hence providing prolonged residence time of drugs at the site of application. It possesses several advantages over oral administration. First, the uptake of a drug into the systemic circulation through the permeable mucus membranes is relatively rapid due to the massive blood supply and sufficient blood flow rate at the mucosal surface. Furthermore, transmucosal delivery helps to avoid drug degradation by some of the body’s natural defense mechanisms thus enhancing bioavailability. In addition, the intimate contact with the absorption site can provide a passive drug uptake by a high concentration gradient leading to faster diffusion (Alexander et al., 2011; BernkopSchnürch, 2005). Mucoadhesive materials can be in forms of tablets, patches, tapes, films, semisolids, powders, in situ gelling systems and nanoparticales (Cook and Khutoryanskiy, 2015; Sosnik et al., 2014). Combining mucoadhesive properties with other polymeric drug carriers offering the advantages of, for example, sustained release rate, protection of pharmaceuticals from hydrolysis and chemical or enzymatic 3
degradation, reduction of drug toxicity, and improvement of drug solubility and availability, has opened the way for the design of powerful non-invasive drug delivery vehicles. The most common group of mucoadhesive polymers are hydrophilic materials that can create weak non-covalent bonds such as hydrogen bonds, Van-Der Waals forces, ionic interactions and chain entanglements with the mucosa (Andrews et al., 2009; Lee et al., 2000). A more recent development has produced modified materials with functional groups capable of forming strong bonds with mucous components. Thiolated polymers, also termed thiomers, were first developed by Leitner et al. (Leitner et al., 2003). They carry a thiol functional group capable of forming disulphide bridges with cysteine-rich subdomains of mucin glycoproteins. Thiolated polymers have been widely explored in the last decade. They are easy to synthesize, and many studies demonstrated that thiomer has a better adhesion ability than the nonmodified polymer (Bernkop-Schnürch, 2003; Kafedjiiski, Krauland, Hoffer, & Bernkop-Schnürch, 2005; Rahmat et al., 2011, 2012). Davidovich-Pinhas and BiancoPeled (Davidovich-Pinhas and Bianco-Peled, 2010) suggested a novel mucoadhesive system based on polymers carrying an acrylate side group. In this approach, a polymer carrying an acrylate end group associates with the sulfide end group of the mucin type glycoprotein by the Michael type addition reaction. The thiol residue on the mucin glycoprotein backbone, acting as a strong nucleophile, attacks the double bond, a covalent bond is formed and hydroxyl is released. To actualize this concept, a novel biomaterial termed alginate-PEGAc, comprising alginate backbone and PEG-acrylate grafts, was developed and investigated. This polymer displayed a significant increase in the adhesion strength compared to both the non-modified alginate and the thiolated alginate (Davidovich-Pinhas and Bianco-Peled, 2011). 4
In the present study, we expand the knowledge on acrylate-based mucoadhesion polymers by creating an additional polymer and investigating its interactions with mucous. The new mucoadhesive polymer was synthesized from chitosan (CS), a nontoxic, biocompatible and biodegradable natural polysaccharide (No, 2002; Rinaudo, 2006), conjugated to poly(ethylene glycol)diacrylate (PEGDA) via the Michael type reaction.. The synthesized product was verified and characterized using Nuclear magnetic resonance (NMR). The adhesive properties were studied in a semi-hydrated environment by tensile tests and in a hydrated environment using the rotating cylinder method. On the molecular level, rheology measurements of polymer/mucin mixtures provided additional evidence of strong interaction between the synthesized polymer and mucin glycoproteins.
3. Materials and methods 3.1.
Materials
Poly(ethylene glycol) diacrylate (PEGDA) with a molecular weight (Mw) of 10 kDa was purchased from the laboratory of Prof. Seliktar in the Department of Biomedical Engineering, Technion, Israel. PEGDA with an Mw of 0.7 kDa, mucin from porcine stomach, Type II (PGM) and low molecular weight chitosan (CS) were obtained from Sigma-Aldrich MO, USA. The degree of deacetylation (DDA) of CS was determined using Fourier transform infrared (FTIR) spectroscopy and found to be 77.6% (Delmar and Bianco-Peled, 2015). Weight averaged Mw was measured by static light scattering and found to be 207 kDa (Delmar and Bianco-Peled, 2015). Porcine small intestine was purchased from the Pre-Clinical Research Authority, Technion, cut 5
open, spread on a petri dish and stored until further use at -20°C. Acetic acid (99.7% A.R.) was purchased from Gadot Biochemical Industries Ltd., Israel. Sodium hydroxide (NaOH) pearls and 32% hydrochloric acid solution (HCl) were purchased from Bio-Lab Ltd., Israel. 2-Iminothiolane was purchased from Proteo-Chem, Denver, CO, USA. Sodium chloride (NaCl) and dimethyl sulfoxide (DMSO) were purchased from Merck KGaA, Darmstadt, Germany. 3.2.
Synthesis of mucoadhesive polymers
3.2.1. Acrylated chitosan synthesis The synthesis of acrylated chitosan (Chitosan-PEGAc) was accomplished according to a previously published procedure (Ma et al., 2009) with slight modifications. Acrylation of chitosan was performed using PEGDA with an MW of 10 kDa, which produced acrylated chitosan with long PEG chains (chitosan–PEGAc(10)), or PEGDA with an MW of 0.7 kDa, which produced acrylated chitosan with short PEG chains (chitosan–PEGAc(0.7)). Both acrylated chitosan versions were prepared.The amine:PEG ratio was used as the experimental variable since it affects the PEG grafting density and therefore the molecule’s properties. The calculated molar ratios summarized in Table 1 refer to the ratio between the molar concentration of the primary amines in chitosan and the molar concentration of PEG chains. An exemplary calculation can be found in the supplementary materials.
First, chitosan powder was dissolved in 2% (v/v) acetic acid in a dark glass bottle in a total concentration of 1% (w/v) and mixed for 24 h to ensure complete dissolution of the polymer. Then, PEGDA was added to the mixture to obtain the desired molar ratio detailed in Table 1, and the mixture was stirred for an additional 15 min to give a homogenic solution. The bottle was placed in a water bath at 60oC and kept for 3 hr 6
under constant shaking at 100 rpm. To remove unreacted PEGDA residues, the solution was dialized using a cellulose tube having a 12–14 kDa molecular weight cutoff against dionized water for 48 hr. The water was changed at least three times during this period. The final product was freezed for 24 hours at -20oC
and
lyophilized by drying the frozen aqueous polymer solution at -30oC and 0.01 mbar and stored at 4oC until needed for further use.
3.2.2. Synthesis of thiolated chitosan (chitosan-TBA) For the sake of comparison, a thiolated chitosan, which is a known mucoadhesive polymer that forms covalent bonds with mucins, was synthesized according to previously published procedures (Bernkop-Schnürch, 2003). In brief, 1 g of chitosan powder was dissolved in 1L of 2% (v/v) acetic acid. 0.4 g of 2-iminothiolane was added to the solution while adjusting the pH to 6.3 using 1M NaOH, and then mixed for 24 h. To remove unreacted thiol residues, the product was dialyzed for three days using a cellulose tube having a 12–14 kDa molecular weight cutoff against 5 mM HCl, two times against 5 mM HCl containing 1% NaCl, once against 5 mM HCl, and once against 1 mM HCl. The final sample was freezed for 24 hours at -20oC and lyophilized by drying the frozen aqueous polymer solution at -30oC and 0.01 mbar and stored at 4oC until needed for further use. 3.3.
Tablet preparation
Samples for assessment of mucoadhesion properties were prepared by compressing 100 mg of a dry polymer sample into a 11 mm diameter plate disc using pressure of 2 ton applied on a compression molding device. The polymers used were chitosan, chitosan-PEGAc and chitosan-TBA.
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3.4.
Polymer characterisation
3.4.1. Nuclear magnetic resonance (NMR) 1
H NMR (400 MHz) spectra were recorded on a Bruker-ARX 400 instrument.
Samples used for the NMR experiments to verify the desired product were performed by preparing a 1% (w/v) polymer solution in CD3COOD at room temperature the night before the measurements, to ensure complete dissolution. 3.4.2. Ninhydrin test for quantitative determination of acrylate groups A ninhydrin reagent was prepared by dissolving 2% (w/v) of ninhydrin in dimethyl sulfoxide (DMSO) in a dark bottle. A set of polymer solutions was prepared by dissolving studied polymer at concentrations of 0.1-0.6% (w/v) in a mixture of 3% (v/v) acetic acid and 1% (v/v) HCl and mixing for 24 hr. 0.1 ml of each solution was mixed with 0.25 ml of 4M phosphate buffer (PB) (pH=5.4) and 1 ml of ninhydrin solution. The final mixtures were incubated in a 100°C bath and shaken at a rate of 60 rpm for 30 min. 0.2 ml from each mixture were analyzed using a Bio-Tek Inc. spectrophotometer at a wavelength of 500 nm. Curves were plotted using the average of at least three independent measurements (n ≥ 3). 3.5.
