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Development of a novel pH sensitive silane crosslinked injectable hydrogel for controlled release of neomycin sulfate
Accepted Manuscript Title: Development of a novel pH sensitive silane crosslinked injectable hydrogel for controlled release of neomycin sulfate Author: Sehrish Jabeen Atif Islam Abdul Ghaffar Nafisa Gull Ayesha Hameed Anbreen Bashir Tahir Jamil Tousif Hussain PII: DOI: Reference:
S0141-8130(16)31681-6 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.01.014 BIOMAC 6920
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
18-9-2016 29-12-2016 3-1-2017
Please cite this article as: Sehrish Jabeen, Atif Islam, Abdul Ghaffar, Nafisa Gull, Ayesha Hameed, Anbreen Bashir, Tahir Jamil, Tousif Hussain, Development
of a novel pH sensitive silane crosslinked injectable hydrogel for controlled release of neomycin sulfate, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2017.01.014 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.
Development of a novel pH sensitive silane crosslinked injectable hydrogel for controlled release of neomycin sulfate Sehrish Jabeena,b, Atif Islamb, Abdul Ghaffara,b, Nafisa Gullb, Ayesha Hameedb, Anbreen Bashira,b, Tahir Jamilb, Tousif Hussainc a
Department of Chemistry, University of Engineering and Technology, Lahore, Pakistan.
b
Department of Polymer Engineering and Technology, University of the Punjab, Lahore, Pakistan.
c
Center for Advanced Studies in Physics (CASP), Government College University, Lahore,
Pakistan.
Corresponding author: A Islam; Email: [email protected], +092-300-6686-506
Graphical abstract
Highlights
Biopolymers based injectable hydrogels were fabricated by solution casting method
Fabricated hydrogels were analyzed by FTIR, TGA, SEM and antimicrobial properties
Swelling studies in different solvent media were also analyzed
Release study of NMS loaded hydrogel in SIF and SGF solutions were examined
NMS was released (83%) in a controlled manner confirming it as injectable hydrogel
Abstract Silane crosslinked biopolymer based novel pH-responsive hydrogels were fabricated by blending the cationic (chitosan) and anionic (alginate) polymers with poly (vinyl alcohol). Tetraethoxysilane (TEOS) was used, as a crosslinker in different amounts due to its nonhazardous nature, to study its impact on physical and chemical properties of the prepared injectable hydrogels along with the controlled release of drug. The swelling response of the prepared hydrogels was examined in different solvent media which exhibited decreased swelling ratio with increase in the amount of TEOS. All the fabricated hydrogels represented highest swelling at acidic pH while low swelling at basic and neutral pH. This specific pH sensitive behavior at pH 7 made them an appropriate
candidate for the injectable controlled drug delivery in which Neomycin Sulfate (NMS) was successfully loaded on suitable hydrogel (comprising 50 μL TEOS) to study its release mechanism. The results revealed that in simulated gastric fluid (SGF), hydrogel released the entire drug (NMS) in initial 30 min while in simulated intestinal fluid (SIF), NMS was released in a controlled way up to 83 % in 80 min. These results endorsed that the hydrogels could be practiced as a smart intelligent material for injectable controlled drug delivery as well as for other biomedical applications at physiological pH. Key Words: Chitosan; Alginate; Injectable hydrogels; Neomycin sulfate; Tetraethoxysilane; Swelling. 1.
Introduction
Hydrogels are hydrophilic, 3D-crosslinked polymeric network and superabsorbent when placed in water or other physiological fluids due to their swelling ability (Ju et al., 2001). Crosslinking in hydrogels occurred through physical interactions or covalent bonding. Hydrophilic groups like OH, -NH2, -COOH, etc. are responsible to generate hydrophilicity in hydrogels. In swollen form due to their softness and rubber like structure, hydrogels appear like a living tissue. Hydrogels are prone to sense a minor change in external stimuli like (pH, temperature, ionic concentration, electric and magnetic field) by displaying variation in swelling response, mechanical properties and network structure (Qu et al., 2000). pH-responsive smart hydrogels are one of the most premium type of hydrogels that has been widely used in medicine for controlled/targeted drug release. The significance of pH-responsive hydrogels is due to their performance in different pH of internal body fluids and organs (Gao et al., 2012). These hydrogels are mainly based on polymers with ionic pendant groups distributed along the back bones of such polymers. These pendant ionic groups are responsible for the pH-sensitivity
of the hydrogels. Hydrogels based on natural/biopolymers are ecofriendly, biodegradable, biocompatible, nontoxic, superabsorbent, hydrophilic, cost effective and have ability to expand making it favorable to be used in biomedical, cosmetics, biotechnology mainly for drug delivery and agricultural applications (Chen & Park, 2000; Pappas et al., 2000; Ghaffar, et al., 2011). Among biopolymers, the polysaccharides (cellulose, agar, pectin, carrageenan and alginic acid) are the most abundant and renewable natural polymers and are neutral or acidic in nature. Chitosan is a cationic biopolymer acquired through the alkaline N-deacetylation of naturally occurring chitin. Chitin is an important constituent of crustaceans, insects and fungal mycelia consists of 2acetamido-2-deoxy-β-D-glucose attached through a β (1,4) linkage (Muzzarelli, 1973). Glucosamine are the major units in chitosan having one primary amino and two free hydroxyl groups (Yang et al., 2016). The degree of N-deacetylation is the main factor which decides its properties (Zikakis, 1984). Chitosan is a biocompatible (Shigemasa & Minami, 1996), biodegradable (Dong, 2001), antifungal, antibacterial (Payne et al., 1992) and having gel forming properties (Ma et al., 2010), so, readily processable to prepare thin films, hydrogels, membranes, beads, nanofibers, etc. Chitosan and its derivatives have wide applications in different fields including: pharmaceutics, biotechnology, drug delivery, textile, food, cosmetics etc. (Gorochovceva & Makuška, 2004; Hu et al., 2005; Wang et al., 2008; Ito, Yoshida & Murakami, 2013; Li and Huang, 2012; Hu et al., 2011). Alginic acid (alginate) is a linear anionic polysaccharide found mainly in the cell wall of brown algae. Alginic acid is a copolymer composed of two types of uronic acid; 1,4-linked β-Dmannuronic acid and α-L-guluronic acid units which form three kinds of polymer segments of blocks like M block, G block and MG block which may place randomly or as consecutively (Atkins et al., 1970; Kim et al., 1990). The viscosity of alginate solution depends on temperature