Adhesion assays
3.5.1. Tensile assays Tensile assays were performed using a technique previously developed for soft substrates (Davidovich-Pinhas and Bianco-Peled, 2010). Adhesion assays were performed by attaching a fresh small intestine sample to a 25 mm stainless steel grid connected to a vacuum system, which attaches the lower surface of the tissue to the grid without the need to use glue. Since the upper surface of the tissue was not exposed to the vacuum, the naturally occurring mucus surface was not dehydrated. This 25 mm grid was then fixed to the lower arm of a Lloyed Tensile machine equipped with a 50-N load cell. The hydration of the intestine surface was 8
standardized by pouring 20 µl of double distilled water (DDW) onto the substrate surface prior to the measurement. It should be noted that intestine washing is avoided in order to preserve the mucus layer. A compressed polymer tablet (11 mm in diameter) was attached to the upper arm of the tensile machine using double-sided adhesive tape. The upper arm carrying the tablet was lowered at 5 mm/min until a force of 0.1 N was recorded between the polymer tablet and the substrate. The force was maintained for 10 minutes. In order to measure the maximum adhesion force, the machine was set to an extension mode where the upper arm was pulled at a constant rate of 0.01 mm/sec and the force was recorded until detachment was observed. The substrate was replaced before each test. The reported values of maximal detachment stress (MDS), calculated by dividing the recorded maximum detachment force by the tablet area, are an average of at least five independent measurements (n ≥ 5) for each substrate and polymer pair. Statistical data analysis is performed using a standard ttest with P < 0.05 considered the minimal level of significance.
3.5.2. Rotating system A homemade system based on the principles of a rotating cylinder ( BernkopSchnürch & Steininger, 2000) was used to measure the time required to detach a dosage form from the mucous surface in simulated intestinal fluid (pH=7.4). A piece of porcine intestine was fixed on a Mylar sheet using cyanoacrylate glue. Dry compressed polymer tablets were attached to the surface using mild pressure. The Mylar carrying the tablets samples was attached to a Styrofoam float and placed in a beaker containing 0.1 M phosphate buffer saline (PBS, pH=7.4) at 37°C. The solution was stirred at 600 rpm using a magnetic stirrer, which led to rotation of the float. The
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detachment of the tablets was monitored. The reported detachment time is the average of at least four independent measurements (n ≥ 4). 3.6.
Rheology
Rheological measurements were carried out using a TA Instruments’ AR-G2 rheometer (New Castle, DE, USA) equipped with a Peltier plate temperature controlled base. The experiments were performed using a parallel plate geometry having a 40 mm radius at a constant temperature of 37°C. 2% (w/v) polymer stock solutions in 0.1M HCl (pH 1) were prepared and stored at room temperature for 24 h, to ensure complete dissolution. 10% (w/v) mucin stock solution in PB pH=7.4 was prepared on the day of measurement to ensure its freshness. 1 ml mucin stock solution was mixed together with 1 ml polymer stock solution; the final composition of the resulting mixture was 5mg/ml mucin and 10 mg/ml polymer and it pH was 4.8. The mucin solution and polymer solution were mixed together at an appropriate ratio to a final polymer:mucin weight ratio of 1:5 and stirred vigorously for 1 h prior to analysis. Measurements were performed in triplicate. Shear rates were varied from 10 to 300 s−1. Following Rossi et al. (Rossi et al., 2001), we used a 1:5 (w/w) polymer:mucin weight ratio for rheology measurements.
4. Results and Discussion 4.1.
Synthesis of chitosan–PEGAc
In order to broaden the range of available covalently associating polymers, we explored a new polymer, chitosan–PEGAc. Conjugating the chitosan’s amine to PEGDA was achieved by Michael type reaction, which is a well-known process to add various amines onto unsaturated carbonyl compounds. Since amines can act as 10
both nucleophiles and bases, in the case of chitosan no additional base is typically needed, makings this reaction a simple and spontaneous one with no further materials needed to be removed after it (Sashiwa et al., 2003). The primary amine on the chitosan backbone acts as a nucleophile and attacks the double bond on the PEGDA. Having two vinyl end groups on the PEGDA enables, on the one hand, the Michael reaction between chitosan and PEGDA, and on the other hand, the final product contains another free vinyl end group for further interaction with the mucous glycoproteins (Figure 1). The calculation of the molar ratios is detailed in the supplementary materials; the calculated molar ratios summarized in Table 1 refer to the ratio between the molar concentration of the primary amines in chitosan and the molar concentration of PEG chains.
3.1.1 Synthesis using PEGDA with an Mw of 10 kDa Initial attempts to synthesize acrylated chitosan were performed by conjugating it to PEGDA with an Mw of 10 kDa. According to previous publications (Almany and Seliktar, 2005; Davidovich-Pinhas and Bianco-Peled, 2011; Lutolf and Hubbell, 2003), it is beneficial to use large amounts of PEGDA to prevent attachment of the two vinyl groups carried by one PEGDA molecule to two amine groups on the same chitosan molecules, a situation that will create “loops” and consume the free acrylate groups required for mucoadhesion. Further, a large molar excess of PEGDA is expected to improve the rate and yield of the spontaneous reaction by providing a large amount of vinyl end groups available for the nucleophilic attack by the primary amine groups of chitosan backbone. When using PEGDA with a large Mw, achieving 11
a molar excess of the vinyl end group, however, requires very large weights of PEGDA. For example, to achieve five times the molar excess of the vinyl end group, which was previously used to synthesize acrylated alginate (Davidovich-Pinhas and Bianco-Peled, 2011), 99.6% (w/v) of PEGDA and 1% (w/v) chitosan was required, making the synthesis very expensive. Therefore, we attempted to synthesize chitosan– PEGAc(10) with lower PEGDA concentrations. As detailed in Table 1, three products with different molar ratios were prepared. The highest PEGDA concentration used was 9/1 (nine primary amine groups of chitosan to one PEGDA chain). While preparing samples for further experiments, we noticed that the polymer has very high solubility in distilled water—which can be expected, as increasing the concentration of PEGDA with a high molecular weight makes the molecule more hydrophilic. It is note that the solubility of chitosan derivatives is expected to be pH dependent and vary between body targets. We did not attempt to optimize the solubility however the high solubility made this molecule irrelevant for mucoadhesion studies since it dissolved in the medium immediately. Further, very soluble molecules are not suitable for drug release applications since they lack the ability to sustain diffusion rates. We, therefore, focused on a molar ratio of 47/1, in which one PEGDA chain is used for every 47 primary amine groups. This ratio produced a polymer from which tablets that are stable in an aqueous medium could be prepared. 1.1.2 Synthesis using PEGDA with an Mw of 0.7 kDa. Given that it was impossible to achieve a molar excess with PEGDA with an Mw of 10 kDa, we also used a shorter PEGDA with an MW of 0.7 kDa. 6.6% (w/v) of PEGDA with a molar mass of 0.7 kDa is needed to achieve a molar ratio of 1:2, i.e., two PEGDA chains for each primary amine group of chitosan, and 13.4% (w/v) for a 12
molar ratio of 1:4. Preliminary experiments have shown that the final product obtained for both molar ratios, chitosan–PEGAc(0.7), can be used to form tablets that are stable in a buffer, making it a good candidate for preparing drug delivery vehicles.
3.2 Characterization of acrylated chitosan 3.2.1 Product verification by NMR The molecular structure of the starting material, chitosan, and the resulted product chitosan–PEGAc were analyzed using NMR spectra. The typical peaks of chitosan are seen in the range of δ=3 to δ=4 ppm (Figure 2 (c)). The desired product, chitosan– PEGAc, contains several new types of protons (Figure 2 (a)): the PEG repeating unit (methylene) protons, located at δ=4.3 and at δ=3.6 ppm and the vinyl end group protons, located at δ=5.9-6.5 ppm (Dust, Fang, & Harris, 1990). Previously studied mucoadhesive systems based on acrylated polymers also display similar peaks (Davidovich-Pinhas and Bianco-Peled, 2011, 2010). A new peak attributed to the methylene protons adjacent to the 2-amino group of the glucose–amine residue was observed at δ=2.88 ppm. The peak located at δ=4.87 ppm is ascribed to water protons. Similar results were presented in another study that examined photo-polymerized chitosan with PEGDA grafts (Ma et al., 2009). An interesting observation is that a singlet at δ=3.1 ppm, which is assigned to primary amino group protons, totally disappeared on the chitosan–PEGAc(0.7) product (Figure 2 B(a)), suggesting that the reaction had a high conversion rate in this case.