and pH. At pH 5 to 11, the thickness of alginate gel remains unaffected. It gives fine gel at pH 4 to 5 but as pH drops from 6 to 2, precipitation will occur while the viscosity of the alginate solution drops with the rise in temperature. Alginate is also a biocompatible and has an outstanding functionality as a stabilizer, emulsifier, gelling and thickening agent. Nowadays, based on these characteristic and remarkable properties, alginate is useful for many kinds of food, cosmetic, textile, pharmaceutical and other biomedical applications (McDowell, 1969; Atkin et al., 1971). Natural polymers based hydrogels have poor mechanical strength due to low crystallinity and amorphous structure but many other factors are also involved like; hydrophilic nature, crosslinking density, degree of swelling, polymerization conditions, etc. Generally, with increasing the concentration of crosslinker, polymer chains come closer to each other, thus reducing the diffusivity and swelling behaviour and hence improved mechanical properties. Beyond the optimum concentration of crosslinker, the number of crosslinks is increased, the polymers become less elastic (polymer chain segments are constrained in their movement as they are hooked) resulting in a brittle structure (Chippada, 2010; Maitra & Singh, 2014; Maitra & Shukla, 2014). Furthermore, to enhance the mechanical properties of natural polymer based hydrogel, these are blended with synthetic polymers which are excellent in mechanical properties and thermal stability (Xu et al., 2010). PVA is a commonly used synthetic polymer with excellent mechanical properties and extensively used in the preparation of films (Chifiriuc et al., 2012; Grumezescu et al., 2013). PVA based hydrogels and membranes have excellent characteristics like non-carcinogenicity, biocompatibility, nontoxicity, high swelling in aqueous solutions and desirable physical and thermal properties (McNeely & Kovacs, 1975; Torres et al., 2013). PVA is also used for the preparation of skin replacement materials, contact lenses, reconstruction of vocal cord (Gao et al., 2012), replacement of artificial cartilage, and hydrogels for biomedical applications (Liang et al.,
2009) such as wound healing/wound dressing (Rasool et al., 2010), drug delivery, membranes form for reverse osmosis/waste water treatment, removal of heavy metals, controlled release of agricultural fertilizers and coating materials for industrial applications (Islam et al., 2011). To develop the coordination among natural and synthetic polymers, different crosslinking agents are available like: formaldehyde, acetaldehyde, glutaraldehyde, TEOS, etc. But TEOS is a preferred crosslinker because silane crosslinkers are mainly suitable to form inorganic crosslinks in the amorphous zone of polymers in order to deliver the covalent bonding between inorganic content and polymer chains, usually used in biomaterials and other applications. It is easy to bind via condensation reactions and nontoxic in comparison with formerly reported crosslinkers (glutaraldehyde, epichlorhydrine, borate and tripolyphosphate) (Jena & Raju, 2008; Liang et al., 2009; Islam et al., 2011). The main issue in the conventional drug release systems is that mostly they are not site oriented or targeted and release the drug in bulk amount, unable to maintain its concentration. So, a diminute amount is absorbed by the body and the rest is excreted without any change. Thus, to deal with such drugs which are not easily absorbed by the body, it is necessary to use alternative ways to maintain drug dosage in actual range otherwise the given prescription becomes useless for the patients who is unable to achieve the desired results. For this issue, the injectable hydrogels are more attractive for releasing drug in a controlled way with optimum/required delivery periods. These are also more suitable for biomedical applications as they can lessen patient distress, infection risk and reduce operation cost of hydrogel implantation (Hou et al., 2004; Lee & Tae, 2007). The most commonly used polymers for the fabrication of injectable hydrogel are cellulose, carboxymethyl cellulose, poly(vinyl pyrollidone), PVA, chitosan, poly(ethylene glycol), etc. (Hoare & Kohane, 2008; Kadajji & Betageri, 2011; James et al., 2014).
Neomycin sulfate (NMS), is a water soluble aminoglycoside. It is an antibiotic obtained through fermentation of Streptomyces fradiae. It is basic in nature and thermally stable compound. It retains its effectiveness in aqueous solution and other vehicles compared to other antibiotics. It also retards the growth of Gram-positive and Gram-negative bacteria (Livingood et al., 1952; Adams et al., 1996; Clarot et al., 2005). In this study, cationic and anionic polymers are blended with PVA and crosslinked via TEOS to develop a novel pH-responsive hydrogel for controlled drug release. The swelling trend of the prepared hydrogels is evaluated in different solvent media. All the hydrogels represented the highest swelling at acidic pH while low at basic and at pH 7. This specific pH-sensitive behavior at pH 4 recommended them as injectable controlled drug delivery system. Hydrogel with the best response is preferred to attain the objective of this research. NMS is an aminoglycoside antibiotic, chosen as a model drug and its released mechanism is evaluated in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). The prepared NMS loaded hydrogel showed release in a controlled way in SIF solution. The amount of NMS release is examined through UV-Vis spectroscopy. 2.