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3.2.2 Quantitative determination of acrylate groups The NMR results described above suggest that higher conversion occurred when using PEGDA with an MW of 0.7 kDa. In order to quantify the acrylation degree, we developed a new procedure based on reaction of ninhydrin with the primary amino group of chitosan to form a colored reaction product that can be detected by spectroscopy (Curotto and Aros, 1993; Prochazkova et al., 1999). The full derivation of the procedure is detailed in the supplementary materials and only the final result is given below. When solutions of chitosan are reacted with ninhydrin, and the absorbance of each solution after reaction is measured and plotted against the chitosan concentration, a straight line is obtained. Denoting the slope of the adsorption vs. concentration curve for non-modified chitosan as acs, the slope of the adsorption vs. concentration curve for acrylated chitosan as apol, and defining the acrylation percentage as (1)
acrylation percentage= NPEG/ NGlcN*100,
where NPEG is the number of chitosan residues carrying PEG chains, and NGlcN is the number of GlcN chitosan residues carrying a primary amine, one gets (2)
acrylation percentage=(1-apol/acs)*100.
Using Eq. (2), we found that the acrylation percentage of chitosan–PEGAc(0.7) with a molar ratio of 1:4 is 98% while that of chitosan–PEGAc(10) was only 30% acrylated. These results are in line with the qualitative NMR observations, suggesting that the shorter PEGDA is much more efficient in this synthesis. The higher efficiency could be due to the large molar excess of acrylate groups, which ensure that acrylate end groups are available for the reaction. The acrylation percentage of chitosan– PEGAc(0.7) with a molar ratio of 1:2 was 45%, indicating that it is less grafted
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compared to the molecule resulting from the synthesis carried out with a molar ratio of 1:4.
4.2.
Mucoadhesion assessment
3.2.3 Tensile strength Mucoadhesion was first characterized by a tensile study. Figure 3A demonstrates that the maximum detachment strength (MDS) of chitosan– PEGAc(10) is significantly higher than that of the thiolated chitosan (chitosan–TBA) (p<0.01), which in turn is significantly higher than that of chitosan (p<0.01). The improved mucoadhesion ability of chitosan–TBA versus chitosan was previously demonstrated by Bernkop-Schnurch et al. (Bernkop-Schnürch et al., 2003). The new polymer, chitosan–PEGAc(10), presents further improvement in mucoadhesive properties compared to the other two polymers. The improved adhesion can be attributed to the unsaturated functional group carried by PEGAc, which increases the probability of creating covalent bonds between the polymer and the mucin on the tissue. These results are in line with previous acrylated polymers that displayed improved adhesion capability. For example, Davidovich-Pinhas and Bianco-Peled (Davidovich-Pinhas and Bianco-Peled, 2010) characterized the adhesion of PEGDA to a fresh small intestine and compared it to thiolated alginate by tensile study. The maximum detachment force (MDF) obtained with PEGDA was of the same order of magnitude as the one obtained with thiolated alginate. In another study by Davidovich-Pinhas and Bianco-Peled (Davidovich-Pinhas and Bianco-Peled, 2011), the adhesion ability of alginate–PEGAc tablets was assessed by the same method and compared to non-modified alginate and thiolated alginate. The MDF of alginate– PEGAc was found to be significantly higher compared to both other polymers. 15
Surprisingly, although chitosan–PEGAc(0.7) has a high degree of acrylation, its MDS value was not significantly different from that of chitosan (p<0.5), whereas chitosan– PEGAc(10) with only 30% acrylation is five times more adhesive than chitosan. The high adhesion of chitosan–PEGAc(10) could be visually observed in a photograph taken during the detachment of the polymeric tablet from the tissue; see Figure 3B. There are two possible explanations for why the mucoadhesion properties of chitosan–PEGAc(0.7) are not improved compared to chitosan, despite the high degree of acrylation. First, the PEG chains are very densely grafted onto these molecules. It is known that a high grafting density leads to a brush-like conformation of the chains (Luckham, 1991; Panyukov et al., 2009). When the PEG chains adopt this conformation, steric effects are increased, hence decreasing the probability that acrylate groups will interact with the cysteine residues on the mucous surface. To further investigate the influence of grafting density we measured the MDS value of a less brunched version of chitosan–PEGAc(0.7) obtained from a synthesis with molar ratio of 1/2 (45% acrylation percentage). As can be seen in figure 3A, the MDS value of chitosan–PEGAc(0.7) with ½ 45% acrylation was not significantly different from that of chitosan–PEGAc(0.7) with 98% acrylation percentage. These results suggest that the density of the grafted PEG chains is not playing a major role in the interaction with the mucous surface. A more
probable reason for the relatively low
mucoadhesion is that the PEG chains might be too short to interact with the mucosa. Short chains are less likely to penetrate into the mucous layer; therefore, the number of cysteine residues with which they interact is smaller. Furthermore, physical entanglements between PEG and mucin chains, which could also contribute to the strength of the adhesives bond, are less pronounced when short chains are involved. A study that examined various aspects of polymer bioadhesion found that the adhesive 16
strength increases as the molecular weight of the polymer increases. Moreover, a critical macromolecular length necessary to produce an interpenetrating layer and entanglements was detected (Peppas and Buri, 1985). Another study that investigated the interaction of porcine gastric mucin with poly (acrylic acid) (PAA) reported a similar lack of interaction of low Mw PAA while high Mw PAA lead to more pronounced interaction (Albarkah et al., 2015)
3.2.4 Rotating system To be of practical use as mucoadhesive drug delivery vehicles, the polymer must survive and function in a hydrated environment. We, therefore, also assessed mucoadhesion using a rotating system. Table 2 summarizes the retention time of dry compressed polymer tablets on porcine small intestinal mucous. Due to technical limitations, it was not possible to observe the tablets for more than 6 h. We found that the detachment time of both chitosan–PEGAc(10) and chitosan–TBA from porcine small intestinal mucous was higher than this limit. The corresponding detachment time of chitosan was 1.1 h±0.2. Chitosan–PEGAc(0.7) disintegrated in the medium after only one minute, probably due to the high grafting density of short PEG chains, which on one hand are too short to form a network, and on the other hand, may interrupt the entanglements of chitosan and make it less stable in the physiological medium. Based on these results, it can be concluded that the mucoadhesion of both chitosan–PEGAc(10) and chitosan–TBA is larger than that of chitosan. These findings are in line with those of the tensile studies. However, the maximum observation time limitation does not allow us to conclude whether chitosan or chitosan–TBA is more efficient in this case. Qualitative support for the enhanced mucoadhesion of chitosan– PEGAc(10) compared to chitosan can be seen in Figure 5, which shows a photograph 17
of chitosan and chitosan–PEGAc(10) tablets after 30 minutes of experiment. It is apparent that the chitosan tablet retained its original shape and size while the chitosan–PEGAc(10) tablet integrated with the tissue, possibly indicating better interactions with the tissue’s mucous layer. Since the tablets of chitosan–PEGAc(10) preserve their original form and demonstrate high adhesion in a hydrated environment, this polymer can be tablets or particles. In order to consider the use of this material as hydrogel or paste, it is important to investigate the mucin/polymer interactions in wet conditions as will be described in the next chapter
3.3 Assessment of mucin/polymer interaction Rheology measurements were performed to provide information on the type of the interactions between mucin glycoproteins and chitosan–PEGAc. Synergistic increase in viscosity can be observed in Figure 6a, where the viscosity of the chitosan–PEGAc(10)/mucin mixture is higher than the additive viscosity of these components separately. On the other hand, as can be seen in Figure 6b, the viscosity of the chitosan–PEGAc(0.7)/mucin mixture is similar to mucin’s viscosity, which means that the synergistic effect is not observed in this case. Previous studies have shown a viscosity increase after mixing mucin with mucoadhesive polymer solutions as a result of molecular interaction (Hassan and Gallo, 1990; Horvát et al., 2015; Rossi et al., 2001). Thus, these observations provide additional evidence of the superiority of chitosan–PEGAc(10) over chitosan–PEGAc(0.7). It is noted that the viscosity of chitosan–PEGAc is pH dependent since the solubility of the chitosan backbone depends on pH. However, both chitosan–PEGAc /mucin mixtures have 18
similar pH values therefore changes is viscosity can be attributed to different degrees of mucoadhesiveness.
It can be also seen from Figure 5 that the viscosity of mucin solutions decreases when the shear rate increases, indicating that mucin displays shear-thinning behavior. On the contrary, the studied acrylated chitosan solutions display an almost Newtonian behavior, as apparent from the very small dependence of the viscosity on the shear rate (Figure 5). This behavior is typical of non-entangled polymer solutions (Jiang and Zukoski, 2012; Tomioka and Matsumura, 1987) and could indicate that the selected concentration is below the critical concentration of polymer entanglements (Caramella et al., 1989; Rossi et al., 2001). The volume of the polymeric chain of chitosan– PEGAc(10) in the solution is larger than that of chitosan–PEGAc(0.7), leading to increased viscosity. As for mucin-acrylated chitosan mixtures, while the chitosan– PEGAc(10)/mucin mixture displayed pronounced shear-thinning behavior (Figure 5a), the viscosity of chitosan–PEGAc(0.7)/mucin is almost Newtonian (Figure 5b). In case of chitosan–PEGAc(10), the high weight concentration and the high molecular weight increases the molecular volume of the polymer/mucin complex hence leading to shear-thinning behavior.