Materials and methods
2.1
Materials
Chitosan (Mw = 17103.41 g/mol, degree of deacetylation (DDA) = 90.28%; viscosity = 200 cp) was received from Biolog (GmbH) Trademark. Alginate (Mw = 60275 g/mol) extra pure food grade, poly (vinyl alcohol) (Mw = 72,000 g/mol; D-85662; degree of hydrolyzation ≥ 98 %), acetic acid (Mw = 60.05 g/mol (100%)) extra pure, absolute ethanol and glacial acetic acid were purchased from Merck, Germany. TEOS (98.5 %) was purchased from DAEJUNG chemical & metals Co. Ltd., Korea. Hydrochloric acid (37%) was purchased from Labscan Asia, co. Ltd.,
Thialand. NaCl and CaCl2 were purchased from sigma Aldrich St. Louis, Missouri, United States. NMS (ATTC B05CA09) was obtained locally and used as a model drug. E-coli gram negative bacteria (ATTC 25922), yeast extract and tryptone were also used. Fluorescein diacetate, dimethyl sulphoxide, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium chloride and potassium chloride were obtained from sigma Aldrich. HeLa cell lines were purchased from ATTC. All chemicals were of analytical grade and used without any purification. 2.2
Fabrication of hydrogels
Chitosan (0.60 g) was dissolved in 50 mL of 2 % acetic acid solution with continuous stirring on a hot plate at 50 ˚C. Alginate (0.20 g) was soaked overnight in 25 mL distilled water in a separate beaker and magnetically stirred for 1 h at room temperature. The prepared chitosan and alginate solutions were blended and stirred for 1 h at 50 ˚C. Preheated distilled water (25 mL) at 80 ˚C was taken and 0.2 g of PVA was added in it and stirred until it dissolved. This PVA solution was added in previously made chitosan-alginate solution and stirred for 1 h at 50 ˚C (referred as CPA). 25 µL (0.5 drop) TEOS was dissolved in 5 mL ethanol having one drop of 0.1 M HCl solution and added drop wise in CPA solution at 50 ˚C during continuous stirring on a hot plate for 2 h. This blended solution was poured on a glass petri dish and dried at room temperature. After complete drying, it was peeled off to get dried hydrogel and finally it was vacuum dried at 60 ˚C for 6 h in drying oven (LVO- 2040, Lab Tech, Korea). The dried hydrogel was stored in polythene bags in a desiccator. CPA was the control hydrogel contained chitosan-PVA-alginate without crosslinker while CPA1, CPA2 and CPA3 hydrogels have 25 µL (0.5 drop), 50 µL (1 drop), and 75 µL (1.5 drop) TEOS (silanol) along with the same amount of chitosan, PVA and alginate, respectively. 3.
Characterization
3.1
Fourier transform infrared (FTIR) analysis
FTIR spectra of chitosan, PVA, alginate and all CPA hydrogels were taken on Shimadzu IRPrestige-21, Kyoto, Kyoto Prefecture, Japan, with attenuated total reflectance (ATR) mode at a resolution of 4 cm-1 with programmed range of 4000-400 cm-1. 3.2
Thermogravimetric analysis (TGA)
TGA was carried on TA instrument SDT Model Q600; TA Instrument, New Castle, DE, USA, with nitrogen flow of (15 mL/min). The temperature ramp was kept at 20 ˚C/min from ambient temperature to 600 °C. 3.3
Scanning electron microscopy (SEM)
The surface morphology of hydrogels was determined through SEM Model JEOL/EO JSM 6480 (LA) Akishima, Tokyo, Japan. The images were collected at different magnifications. 3.4
Swelling experiment
The swelling experiment was conducted in distilled water, buffer and ionic solutions by following procedure; vacuum dried hydrogels were cut into tiny fragments (25 mg), weighed and dipped in a glass vial contained 40 mL of respective solvent. After a specific time interval (20 min), the excess of external solution was discarded from vial and cleaned the vial with tissue paper and swollen weight of hydrogel sample was measured. The experiment was revised at the same time interval until the equilibrium was attained. This experiment was repeated thrice for each hydrogel to obtain mean value for more accurate results. Degree of swelling was determined by equation 1. Swelling (g/g) =
(1)
Where, Wd is the weight of dried hydrogel and Ws is the weight of swollen hydrogel at time t. Buffer solutions of different pH (2, 4, 6, 7, 8 and 10) were used to study the pH response. Ionic solutions of NaCl and CaCl2 of different concentrations (0.1, 0.3, 0.5, 0.7, 0.9 and 1M) were prepared and swelling of hydrogel samples was determined in each solution. To find the crosslinking degree of the hydrogels, the samples were cut into small pieces and known weight of hydrogel samples (0.1g) was put into a stainless steel cloth according to the ASTM 2765 (Mir et al., 2014). Soxhlet extractor was used to extract the crosslinked material in the presence of distilled water at 100 ˚C for 10 h. After extraction, the samples were dried in a drying oven until the weight become constant. The degree of crosslinking was calculated by weighing the dried crosslinked samples using equation 2. (2) Where, Wg is the weight of dried crosslinked sample and Wo is the initial weight of the sample. 3.5
Antimicrobial test
Antimicrobial activity of the hydrogels was investigated using liquid diffusion method. P.lice strains of E.Coli (gram -) were used as bacterial modal. The method involves the following steps; LB-media was prepared by taking 10 g NaCl, 5 g yeast extract and 10 g Tryptone in 800 mL distilled water and the solution was maintained at pH 7. After this, the volume of the solution was raised up to 1000 mL and autoclaved it for 1 h. To prepare the control solution, 20 mL of above prepared LB-media and 20 µL P.lice strain of E.Coli (for the growth of bacteria) were taken in a 50 mL flask and incubated at 38 ˚C for overnight. To prepare the test sample solution, 3 g of hydrogel sample was added in above mixture (20 mL LB-media and 20 µL P.lice strain of E.Coli) and same
procedure was repeated. On the next day, the absorbance of control and test sample solutions at 600 nm was taken from UV visible spectrophotometer, Labomed, Inc. UVD-3500, USA.
3.6
In Vitro Cytotoxicity/Cell Viability
HeLa cell lines were used for cell culturing and cytotoxity analysis. The culture was maintained under standard conditions (incubated at 37 ˚C under 5 % CO2 and 95 % air). The cell viability was assessed with fluorescein diacetate (FDA) staining. 10 μL of 2 mg/mL of FDA stock in dimethyl sulfoxide (DMSO) was diluted in 5 mL of phosphate buffer saline (PBS) and 1 mL of this solution was used to stain each sample in 12 well plates. The cells were incubated for 5-10 min and washed with PBS. Positively stained cells were observed under fluorescent microscope (Nikon TS 100) for green fluorescent filter. The surface of the plate was coated with 100 μL of each hydrogel solution, air dried and then UV sterilized. The cultured cells were seeded in 10,000 cells per cm2 density and cultured over night to ensure proper cell attachment and incubated again for 48 h. After incubation, the media (used for cell culturing) was removed, the cells were washed with PBS, and neutral red media was added and incubated again for 2 h. After two hours, neutral red media was removed and the cells were again washed either PBS. Freshly prepared de-staining solution (49 % deionized water, 50 % absolute ethanol and 1 % glacial acetic acid) was added and culture was incubated for 10 min and its optical density was taken at 570 nm on UV visible spectrophotometer, Labomed, Inc. UVD-3500, USA. 3.7
Controlled drug (NMS) delivery analysis
3.7.1
Preparation of simulated solutions
Simulated gastric fluid (SGF) of pH 1.2 was prepared by taking 1 g NaCl in 3.5 mL of HCl and diluted up to 500 mL with distilled water.
Simulated intestinal fluid (SIF) of pH 6.8 was prepared by adding 0.2 M solution of 250 mL of KH2PO4 and 0.2 M solution of 118 mL of NaOH (Islam et al., 2013). 3.7.2
Drug loading procedure
To prepare the drug loaded hydrogel samples, NMS (100 mg for each blend) in 25 mL of distilled water was loaded into two separate blends of chitosan (0.60 g in 50 mL of 2 % acetic acid solution), PVA (0.20 g in 25 mL of distilled water) and alginate (0.20 g in 25 mL of distilled water) and stirred for 1 h at 50 ˚C. 50 μL (1 drop) of TEOS was added in each of these solutions and stirred for 3 h. These drug loaded blend solution were poured on glass petri dishes and dried at room temperature. After complete drying, these were peeled off to get the dried hydrogels and finally vacuum dried at 60 ˚C for 3 h in drying oven (LVO- 2040, Lab Tech, Korea). The vacuum dried drug loaded hydrogels (1.245 g each) were placed separately in SGF and SIF (100 mL each) solutions at 37 ˚C. After every 10 min, 5 mL solution was collected using a syringe from each SGF and SIF solution having drug loaded hydrogel. Fresh solutions (5 mL) of SGF and SIF were added back every time to each beaker of SGF and SIF solution to keep the volume up to 100 mL. Subsequently, the samples were collected for next 2 h from SGF solution and 3 h from SIF solution. The amount of released NMS by drug loaded hydrogels was estimated spectrophotometrically at 550 nm by UV visible spectrophotometer, Labomed, Inc. UVD-3500, USA. The standard drug solution (100 ppm of NMS drug in SGF and SIF) was taken as reference for UV visible analysis. 4.
Results and discussion
The proposed interactions (covalent and hydrogen bonding) of silane crosslinked blends of CS, PVA and alginate having hydrogel properties are presented in scheme 1. These properties especially the pH sensitivity could be used for the controlled/targeted release of drugs. 4.1
FTIR analysis
FTIR spectroscopy was conducted to investigate the functional groups of pure chitosan, PVA, alginate, CPA (control), and crosslinked CPA1, CPA2 and CPA3 hydrogels as shown in Figure 1. These spectra confirmed the presence of incorporated components. The bands of amide I and amide III were observed at 1642 and 1245 cm-1, respectively while the band of amide II was observed at 1556 cm-1 (Islam et al., 2012). Amide IV and amide V bands were appeared at 600 cm-1 and 660 cm,-1 respectively. A band due to pyranose ring was observed at 898 cm-1 which confirmed the existence of chitosan and alginate in the samples (Islam et al., 2011). Sodium alginate showed the characteristic absorption bands at 1414 and 1543 cm-1 due to carboxyl anions. The C–O stretching bands of C–O–C bridge were observed at 1030 cm-1 in chitosan and alginate. The absorption bands at 1424 and 1090 cm-1 were ascribed to the bending (–OH) and stretching (C–O) of pure PVA, respectively. The asymmetric stretching bands in the range of 1130-1000 cm-1 indicated the presence of both –Si–O–Si and –Si–O–C which confirmed the crosslinking via silanol groups. A broad band between 3570-3100 cm-1 showed -OH stretching of intra- and inter-molecular hydrogen bonds in the prepared hydrogels (Ma et al., 2008). A vibrational band of alkyl group (–CH stretching) was observed at 2921 cm-1. The silanol (–Si–OH) stretching vibration was observed at 3725 cm-1 (Kamoun et al., 2015). 4.2
Thermogravimetric analysis
TGA was carried out to study the effect of crosslinker i.e. TEOS content on the thermal stability of the hydrogels. The weight loss (%) behavior of prepared hydrogel samples are shown in Figure 2.