Another interesting phenomenon is the differences in the viscosities of the polymer/mucin mixtures at high shear rates. While the ultimate viscosity of chitosan– PEGAc(0.7)/mucin is similar to that of mucin, the viscosity of the chitosan– PEGAc(10)/mucin mixture is higher than that of mucin. To quantitatively determine the high shear rate viscosity, the flow curves were fitted to a Cross model using Eq. 3 (Cross, 1965; Macosko, 1994; Picout & Ross-Murphy, 2003) 19
(3)
𝜂 = 𝜂∞ +
𝜂0 −𝜂∞ 1+(𝜆𝛾)̇1−𝑛
,
where 𝜂0 is the low shear rate viscosity, 𝜂∞ is the high shear rate viscosity, 𝜆 is time constant related to the relaxation time of the polymer in solution and n is the power law flow behavior index. The most often used equation to set low and high shear rate viscosity is Cheng-Evans equation (Bonferoni et al., 1992; Caramella et al., 1989; Rossi et al., 2000), however our results fit better to Cross model which is used here at the first in the context of mucoadhesion. Fitting was performed using a non-linear regression analysis in Excel. Equation parameters (𝜂0 , 𝜂∞ ) and their coefficients of variations were estimated and the goodness-of-fit was assessed by least squares analysis. Table 3 summarizes the Cross model best-fit parameters. It can be seen that the high shear rate viscosity of the mucin solution and of the chitosan– PEGAc(0.7)/mucin mixture is zero, while the high shear rate viscosity of the chitosan–PEGAc(10)/mucin mixture is higher. At high shear rates, molecular chain alignments and disentanglements occur since the rate of destruction of entanglements is higher than the formation of new entanglements, leading to orientation of polymer chains in the direction of the flow and decreasing the viscosity (Menchicchi et al., 2014; Tomioka and Matsumura, 1987). It is likely that polymer/mucin complexation is based both on physical and chemical interactions. A high shear rate may be capable of breaking physical interactions and disentangling the polymer; covalent bonds, however, are much more difficult to destroy. The viscosity of the chitosan– PEGAc(10)/mucin mixture decreases dramatically but still stays high even at high shear rates, which may be indicative of strong chemical bonds between the mixture’s components. It is important to stress out that chain penetration into the mucus layer effect both tensile and rotating cylinder results. In rheology measurements, on the other hand, only the interactions between mucin glycoproteins and chitosan–PEGAc 20
play a role. Therefore these measurements provide means to isolate the effect of chain-chain interactions and provide a clear indication it occurs through strong bond such as covalent bond. From the practical point of view, the high mucoadhesion ability of the completely hydrated chitosan–PEGAc(10) used in rheology studies emphasize that future application of this polymer as hydrogel or paste can also be considered.
It is noted that covalent bonds are permanent and irreversible, however the mucosal clearance time ranges from few minutes to few hours (Lai et al., 2009; Pepić and Lovrić, 2013) and the polymer is expected to be removed from the body with the mucosa.
5. Conclusions A novel mucoadhesive polymer was designed and synthesized by conjugating chitosan with PEGDA through its primary amino group. Two types of PEGDA with different molecular weights were used for the synthesis: PEGDA with an Mw of 10 kDa was performed to obtain Chitosan–PEGAc(10), and PEGDA with an Mw of 0.7 kDa was performed to obtain Chitosan–PEGAc(0.7). NMR results and quantitative determination using ninhydrin reactions showed that higher conversion occurred when using PEGDA with an Mw of 0.7 kDa due to the large molar excess of PEGDA, which ensures that acrylate end groups are available for the reaction. Mucoadhesion was characterized directly by tensile study and rotating system. Chitosan–PEGAc(10) presented improvement in mucoadhesive properties that can be attributed to the unsaturated functional group carried by PEGAc, which increases the probability of 21
creating covalent bonds between the polymer and the mucin on the tissue. Rheology measurements detected a synergistic increase in viscosity in the chitosan–PEGAc(10) solution upon mucin addition, a behavior that was not observed when mixing mucin with
the
chitosan–PEGAc(0.7)
solution.
The
viscosity
of
the
chitosan–
PEGAc(10)/mucin mixture decreased dramatically but remained high even at high shear rates, which may be also indicative of strong chemical bonds between the mixture’s components. This result supports our hypothesis that chitosan–PEGAc(10) interacts in a better way with mucin glycoproteins than chitosan or chitosan– PEGAc(0.7). The new polymer described here shows strong potential to act as a delivery vehicle to mucus-covered tissues due to its ability to enhance bioadhesion by covalent interaction with mucous glycoproteins under physiologic conditions.
6. Acknowledgement The authors wish to thank Mrs. Shany Otmazgin for her help with the rotating cylinder system. This research was partial supported by the Ministry of Science Technology & Space, Israel (grant 3-77111).
22
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26
Figure caption
Figure 1 – Synthesis of chitosan–PEGAc
27
1
Figure 2 – H NMR spectra of 15 mg/ml. A: (a) chitosan–PEGAc(10) (b) PEGDA 10 kDa and (c) chitosan; and B: (a) chitosan–PEGAc(0.7) (b) PEGDA 0.7 kDa and (c) chitosan at room temperature in CD3COOD.
28
Figure 3 – A: MDS of the dry compressed polymer tablets from fresh intestine surfaces. B: Pulling a chitosan–PEGAc(10) tablet from fresh small intestine surface during a tensile test. (**) refers to statistically significant difference (p<0.01).
29
Figure 4 – Chitosan (A) and chitosan–PEGAc(10) (B) samples on porcine small intestinal mucous after 30 minutes of experiment.
30
Figure 5 – Viscosity vs. shear rate for (A) chitosan–PEGAc(10), mucin (5%) and their mixture and (B) chitosan–PEGAc(0.7), mucin (5%) and their mixture in 0.1 M PBS at 37°C. Symbols: (♦) chitosan–PEGAc/mucin mixture, (■) mucin and (▲) chitosan– PEGAc. Dashed line represents summation of the viscosity of chitosan–PEGAc and that of mucin (5%).
31
Table 1 – Weight and mole concentrations of PEGDA (10 kDa) and PEGDA (0.7 kDa) chosen for the synthesis, which led to different mole ratios. Weight conc. of PEGDA Molar conc. of PEGDA Molar [mg/ml]
[mol/ml]
amine/PEGDA
10
1.00E-06
47
50
5.00E-06
9
66.1
9.44E-05
0.5
134
1.91E-04
0.25
ratio
PEGDA 10 kDa
PEGDA 0.7 kDa
32
Table 1 – Retention time of dry compressed polymer samples on porcine small intestinal mucous. Mucoadhesion studies were performed in 0.1 M PBS pH 7.4 at 37°C. Indicated values are means ±SD (n≥4). Polymer
Retention time [h]
Chitosan
1.1±0.2
Chitosan–TBA
>6
Chitosan–PEGAc(10)
>6
Chitosan–PEGAc(0.7)
0.01±0.005
33
Table 2 – Non-linear regression parameters for the Cross model of 5% mucin and of polymer/mucin mixtures as calculated by the Cross model. Sample Mucin 5%
𝜂0 [𝑃𝑎 ∗ 𝑠] 0.046
𝜂∞ [𝑃𝑎 ∗ 𝑠]
𝜆
n
R2
0
0.215
0.618
0.011
Chitosan–PEGAc(0.7)/mucin 0.02
0
0.027
0.414
0.009
Chitosan–PEGAc(10)/mucin
0.02
0.139
0.024
0.001
0.087
34
S0378-5173(16)31165-6 http://dx.doi.org/doi:10.1016/j.ijpharm.2016.12.023 IJP 16294
To appear in:
International Journal of Pharmaceutics
Received date: Revised date: Accepted date:
25-7-2016 13-11-2016 10-12-2016
Please cite this article as: Shitrit, Yulia, Bianco-Peled, Havazelet, Acrylated Chitosan for Mucoadhesive Drug Delivery Systems.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2016.12.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Acrylated Chitosan for Mucoadhesive Drug
Delivery Systems
Yulia Shitrit and Havazelet Bianco-Peled*
Department of Chemical Engineering, Technion – Israel Institute of Technology,
Haifa 32000, Israel
*Corresponding author: Professor Havazelet Bianco-Peled, Department of Chemical Engineering, Technion – Israel Institute of Technology, Haifa 32000, Israel. Tel.