TGA of pure chitosan, alginate and prepared hydrogels showed that the degradation was occurred in two differentiated steps whereas in PVA it took place in three well differentiated steps. The thermogram of PVA exhibited three regions of mass loss. After the water loss step, the decomposition occurred mainly in the second and third degradation steps that occurred at 281 and 420˚C, respectively and reflected the decomposition of pendant groups and backbone chains of PVA (83 % weight loss). At 600 ˚C, a residue of 4.0 % could be found (Santos et al., 2014). For chitosan, CPA, CPA1, CPA2 and CPA3, the first weight loss was observed at 260 °C while for alginate it was observed at 285 °C due to dehydration. For chitosan and alginate, the onset degradation was observed at 260 and 303 °C and offset was found at 349 and 362 °C, respectively. All hydrogel thermograms showed two steps degradation; first degradation was attributed to the breakdown of side chains and pendent groups while second phase was corresponded to the scission of backbone polymer chains (Hench, 1998, Buranachai et al., 2010; Honary et al., 2010). In the controlled hydrogel (CPA) having two stages for degradation, the onset of degradation started earlier having no crosslinker as CPA showed 30 % weight loss at 280 ˚C while second step was due to main degradation and offset occurred at 400 ˚C. The 30 % weight loss was increased up to 289 °C in CPA3. Whereas, 60 % weight loss was observed at 350 ˚C in CPA but with the addition of crosslinker, the same weight loss (60 %) was observed at 370 °C for CPA1 and for CPA3 it was at 410 °C. This increase in temperature (at 30 % and 60 % weight loss) confirmed the increase in thermal stability with the addition of TEOS. The higher degree of crosslinking with increase in the amount of TEOS resulted in hindrance in the chains movement. 4.3
Scanning electron microscopy
The morphology of the prepared chitosan/PVA/alginate hydrogels are shown in Figure 3. The hydrogels without crosslinker showed roughness and surface irregularities (Pieróg et al., 2012;
Kamoun et al., 2015) with the formation of visible depressions (Figure 3a and 3b) (Eldin et al., 2015) while with the addition of TEOS (CPA1), uniform and smoother surface was observed. SEM images clearly showed that the prepared hydrogels have defined porous structure (Straccia et al., 2015) as the addition of crosslinker imparted a key role in the development of pores having size ranged from 2-30 µm in CPA2. These micrographs showed that by increasing the amount of crosslinker (50 μL), number of pores with good distribution on the surface was observed (Fig. 3e and 3f). In fact, the number of –OH groups were increased with increase in the crosslinker amount instigated the development and more expansion of hydrogel network among the polymer chains. This might be the reason of more water absorption in CPA2 and make it suitable for gaseous exchange and encapsulation of biochemical agents (Rashidzade et al., 2014; Rasool et al., 2010). 4.4
Study of swelling behavior in different media
4.4.1
Swelling in distilled water
The hydrogels swell by the absorption of water through diffusion process. This diffusion occurred due to the movement between external solvent and polymer chains. The swelling behavior of the developed CPA hydrogels in distilled water with respect to time is shown in Figure 4a. The swelling performance of all the hydrogels was different, a linear and continuous increase in the swelling was seen which increased with increase in time (Islam et al., 2012). The equilibrium time for CPA, CPA1 and CPA2 was observed at 80 min and for CAP3 it was 60 min. Maximum swelling was observed in CPA2 (68 g/g) while minimum swelling was noticed for CPA3 (38 g/g). Initially, an increase in swelling with the addition of TEOS (silanol) was observed in contrary to the normal trend of decreased swelling of hydrogels with the increase in amount of crosslinker. First trend of increase in swelling was due to the generation of pores and more diffusion of water molecules as discussed in section 4.3. The number of –OH groups were increased with increase in
the crosslinker amount caused the development of hydrogel network among the polymer chains which might be the reason of more water absorption at a higher crosslinker amount (50 µL) as the swelling analysis was repeated for three times and found the same results. The swelling was decreased with the maximum amount of TEOS (75 µL) which caused to shrink the spaces in the hydrogel network by the generation of more crosslinking points and made it more tough to be swollen by water. Also, the highest amount of crosslinker (75 µL) created more compact network resulted in the reduced swelling. This trend was in accordance with the normal trend of decrease in swelling at higher crosslinker amount (Rasool et al., 2010; Rashidzadeh et al., 2014). Generally, the swelling of hydrogels depends on the diffusion of solvent from extracellular matrix to its structure. The mechanism of swelling process was determined by equation 3. F=
(3)
Where ‘k’ is the swelling rate constant, n is the swelling exponent, F is the fractional swelling estimated through swelling ratio of Wt and Weq. Where, Wt and Weq are the swelling ratio at time t and at equilibrium time (min), respectively. The swelling data of CPA hydrogels in water was used to determine the values of n and k parameters. A plot of ln(F) vs ln(t) shows in Figure 4b and values of diffusion parameter are given in Table 1. For CPA, CPA1, CPA2 and CPA3, n ˂ 0.5; corresponded to the Fickian transport which showed that the rate of diffusion was lower than the rate of relaxation. The crosslinking degree of hydrogels represented the crosslinked portion or undissolved part of the CPA blends which was observed to be increased with increase in the concentration of crosslinker. The crosslinking degree of CPA hydrogels are given in Table 1. 4.4.2
Swelling in buffer solutions