+972-4-829-3588, Fax. +972-4-829-5672, email [email protected]
1
Graphical abstract
Abstract A new mucoadhesive polymer was synthesized by conjugating chitosan to poly(ethylene glycol)diacrylate (PEGDA) via the Michael type reaction. The product was characterized using NMR. Higher PEGDA grafting efficacy was observed with low molecular weight PEGDA (0.7 kDa), compared to long 10 kDa PEGDA. The acrylation percentage was calculated based on the reaction of ninhydrin with chitosan, and supported the qualitative NMR findings. The adhesive properties were studied by tensile test and rotating system involving detachment of polymer tablets from a fresh intestine sample. Chitosan modified with high molecular weight PEGDA presented improvement in mucoadhesive properties compared to both non-modified and thiolated chitosan. On the molecular level, rheology measurements of polymer/mucin mixtures provided additional evidence of strong interaction between modified chitosan and mucin glycoproteins. This new polymer shows promise as a useful polymeric carrier matrix for delivery systems, which could provide prolonged residence time of the vehicle on the mucosa surface. 2
Keywords Chitosan; PEGDA (poly(ethylene glycol) diacrylate); mucoadhesion 1.
2. Introduction The mucous surface is the first barrier that interacts with nutrients and drugs before they can be absorbed by diffusion and reach their target organs (Bansil and Turner, 2006; Cone, 2009). Over the last three decades, researchers have been studying mucoadhesion due to its potential to optimize localized drug delivery. This delivery method utilizes vehicles capable of attaching to mucosal surfaces, hence providing prolonged residence time of drugs at the site of application. It possesses several advantages over oral administration. First, the uptake of a drug into the systemic circulation through the permeable mucus membranes is relatively rapid due to the massive blood supply and sufficient blood flow rate at the mucosal surface. Furthermore, transmucosal delivery helps to avoid drug degradation by some of the body’s natural defense mechanisms thus enhancing bioavailability. In addition, the intimate contact with the absorption site can provide a passive drug uptake by a high concentration gradient leading to faster diffusion (Alexander et al., 2011; BernkopSchnürch, 2005). Mucoadhesive materials can be in forms of tablets, patches, tapes, films, semisolids, powders, in situ gelling systems and nanoparticales (Cook and Khutoryanskiy, 2015; Sosnik et al., 2014). Combining mucoadhesive properties with other polymeric drug carriers offering the advantages of, for example, sustained release rate, protection of pharmaceuticals from hydrolysis and chemical or enzymatic 3
degradation, reduction of drug toxicity, and improvement of drug solubility and availability, has opened the way for the design of powerful non-invasive drug delivery vehicles. The most common group of mucoadhesive polymers are hydrophilic materials that can create weak non-covalent bonds such as hydrogen bonds, Van-Der Waals forces, ionic interactions and chain entanglements with the mucosa (Andrews et al., 2009; Lee et al., 2000). A more recent development has produced modified materials with functional groups capable of forming strong bonds with mucous components. Thiolated polymers, also termed thiomers, were first developed by Leitner et al. (Leitner et al., 2003). They carry a thiol functional group capable of forming disulphide bridges with cysteine-rich subdomains of mucin glycoproteins. Thiolated polymers have been widely explored in the last decade. They are easy to synthesize, and many studies demonstrated that thiomer has a better adhesion ability than the nonmodified polymer (Bernkop-Schnürch, 2003; Kafedjiiski, Krauland, Hoffer, & Bernkop-Schnürch, 2005; Rahmat et al., 2011, 2012). Davidovich-Pinhas and BiancoPeled (Davidovich-Pinhas and Bianco-Peled, 2010) suggested a novel mucoadhesive system based on polymers carrying an acrylate side group. In this approach, a polymer carrying an acrylate end group associates with the sulfide end group of the mucin type glycoprotein by the Michael type addition reaction. The thiol residue on the mucin glycoprotein backbone, acting as a strong nucleophile, attacks the double bond, a covalent bond is formed and hydroxyl is released. To actualize this concept, a novel biomaterial termed alginate-PEGAc, comprising alginate backbone and PEG-acrylate grafts, was developed and investigated. This polymer displayed a significant increase in the adhesion strength compared to both the non-modified alginate and the thiolated alginate (Davidovich-Pinhas and Bianco-Peled, 2011). 4
In the present study, we expand the knowledge on acrylate-based mucoadhesion polymers by creating an additional polymer and investigating its interactions with mucous. The new mucoadhesive polymer was synthesized from chitosan (CS), a nontoxic, biocompatible and biodegradable natural polysaccharide (No, 2002; Rinaudo, 2006), conjugated to poly(ethylene glycol)diacrylate (PEGDA) via the Michael type reaction.. The synthesized product was verified and characterized using Nuclear magnetic resonance (NMR). The adhesive properties were studied in a semi-hydrated environment by tensile tests and in a hydrated environment using the rotating cylinder method. On the molecular level, rheology measurements of polymer/mucin mixtures provided additional evidence of strong interaction between the synthesized polymer and mucin glycoproteins.
3. Materials and methods 3.1.
Materials
Poly(ethylene glycol) diacrylate (PEGDA) with a molecular weight (Mw) of 10 kDa was purchased from the laboratory of Prof. Seliktar in the Department of Biomedical Engineering, Technion, Israel. PEGDA with an Mw of 0.7 kDa, mucin from porcine stomach, Type II (PGM) and low molecular weight chitosan (CS) were obtained from Sigma-Aldrich MO, USA. The degree of deacetylation (DDA) of CS was determined using Fourier transform infrared (FTIR) spectroscopy and found to be 77.6% (Delmar and Bianco-Peled, 2015). Weight averaged Mw was measured by static light scattering and found to be 207 kDa (Delmar and Bianco-Peled, 2015). Porcine small intestine was purchased from the Pre-Clinical Research Authority, Technion, cut 5
open, spread on a petri dish and stored until further use at -20°C. Acetic acid (99.7% A.R.) was purchased from Gadot Biochemical Industries Ltd., Israel. Sodium hydroxide (NaOH) pearls and 32% hydrochloric acid solution (HCl) were purchased from Bio-Lab Ltd., Israel. 2-Iminothiolane was purchased from Proteo-Chem, Denver, CO, USA. Sodium chloride (NaCl) and dimethyl sulfoxide (DMSO) were purchased from Merck KGaA, Darmstadt, Germany. 3.2.
Synthesis of mucoadhesive polymers
3.2.1. Acrylated chitosan synthesis The synthesis of acrylated chitosan (Chitosan-PEGAc) was accomplished according to a previously published procedure (Ma et al., 2009) with slight modifications. Acrylation of chitosan was performed using PEGDA with an MW of 10 kDa, which produced acrylated chitosan with long PEG chains (chitosan–PEGAc(10)), or PEGDA with an MW of 0.7 kDa, which produced acrylated chitosan with short PEG chains (chitosan–PEGAc(0.7)). Both acrylated chitosan versions were prepared.The amine:PEG ratio was used as the experimental variable since it affects the PEG grafting density and therefore the molecule’s properties. The calculated molar ratios summarized in Table 1 refer to the ratio between the molar concentration of the primary amines in chitosan and the molar concentration of PEG chains. An exemplary calculation can be found in the supplementary materials.
First, chitosan powder was dissolved in 2% (v/v) acetic acid in a dark glass bottle in a total concentration of 1% (w/v) and mixed for 24 h to ensure complete dissolution of the polymer. Then, PEGDA was added to the mixture to obtain the desired molar ratio detailed in Table 1, and the mixture was stirred for an additional 15 min to give a homogenic solution. The bottle was placed in a water bath at 60oC and kept for 3 hr 6
under constant shaking at 100 rpm. To remove unreacted PEGDA residues, the solution was dialized using a cellulose tube having a 12–14 kDa molecular weight cutoff against dionized water for 48 hr. The water was changed at least three times during this period. The final product was freezed for 24 hours at -20oC
and
lyophilized by drying the frozen aqueous polymer solution at -30oC and 0.01 mbar and stored at 4oC until needed for further use.
3.2.2. Synthesis of thiolated chitosan (chitosan-TBA) For the sake of comparison, a thiolated chitosan, which is a known mucoadhesive polymer that forms covalent bonds with mucins, was synthesized according to previously published procedures (Bernkop-Schnürch, 2003). In brief, 1 g of chitosan powder was dissolved in 1L of 2% (v/v) acetic acid. 0.4 g of 2-iminothiolane was added to the solution while adjusting the pH to 6.3 using 1M NaOH, and then mixed for 24 h. To remove unreacted thiol residues, the product was dialyzed for three days using a cellulose tube having a 12–14 kDa molecular weight cutoff against 5 mM HCl, two times against 5 mM HCl containing 1% NaCl, once against 5 mM HCl, and once against 1 mM HCl. The final sample was freezed for 24 hours at -20oC and lyophilized by drying the frozen aqueous polymer solution at -30oC and 0.01 mbar and stored at 4oC until needed for further use. 3.3.
Tablet preparation
Samples for assessment of mucoadhesion properties were prepared by compressing 100 mg of a dry polymer sample into a 11 mm diameter plate disc using pressure of 2 ton applied on a compression molding device. The polymers used were chitosan, chitosan-PEGAc and chitosan-TBA.