The hydrogels are sensitive to change in pH therefore the swelling medium imparted a main role in the swelling of hydrogels. A slight change in pH of external solvent disturbed the charge balance in the complex which altered the interactions of polymer chains. The effect of pH on the swelling of CPA, CPA1, CPA2 and CPA3 hydrogels in buffer media is shown in Figure 5. The hydrogels expressed maximum swelling at acidic whereas least at neutral and basic pH. To study the swelling response at different pH buffers (2, 4, 6, 7, 8, and 10), all CPA hydrogels were soaked in specific buffer solutions up to their equilibrium time. The amino (-NH2+) group of chitosan and carboxyl (-COO-) group of alginate are the two basic groups existed in the prepared hydrogel. The amino group was stronger base and have greater tendency to accept the hydrogen ions first, therefore, the control hydrogel without TEOS and CPA1 with the least amount of TEOS exhibited highest swelling at pH 4. While in CPA2 and CPA3, the swelling was decreased with the increase in crosslinker amount. In acidic buffers, higher concentration of charged entities created charge repulsion and the entrance of solvent through diffusion resulted in the increased swelling ratio (Atta et al., 2015). CPA1 and CPA2 showed maximum swelling of 32 g/g at pH 2 while the lowest values of 4 and 6 g/g were observed at pH 7, respectively. All the hydrogels displayed a steady decrease in swelling from pH 4 to pH 6. At neutral pH, carboxyl group become unprotonated as it was strong acid than amino group, so, low swelling was observed. At basic pH, due to decrease in the degree of ionization of charged entities or deprotonation of ammonium ions (-NH3+), water was released from hydrogel resulted in the shrinkage of polymer chains and hence the reduction in swelling ratio while with further increase in pH up to 10, it was remained almost the same. 4.4.3
Swelling in ionic solutions
The nature of salt and its concentration have prominent impact on the swelling response of hydrogels. Therefore, NaCl and CaCl2 were used to prepare the ionic solutions to study the swelling behaviour. The two ionic solutions have same anion (Cl-1) but different cation charges on it. The swelling of control and crosslinked hydrogels was evaluated and shown in Figure 6. The swelling was reduced with enhanced concentration of both electrolytes. A high amount of salts in the external swelling solvent generated the charge screening effect due to excessive electrolytes which ultimately reduced the developed osmotic pressure between the external solvent and hydrogel. So, the diffusion was decreased and resulted in less swelling. Maximum swelling was observed at 0.1 M CaCl2 compared to the NaCl (0.1 M). The swelling value for CPA hydrogels in 0.1 M CaCl2 was 21 g/g, whereas, 18 g/g was determined in 0.1 M solution of NaCl. At lower concentration, Ca2+ ions formed complex with polymer so pore size was increased resulted in the increased swelling ratio (Islam & Yasin, 2012). 4.5
Antimicrobial results
Antimicrobial activity of the hydrogels was investigated using liquid diffusion method and results are given in Table 2. The absorbance value of the controlled solution was observed at 1.287 but on the other hand the absorbance values of test samples were below 1.287 which confirmed their good inhibition, because, if the absorbance values of test samples were same as the controlled solution, then these were not capable of inhibition of the bacterial growth. Particularly, CPA2 is -0.025 which showed excellent bacterial inhibition properties. The test samples contained chitosan having positive charged (cationic) and interact with negatively charged bacterial cell wall, which in turn caused the disruption onto the cell wall of bacterium, penetrated in bacterial cell and retarded its growth by avoiding DNA conversion into RNA.
4.6
In Vitro Cytotoxicity Analysis
The cell culture methods, also known as cytotoxicity analysis, were used to examine the toxic nature of the hydrogels. In vitro cytotoxicity of fabricated hydrogels was carried out using HeLa cell line (model cell line) and Neutral red assay by indirect method as shown in Figure 7. This figure shows that only few cells were attached to the CPA compared to standard and had round morphology. The cells were also appeared as clusters which showed that cell to cell adhesion forces were established before cell to matrix adhesion. In CPA1, the cells were uniformly distributed compared to CPA but unlike standard very few cells were attached. In CPA2, the cells were attached to the most of the surface area of hydrogel sample. The cell viability was good with round morphology and the cells were separated due to the nontoxic behaviour of material with higher cell count compared to CPA, CPA1 and CPA3 in which cells were not evenly distributed and few in numbers. Neutral Red is a supra-vital dye and all actively growing cells uptake the Neutral red from surrounding and bind it to lysosomes of cells. This test was widely used to check the cell viability and also for cytotoxicity assays (Repetto et al. 2008). The cell viability of the prepared hydrogel samples was compared with the standard sample (cell line without hydrogel sample) and found that CPA2 hydrogel containing 50 µL crosslinker have the highest cell viability i.e. the lowest cytotoxicity with cell survival rate of approximately 60 % compared to the other developed hydrogels (Figure 8). This maximum cell growth rate was attributed to the fact that 50 µL was the optimum concentration of crosslinker for the fabrication of CPA hydrogels used for the injectable drug delivery system. These results showed the nontoxic behaviour towards cells growth which performed their normal activities and undergone their normal proliferation and life cycle. This
outcome was strongly in accordance with the antimicrobial analysis of these hydrogels discussed in section 4.5. 4.7
Release Analysis of NMS
CPA2 was loaded by NMS drug and its release (%) mechanism as a function of time was studied in SGF and SIF. The entire drug was released in half an hour in SGF but according to the US pharmacopeia standard, the release should not be more than 10 % in SGF. This was the reason that this hydrogel could not be used for oral drug administration. NMS was released in a controlled manner in SIF (Figure 9) i.e. 83 % in 80 min, which was in accordance with the US pharmacopeia standard and the left amount could not be determined because hydrogel was broken down into fragments. The developed hydrogel possessed the property of controlled drug release and could be used as intravenous (IV) medication. These consequences specified that the hydrogels could be a suitable candidate for the injectable drug delivery and other biomedical applications. 5.
Conclusions
Biopolymer based novel, pH-sensitive, crosslinked CPA hydrogels were formed by solution casting technique. CPA2 showed maximum swelling degree (68 g/g) in distilled water. The degree of swelling in distilled water was reduced with increase in the quantity of TEOS. CPA3 showed maximum thermal stability compared to the other specimens. The swelling behavior of the hydrogels for different pH showed high swelling in acidic and low in basic and neutral pH. This specific pH-responsive behavior at pH 7 recommended these hydrogels for the injectable controlled release of drug. In vitro cytotoxicity analysis exhibited the nontoxic behaviour toward cells growth executed their normal actions and experienced normal production and life cycle. The controlled release study of NMS (model drug) loaded CPA2 hydrogel exhibited that in SIF, 83% of drug was
released in a controlled manner within 80 min. This confirmed that these hydrogels could be a striking candidate for the injectable drug delivery and other biomedical applications. Acknowledgments The authors are highly thankful to Higher Education Commission of Pakistan for funding under the project (HEC-USAID) of “Development of Innovative Technical and Medical Textile Products” and Prof. Dr. Amtul Jamil Sami, Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore, Pakistan for cytotoxicity analysis.
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Figure 1. FTIR spectra of pure chitosan, PVA, alginate, CPA (control), CPA1, CPA2 and CPA3 hydrogels.
Figure 2. The thermograms of pure chitosan, PVA, alginate, CPA (control), CPA1, CPA2 and CPA3 hydrogels.
Figure 3. SEM micrographs of CPA (control) (a Å~ 250), (b Å~ 1000) CPA1 (c Å~ 90), (d Å~ 500) CPA2 (e Å~ 250) (f Å~ 1000) and CPA3 (d g~ 250) (d h~ 1000).
Figure 4(a). Time (min) vs swelling (g/g) of CPA (control), CPA1, CPA2 and CPA3 hydrogels in water at room temperature.
Figure 4(b). ln (F) vs ln (t) for CPA (control), CPA1, CPA2 and CPA3 hydrogels.
Figure 5. A plot of pH vs swelling (g/g) of CPA (control), CPA1, CPA2 and CPA3 hydrogels at different pH (2-10) of buffer solutions.
Figure 6. A plot of concentration (mol/L) vs swelling (g/g) of CPA (control), CPA1, CPA2 and CPA3 hydrogels in different molar concentration of NaCl (solid) and CaCl2 (hollow) solutions.
Figure 7. Optical micrographs obtained after 24 h incubation of prepared hydrogels and standard sample.
Figure 8. A comparison of cell viability of hydrogel samples with the standard sample.
Figure 9. Release analysis of NMS in SIF (PH 6.8) with time.
Scheme 1: The proposed interactions of chitosan, PVA and alginate via TEOS of prepared crosslinked hydrogels.
Table 1. Diffusion parameters and crosslinking degree of different CPA hydrogels. Parameters
CPA
CPA1
CPA2
CPA3
n
0.35
0.38
0.44
0.19
Intercept
-1.77
-1.82
-1.93
-1.22
k
0.170
0.162
0.145
0.295
Regression
98
97
93
87
45
71
80
87
(R %) Crosslinking degree (%)
Table 2. Absorbance data of controlled, CPA (control), CPA1, CPA2 and CPA3 hydrogel samples along with trends representing their antimicrobial behavior. Sample
Absorbance at
Trends
600 nm Controlled
1.287
----
CPA
0.211
Good
CPA1
0.004
Good
CPA2
-0.025
Excellent
CPA3
0.014
Good
S0141-8130(16)31681-6 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.01.014 BIOMAC 6920
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
18-9-2016 29-12-2016 3-1-2017
Please cite this article as: Sehrish Jabeen, Atif Islam, Abdul Ghaffar, Nafisa Gull, Ayesha Hameed, Anbreen Bashir, Tahir Jamil, Tousif Hussain, Development
of a novel pH sensitive silane crosslinked injectable hydrogel for controlled release of neomycin sulfate, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2017.01.014 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.
Development of a novel pH sensitive silane crosslinked injectable hydrogel for controlled release of neomycin sulfate Sehrish Jabeena,b, Atif Islamb, Abdul Ghaffara,b, Nafisa Gullb, Ayesha Hameedb, Anbreen Bashira,b, Tahir Jamilb, Tousif Hussainc a
Department of Chemistry, University of Engineering and Technology, Lahore, Pakistan.
b
Department of Polymer Engineering and Technology, University of the Punjab, Lahore, Pakistan.
c
Center for Advanced Studies in Physics (CASP), Government College University, Lahore,
Pakistan.