7
3.4.
Polymer characterisation
3.4.1. Nuclear magnetic resonance (NMR) 1
H NMR (400 MHz) spectra were recorded on a Bruker-ARX 400 instrument.
Samples used for the NMR experiments to verify the desired product were performed by preparing a 1% (w/v) polymer solution in CD3COOD at room temperature the night before the measurements, to ensure complete dissolution. 3.4.2. Ninhydrin test for quantitative determination of acrylate groups A ninhydrin reagent was prepared by dissolving 2% (w/v) of ninhydrin in dimethyl sulfoxide (DMSO) in a dark bottle. A set of polymer solutions was prepared by dissolving studied polymer at concentrations of 0.1-0.6% (w/v) in a mixture of 3% (v/v) acetic acid and 1% (v/v) HCl and mixing for 24 hr. 0.1 ml of each solution was mixed with 0.25 ml of 4M phosphate buffer (PB) (pH=5.4) and 1 ml of ninhydrin solution. The final mixtures were incubated in a 100°C bath and shaken at a rate of 60 rpm for 30 min. 0.2 ml from each mixture were analyzed using a Bio-Tek Inc. spectrophotometer at a wavelength of 500 nm. Curves were plotted using the average of at least three independent measurements (n ≥ 3). 3.5.
Adhesion assays
3.5.1. Tensile assays Tensile assays were performed using a technique previously developed for soft substrates (Davidovich-Pinhas and Bianco-Peled, 2010). Adhesion assays were performed by attaching a fresh small intestine sample to a 25 mm stainless steel grid connected to a vacuum system, which attaches the lower surface of the tissue to the grid without the need to use glue. Since the upper surface of the tissue was not exposed to the vacuum, the naturally occurring mucus surface was not dehydrated. This 25 mm grid was then fixed to the lower arm of a Lloyed Tensile machine equipped with a 50-N load cell. The hydration of the intestine surface was 8
standardized by pouring 20 µl of double distilled water (DDW) onto the substrate surface prior to the measurement. It should be noted that intestine washing is avoided in order to preserve the mucus layer. A compressed polymer tablet (11 mm in diameter) was attached to the upper arm of the tensile machine using double-sided adhesive tape. The upper arm carrying the tablet was lowered at 5 mm/min until a force of 0.1 N was recorded between the polymer tablet and the substrate. The force was maintained for 10 minutes. In order to measure the maximum adhesion force, the machine was set to an extension mode where the upper arm was pulled at a constant rate of 0.01 mm/sec and the force was recorded until detachment was observed. The substrate was replaced before each test. The reported values of maximal detachment stress (MDS), calculated by dividing the recorded maximum detachment force by the tablet area, are an average of at least five independent measurements (n ≥ 5) for each substrate and polymer pair. Statistical data analysis is performed using a standard ttest with P < 0.05 considered the minimal level of significance.
3.5.2. Rotating system A homemade system based on the principles of a rotating cylinder ( BernkopSchnürch & Steininger, 2000) was used to measure the time required to detach a dosage form from the mucous surface in simulated intestinal fluid (pH=7.4). A piece of porcine intestine was fixed on a Mylar sheet using cyanoacrylate glue. Dry compressed polymer tablets were attached to the surface using mild pressure. The Mylar carrying the tablets samples was attached to a Styrofoam float and placed in a beaker containing 0.1 M phosphate buffer saline (PBS, pH=7.4) at 37°C. The solution was stirred at 600 rpm using a magnetic stirrer, which led to rotation of the float. The
9
detachment of the tablets was monitored. The reported detachment time is the average of at least four independent measurements (n ≥ 4). 3.6.
Rheology
Rheological measurements were carried out using a TA Instruments’ AR-G2 rheometer (New Castle, DE, USA) equipped with a Peltier plate temperature controlled base. The experiments were performed using a parallel plate geometry having a 40 mm radius at a constant temperature of 37°C. 2% (w/v) polymer stock solutions in 0.1M HCl (pH 1) were prepared and stored at room temperature for 24 h, to ensure complete dissolution. 10% (w/v) mucin stock solution in PB pH=7.4 was prepared on the day of measurement to ensure its freshness. 1 ml mucin stock solution was mixed together with 1 ml polymer stock solution; the final composition of the resulting mixture was 5mg/ml mucin and 10 mg/ml polymer and it pH was 4.8. The mucin solution and polymer solution were mixed together at an appropriate ratio to a final polymer:mucin weight ratio of 1:5 and stirred vigorously for 1 h prior to analysis. Measurements were performed in triplicate. Shear rates were varied from 10 to 300 s−1. Following Rossi et al. (Rossi et al., 2001), we used a 1:5 (w/w) polymer:mucin weight ratio for rheology measurements.
4. Results and Discussion 4.1.
Synthesis of chitosan–PEGAc
In order to broaden the range of available covalently associating polymers, we explored a new polymer, chitosan–PEGAc. Conjugating the chitosan’s amine to PEGDA was achieved by Michael type reaction, which is a well-known process to add various amines onto unsaturated carbonyl compounds. Since amines can act as 10
both nucleophiles and bases, in the case of chitosan no additional base is typically needed, makings this reaction a simple and spontaneous one with no further materials needed to be removed after it (Sashiwa et al., 2003). The primary amine on the chitosan backbone acts as a nucleophile and attacks the double bond on the PEGDA. Having two vinyl end groups on the PEGDA enables, on the one hand, the Michael reaction between chitosan and PEGDA, and on the other hand, the final product contains another free vinyl end group for further interaction with the mucous glycoproteins (Figure 1). The calculation of the molar ratios is detailed in the supplementary materials; the calculated molar ratios summarized in Table 1 refer to the ratio between the molar concentration of the primary amines in chitosan and the molar concentration of PEG chains.
3.1.1 Synthesis using PEGDA with an Mw of 10 kDa Initial attempts to synthesize acrylated chitosan were performed by conjugating it to PEGDA with an Mw of 10 kDa. According to previous publications (Almany and Seliktar, 2005; Davidovich-Pinhas and Bianco-Peled, 2011; Lutolf and Hubbell, 2003), it is beneficial to use large amounts of PEGDA to prevent attachment of the two vinyl groups carried by one PEGDA molecule to two amine groups on the same chitosan molecules, a situation that will create “loops” and consume the free acrylate groups required for mucoadhesion. Further, a large molar excess of PEGDA is expected to improve the rate and yield of the spontaneous reaction by providing a large amount of vinyl end groups available for the nucleophilic attack by the primary amine groups of chitosan backbone. When using PEGDA with a large Mw, achieving 11
a molar excess of the vinyl end group, however, requires very large weights of PEGDA. For example, to achieve five times the molar excess of the vinyl end group, which was previously used to synthesize acrylated alginate (Davidovich-Pinhas and Bianco-Peled, 2011), 99.6% (w/v) of PEGDA and 1% (w/v) chitosan was required, making the synthesis very expensive. Therefore, we attempted to synthesize chitosan– PEGAc(10) with lower PEGDA concentrations. As detailed in Table 1, three products with different molar ratios were prepared. The highest PEGDA concentration used was 9/1 (nine primary amine groups of chitosan to one PEGDA chain). While preparing samples for further experiments, we noticed that the polymer has very high solubility in distilled water—which can be expected, as increasing the concentration of PEGDA with a high molecular weight makes the molecule more hydrophilic. It is note that the solubility of chitosan derivatives is expected to be pH dependent and vary between body targets. We did not attempt to optimize the solubility however the high solubility made this molecule irrelevant for mucoadhesion studies since it dissolved in the medium immediately. Further, very soluble molecules are not suitable for drug release applications since they lack the ability to sustain diffusion rates. We, therefore, focused on a molar ratio of 47/1, in which one PEGDA chain is used for every 47 primary amine groups. This ratio produced a polymer from which tablets that are stable in an aqueous medium could be prepared. 1.1.2 Synthesis using PEGDA with an Mw of 0.7 kDa. Given that it was impossible to achieve a molar excess with PEGDA with an Mw of 10 kDa, we also used a shorter PEGDA with an MW of 0.7 kDa. 6.6% (w/v) of PEGDA with a molar mass of 0.7 kDa is needed to achieve a molar ratio of 1:2, i.e., two PEGDA chains for each primary amine group of chitosan, and 13.4% (w/v) for a 12
molar ratio of 1:4. Preliminary experiments have shown that the final product obtained for both molar ratios, chitosan–PEGAc(0.7), can be used to form tablets that are stable in a buffer, making it a good candidate for preparing drug delivery vehicles.
3.2 Characterization of acrylated chitosan 3.2.1 Product verification by NMR The molecular structure of the starting material, chitosan, and the resulted product chitosan–PEGAc were analyzed using NMR spectra. The typical peaks of chitosan are seen in the range of δ=3 to δ=4 ppm (Figure 2 (c)). The desired product, chitosan– PEGAc, contains several new types of protons (Figure 2 (a)): the PEG repeating unit (methylene) protons, located at δ=4.3 and at δ=3.6 ppm and the vinyl end group protons, located at δ=5.9-6.5 ppm (Dust, Fang, & Harris, 1990). Previously studied mucoadhesive systems based on acrylated polymers also display similar peaks (Davidovich-Pinhas and Bianco-Peled, 2011, 2010). A new peak attributed to the methylene protons adjacent to the 2-amino group of the glucose–amine residue was observed at δ=2.88 ppm. The peak located at δ=4.87 ppm is ascribed to water protons. Similar results were presented in another study that examined photo-polymerized chitosan with PEGDA grafts (Ma et al., 2009). An interesting observation is that a singlet at δ=3.1 ppm, which is assigned to primary amino group protons, totally disappeared on the chitosan–PEGAc(0.7) product (Figure 2 B(a)), suggesting that the reaction had a high conversion rate in this case.
13
3.2.2 Quantitative determination of acrylate groups The NMR results described above suggest that higher conversion occurred when using PEGDA with an MW of 0.7 kDa. In order to quantify the acrylation degree, we developed a new procedure based on reaction of ninhydrin with the primary amino group of chitosan to form a colored reaction product that can be detected by spectroscopy (Curotto and Aros, 1993; Prochazkova et al., 1999). The full derivation of the procedure is detailed in the supplementary materials and only the final result is given below. When solutions of chitosan are reacted with ninhydrin, and the absorbance of each solution after reaction is measured and plotted against the chitosan concentration, a straight line is obtained. Denoting the slope of the adsorption vs. concentration curve for non-modified chitosan as acs, the slope of the adsorption vs. concentration curve for acrylated chitosan as apol, and defining the acrylation percentage as (1)
acrylation percentage= NPEG/ NGlcN*100,
where NPEG is the number of chitosan residues carrying PEG chains, and NGlcN is the number of GlcN chitosan residues carrying a primary amine, one gets (2)
acrylation percentage=(1-apol/acs)*100.
Using Eq. (2), we found that the acrylation percentage of chitosan–PEGAc(0.7) with a molar ratio of 1:4 is 98% while that of chitosan–PEGAc(10) was only 30% acrylated. These results are in line with the qualitative NMR observations, suggesting that the shorter PEGDA is much more efficient in this synthesis. The higher efficiency could be due to the large molar excess of acrylate groups, which ensure that acrylate end groups are available for the reaction. The acrylation percentage of chitosan– PEGAc(0.7) with a molar ratio of 1:2 was 45%, indicating that it is less grafted
14
compared to the molecule resulting from the synthesis carried out with a molar ratio of 1:4.
4.2.
Mucoadhesion assessment
3.2.3 Tensile strength Mucoadhesion was first characterized by a tensile study. Figure 3A demonstrates that the maximum detachment strength (MDS) of chitosan– PEGAc(10) is significantly higher than that of the thiolated chitosan (chitosan–TBA) (p<0.01), which in turn is significantly higher than that of chitosan (p<0.01). The improved mucoadhesion ability of chitosan–TBA versus chitosan was previously demonstrated by Bernkop-Schnurch et al. (Bernkop-Schnürch et al., 2003). The new polymer, chitosan–PEGAc(10), presents further improvement in mucoadhesive properties compared to the other two polymers. The improved adhesion can be attributed to the unsaturated functional group carried by PEGAc, which increases the probability of creating covalent bonds between the polymer and the mucin on the tissue. These results are in line with previous acrylated polymers that displayed improved adhesion capability. For example, Davidovich-Pinhas and Bianco-Peled (Davidovich-Pinhas and Bianco-Peled, 2010) characterized the adhesion of PEGDA to a fresh small intestine and compared it to thiolated alginate by tensile study. The maximum detachment force (MDF) obtained with PEGDA was of the same order of magnitude as the one obtained with thiolated alginate. In another study by Davidovich-Pinhas and Bianco-Peled (Davidovich-Pinhas and Bianco-Peled, 2011), the adhesion ability of alginate–PEGAc tablets was assessed by the same method and compared to non-modified alginate and thiolated alginate. The MDF of alginate– PEGAc was found to be significantly higher compared to both other polymers. 15
Surprisingly, although chitosan–PEGAc(0.7) has a high degree of acrylation, its MDS value was not significantly different from that of chitosan (p<0.5), whereas chitosan– PEGAc(10) with only 30% acrylation is five times more adhesive than chitosan. The high adhesion of chitosan–PEGAc(10) could be visually observed in a photograph taken during the detachment of the polymeric tablet from the tissue; see Figure 3B. There are two possible explanations for why the mucoadhesion properties of chitosan–PEGAc(0.7) are not improved compared to chitosan, despite the high degree of acrylation. First, the PEG chains are very densely grafted onto these molecules. It is known that a high grafting density leads to a brush-like conformation of the chains (Luckham, 1991; Panyukov et al., 2009). When the PEG chains adopt this conformation, steric effects are increased, hence decreasing the probability that acrylate groups will interact with the cysteine residues on the mucous surface. To further investigate the influence of grafting density we measured the MDS value of a less brunched version of chitosan–PEGAc(0.7) obtained from a synthesis with molar ratio of 1/2 (45% acrylation percentage). As can be seen in figure 3A, the MDS value of chitosan–PEGAc(0.7) with ½ 45% acrylation was not significantly different from that of chitosan–PEGAc(0.7) with 98% acrylation percentage. These results suggest that the density of the grafted PEG chains is not playing a major role in the interaction with the mucous surface. A more
probable reason for the relatively low
mucoadhesion is that the PEG chains might be too short to interact with the mucosa. Short chains are less likely to penetrate into the mucous layer; therefore, the number of cysteine residues with which they interact is smaller. Furthermore, physical entanglements between PEG and mucin chains, which could also contribute to the strength of the adhesives bond, are less pronounced when short chains are involved. A study that examined various aspects of polymer bioadhesion found that the adhesive 16
strength increases as the molecular weight of the polymer increases. Moreover, a critical macromolecular length necessary to produce an interpenetrating layer and entanglements was detected (Peppas and Buri, 1985). Another study that investigated the interaction of porcine gastric mucin with poly (acrylic acid) (PAA) reported a similar lack of interaction of low Mw PAA while high Mw PAA lead to more pronounced interaction (Albarkah et al., 2015)
3.2.4 Rotating system To be of practical use as mucoadhesive drug delivery vehicles, the polymer must survive and function in a hydrated environment. We, therefore, also assessed mucoadhesion using a rotating system. Table 2 summarizes the retention time of dry compressed polymer tablets on porcine small intestinal mucous. Due to technical limitations, it was not possible to observe the tablets for more than 6 h. We found that the detachment time of both chitosan–PEGAc(10) and chitosan–TBA from porcine small intestinal mucous was higher than this limit. The corresponding detachment time of chitosan was 1.1 h±0.2. Chitosan–PEGAc(0.7) disintegrated in the medium after only one minute, probably due to the high grafting density of short PEG chains, which on one hand are too short to form a network, and on the other hand, may interrupt the entanglements of chitosan and make it less stable in the physiological medium. Based on these results, it can be concluded that the mucoadhesion of both chitosan–PEGAc(10) and chitosan–TBA is larger than that of chitosan. These findings are in line with those of the tensile studies. However, the maximum observation time limitation does not allow us to conclude whether chitosan or chitosan–TBA is more efficient in this case. Qualitative support for the enhanced mucoadhesion of chitosan– PEGAc(10) compared to chitosan can be seen in Figure 5, which shows a photograph 17
of chitosan and chitosan–PEGAc(10) tablets after 30 minutes of experiment. It is apparent that the chitosan tablet retained its original shape and size while the chitosan–PEGAc(10) tablet integrated with the tissue, possibly indicating better interactions with the tissue’s mucous layer. Since the tablets of chitosan–PEGAc(10) preserve their original form and demonstrate high adhesion in a hydrated environment, this polymer can be tablets or particles. In order to consider the use of this material as hydrogel or paste, it is important to investigate the mucin/polymer interactions in wet conditions as will be described in the next chapter
3.3 Assessment of mucin/polymer interaction Rheology measurements were performed to provide information on the type of the interactions between mucin glycoproteins and chitosan–PEGAc. Synergistic increase in viscosity can be observed in Figure 6a, where the viscosity of the chitosan–PEGAc(10)/mucin mixture is higher than the additive viscosity of these components separately. On the other hand, as can be seen in Figure 6b, the viscosity of the chitosan–PEGAc(0.7)/mucin mixture is similar to mucin’s viscosity, which means that the synergistic effect is not observed in this case. Previous studies have shown a viscosity increase after mixing mucin with mucoadhesive polymer solutions as a result of molecular interaction (Hassan and Gallo, 1990; Horvát et al., 2015; Rossi et al., 2001). Thus, these observations provide additional evidence of the superiority of chitosan–PEGAc(10) over chitosan–PEGAc(0.7). It is noted that the viscosity of chitosan–PEGAc is pH dependent since the solubility of the chitosan backbone depends on pH. However, both chitosan–PEGAc /mucin mixtures have 18
similar pH values therefore changes is viscosity can be attributed to different degrees of mucoadhesiveness.
It can be also seen from Figure 5 that the viscosity of mucin solutions decreases when the shear rate increases, indicating that mucin displays shear-thinning behavior. On the contrary, the studied acrylated chitosan solutions display an almost Newtonian behavior, as apparent from the very small dependence of the viscosity on the shear rate (Figure 5). This behavior is typical of non-entangled polymer solutions (Jiang and Zukoski, 2012; Tomioka and Matsumura, 1987) and could indicate that the selected concentration is below the critical concentration of polymer entanglements (Caramella et al., 1989; Rossi et al., 2001). The volume of the polymeric chain of chitosan– PEGAc(10) in the solution is larger than that of chitosan–PEGAc(0.7), leading to increased viscosity. As for mucin-acrylated chitosan mixtures, while the chitosan– PEGAc(10)/mucin mixture displayed pronounced shear-thinning behavior (Figure 5a), the viscosity of chitosan–PEGAc(0.7)/mucin is almost Newtonian (Figure 5b). In case of chitosan–PEGAc(10), the high weight concentration and the high molecular weight increases the molecular volume of the polymer/mucin complex hence leading to shear-thinning behavior.
Another interesting phenomenon is the differences in the viscosities of the polymer/mucin mixtures at high shear rates. While the ultimate viscosity of chitosan– PEGAc(0.7)/mucin is similar to that of mucin, the viscosity of the chitosan– PEGAc(10)/mucin mixture is higher than that of mucin. To quantitatively determine the high shear rate viscosity, the flow curves were fitted to a Cross model using Eq. 3 (Cross, 1965; Macosko, 1994; Picout & Ross-Murphy, 2003) 19
(3)
𝜂 = 𝜂∞ +
𝜂0 −𝜂∞ 1+(𝜆𝛾)̇1−𝑛
,
where 𝜂0 is the low shear rate viscosity, 𝜂∞ is the high shear rate viscosity, 𝜆 is time constant related to the relaxation time of the polymer in solution and n is the power law flow behavior index. The most often used equation to set low and high shear rate viscosity is Cheng-Evans equation (Bonferoni et al., 1992; Caramella et al., 1989; Rossi et al., 2000), however our results fit better to Cross model which is used here at the first in the context of mucoadhesion. Fitting was performed using a non-linear regression analysis in Excel. Equation parameters (𝜂0 , 𝜂∞ ) and their coefficients of variations were estimated and the goodness-of-fit was assessed by least squares analysis. Table 3 summarizes the Cross model best-fit parameters. It can be seen that the high shear rate viscosity of the mucin solution and of the chitosan– PEGAc(0.7)/mucin mixture is zero, while the high shear rate viscosity of the chitosan–PEGAc(10)/mucin mixture is higher. At high shear rates, molecular chain alignments and disentanglements occur since the rate of destruction of entanglements is higher than the formation of new entanglements, leading to orientation of polymer chains in the direction of the flow and decreasing the viscosity (Menchicchi et al., 2014; Tomioka and Matsumura, 1987). It is likely that polymer/mucin complexation is based both on physical and chemical interactions. A high shear rate may be capable of breaking physical interactions and disentangling the polymer; covalent bonds, however, are much more difficult to destroy. The viscosity of the chitosan– PEGAc(10)/mucin mixture decreases dramatically but still stays high even at high shear rates, which may be indicative of strong chemical bonds between the mixture’s components. It is important to stress out that chain penetration into the mucus layer effect both tensile and rotating cylinder results. In rheology measurements, on the other hand, only the interactions between mucin glycoproteins and chitosan–PEGAc 20
play a role. Therefore these measurements provide means to isolate the effect of chain-chain interactions and provide a clear indication it occurs through strong bond such as covalent bond. From the practical point of view, the high mucoadhesion ability of the completely hydrated chitosan–PEGAc(10) used in rheology studies emphasize that future application of this polymer as hydrogel or paste can also be considered.
It is noted that covalent bonds are permanent and irreversible, however the mucosal clearance time ranges from few minutes to few hours (Lai et al., 2009; Pepić and Lovrić, 2013) and the polymer is expected to be removed from the body with the mucosa.
5. Conclusions A novel mucoadhesive polymer was designed and synthesized by conjugating chitosan with PEGDA through its primary amino group. Two types of PEGDA with different molecular weights were used for the synthesis: PEGDA with an Mw of 10 kDa was performed to obtain Chitosan–PEGAc(10), and PEGDA with an Mw of 0.7 kDa was performed to obtain Chitosan–PEGAc(0.7). NMR results and quantitative determination using ninhydrin reactions showed that higher conversion occurred when using PEGDA with an Mw of 0.7 kDa due to the large molar excess of PEGDA, which ensures that acrylate end groups are available for the reaction. Mucoadhesion was characterized directly by tensile study and rotating system. Chitosan–PEGAc(10) presented improvement in mucoadhesive properties that can be attributed to the unsaturated functional group carried by PEGAc, which increases the probability of 21
creating covalent bonds between the polymer and the mucin on the tissue. Rheology measurements detected a synergistic increase in viscosity in the chitosan–PEGAc(10) solution upon mucin addition, a behavior that was not observed when mixing mucin with
the
chitosan–PEGAc(0.7)
solution.
The
viscosity
of
the
chitosan–
PEGAc(10)/mucin mixture decreased dramatically but remained high even at high shear rates, which may be also indicative of strong chemical bonds between the mixture’s components. This result supports our hypothesis that chitosan–PEGAc(10) interacts in a better way with mucin glycoproteins than chitosan or chitosan– PEGAc(0.7). The new polymer described here shows strong potential to act as a delivery vehicle to mucus-covered tissues due to its ability to enhance bioadhesion by covalent interaction with mucous glycoproteins under physiologic conditions.
6. Acknowledgement The authors wish to thank Mrs. Shany Otmazgin for her help with the rotating cylinder system. This research was partial supported by the Ministry of Science Technology & Space, Israel (grant 3-77111).
22
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Figure caption
Figure 1 – Synthesis of chitosan–PEGAc
27
1
Figure 2 – H NMR spectra of 15 mg/ml. A: (a) chitosan–PEGAc(10) (b) PEGDA 10 kDa and (c) chitosan; and B: (a) chitosan–PEGAc(0.7) (b) PEGDA 0.7 kDa and (c) chitosan at room temperature in CD3COOD.
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Figure 3 – A: MDS of the dry compressed polymer tablets from fresh intestine surfaces. B: Pulling a chitosan–PEGAc(10) tablet from fresh small intestine surface during a tensile test. (**) refers to statistically significant difference (p<0.01).
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Figure 4 – Chitosan (A) and chitosan–PEGAc(10) (B) samples on porcine small intestinal mucous after 30 minutes of experiment.
30
Figure 5 – Viscosity vs. shear rate for (A) chitosan–PEGAc(10), mucin (5%) and their mixture and (B) chitosan–PEGAc(0.7), mucin (5%) and their mixture in 0.1 M PBS at 37°C. Symbols: (♦) chitosan–PEGAc/mucin mixture, (■) mucin and (▲) chitosan– PEGAc. Dashed line represents summation of the viscosity of chitosan–PEGAc and that of mucin (5%).
31
Table 1 – Weight and mole concentrations of PEGDA (10 kDa) and PEGDA (0.7 kDa) chosen for the synthesis, which led to different mole ratios. Weight conc. of PEGDA Molar conc. of PEGDA Molar [mg/ml]
[mol/ml]
amine/PEGDA
10
1.00E-06
47
50
5.00E-06
9
66.1
9.44E-05
0.5
134
1.91E-04
0.25
ratio
PEGDA 10 kDa
PEGDA 0.7 kDa
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Table 1 – Retention time of dry compressed polymer samples on porcine small intestinal mucous. Mucoadhesion studies were performed in 0.1 M PBS pH 7.4 at 37°C. Indicated values are means ±SD (n≥4). Polymer
Retention time [h]
Chitosan
1.1±0.2
Chitosan–TBA
>6
Chitosan–PEGAc(10)
>6
Chitosan–PEGAc(0.7)
0.01±0.005
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Table 2 – Non-linear regression parameters for the Cross model of 5% mucin and of polymer/mucin mixtures as calculated by the Cross model. Sample Mucin 5%
𝜂0 [𝑃𝑎 ∗ 𝑠] 0.046
𝜂∞ [𝑃𝑎 ∗ 𝑠]
𝜆
n
R2
0
0.215
0.618
0.011
Chitosan–PEGAc(0.7)/mucin 0.02
0
0.027
0.414
0.009
Chitosan–PEGAc(10)/mucin
0.02
0.139
0.024
0.001
0.087
34