Corresponding author: A Islam; Email: [email protected], +092-300-6686-506
Graphical abstract
Highlights
Biopolymers based injectable hydrogels were fabricated by solution casting method
Fabricated hydrogels were analyzed by FTIR, TGA, SEM and antimicrobial properties
Swelling studies in different solvent media were also analyzed
Release study of NMS loaded hydrogel in SIF and SGF solutions were examined
NMS was released (83%) in a controlled manner confirming it as injectable hydrogel
Abstract Silane crosslinked biopolymer based novel pH-responsive hydrogels were fabricated by blending the cationic (chitosan) and anionic (alginate) polymers with poly (vinyl alcohol). Tetraethoxysilane (TEOS) was used, as a crosslinker in different amounts due to its nonhazardous nature, to study its impact on physical and chemical properties of the prepared injectable hydrogels along with the controlled release of drug. The swelling response of the prepared hydrogels was examined in different solvent media which exhibited decreased swelling ratio with increase in the amount of TEOS. All the fabricated hydrogels represented highest swelling at acidic pH while low swelling at basic and neutral pH. This specific pH sensitive behavior at pH 7 made them an appropriate
candidate for the injectable controlled drug delivery in which Neomycin Sulfate (NMS) was successfully loaded on suitable hydrogel (comprising 50 μL TEOS) to study its release mechanism. The results revealed that in simulated gastric fluid (SGF), hydrogel released the entire drug (NMS) in initial 30 min while in simulated intestinal fluid (SIF), NMS was released in a controlled way up to 83 % in 80 min. These results endorsed that the hydrogels could be practiced as a smart intelligent material for injectable controlled drug delivery as well as for other biomedical applications at physiological pH. Key Words: Chitosan; Alginate; Injectable hydrogels; Neomycin sulfate; Tetraethoxysilane; Swelling. 1.
Introduction
Hydrogels are hydrophilic, 3D-crosslinked polymeric network and superabsorbent when placed in water or other physiological fluids due to their swelling ability (Ju et al., 2001). Crosslinking in hydrogels occurred through physical interactions or covalent bonding. Hydrophilic groups like OH, -NH2, -COOH, etc. are responsible to generate hydrophilicity in hydrogels. In swollen form due to their softness and rubber like structure, hydrogels appear like a living tissue. Hydrogels are prone to sense a minor change in external stimuli like (pH, temperature, ionic concentration, electric and magnetic field) by displaying variation in swelling response, mechanical properties and network structure (Qu et al., 2000). pH-responsive smart hydrogels are one of the most premium type of hydrogels that has been widely used in medicine for controlled/targeted drug release. The significance of pH-responsive hydrogels is due to their performance in different pH of internal body fluids and organs (Gao et al., 2012). These hydrogels are mainly based on polymers with ionic pendant groups distributed along the back bones of such polymers. These pendant ionic groups are responsible for the pH-sensitivity
of the hydrogels. Hydrogels based on natural/biopolymers are ecofriendly, biodegradable, biocompatible, nontoxic, superabsorbent, hydrophilic, cost effective and have ability to expand making it favorable to be used in biomedical, cosmetics, biotechnology mainly for drug delivery and agricultural applications (Chen & Park, 2000; Pappas et al., 2000; Ghaffar, et al., 2011). Among biopolymers, the polysaccharides (cellulose, agar, pectin, carrageenan and alginic acid) are the most abundant and renewable natural polymers and are neutral or acidic in nature. Chitosan is a cationic biopolymer acquired through the alkaline N-deacetylation of naturally occurring chitin. Chitin is an important constituent of crustaceans, insects and fungal mycelia consists of 2acetamido-2-deoxy-β-D-glucose attached through a β (1,4) linkage (Muzzarelli, 1973). Glucosamine are the major units in chitosan having one primary amino and two free hydroxyl groups (Yang et al., 2016). The degree of N-deacetylation is the main factor which decides its properties (Zikakis, 1984). Chitosan is a biocompatible (Shigemasa & Minami, 1996), biodegradable (Dong, 2001), antifungal, antibacterial (Payne et al., 1992) and having gel forming properties (Ma et al., 2010), so, readily processable to prepare thin films, hydrogels, membranes, beads, nanofibers, etc. Chitosan and its derivatives have wide applications in different fields including: pharmaceutics, biotechnology, drug delivery, textile, food, cosmetics etc. (Gorochovceva & Makuška, 2004; Hu et al., 2005; Wang et al., 2008; Ito, Yoshida & Murakami, 2013; Li and Huang, 2012; Hu et al., 2011). Alginic acid (alginate) is a linear anionic polysaccharide found mainly in the cell wall of brown algae. Alginic acid is a copolymer composed of two types of uronic acid; 1,4-linked β-Dmannuronic acid and α-L-guluronic acid units which form three kinds of polymer segments of blocks like M block, G block and MG block which may place randomly or as consecutively (Atkins et al., 1970; Kim et al., 1990). The viscosity of alginate solution depends on temperature
and pH. At pH 5 to 11, the thickness of alginate gel remains unaffected. It gives fine gel at pH 4 to 5 but as pH drops from 6 to 2, precipitation will occur while the viscosity of the alginate solution drops with the rise in temperature. Alginate is also a biocompatible and has an outstanding functionality as a stabilizer, emulsifier, gelling and thickening agent. Nowadays, based on these characteristic and remarkable properties, alginate is useful for many kinds of food, cosmetic, textile, pharmaceutical and other biomedical applications (McDowell, 1969; Atkin et al., 1971). Natural polymers based hydrogels have poor mechanical strength due to low crystallinity and amorphous structure but many other factors are also involved like; hydrophilic nature, crosslinking density, degree of swelling, polymerization conditions, etc. Generally, with increasing the concentration of crosslinker, polymer chains come closer to each other, thus reducing the diffusivity and swelling behaviour and hence improved mechanical properties. Beyond the optimum concentration of crosslinker, the number of crosslinks is increased, the polymers become less elastic (polymer chain segments are constrained in their movement as they are hooked) resulting in a brittle structure (Chippada, 2010; Maitra & Singh, 2014; Maitra & Shukla, 2014). Furthermore, to enhance the mechanical properties of natural polymer based hydrogel, these are blended with synthetic polymers which are excellent in mechanical properties and thermal stability (Xu et al., 2010). PVA is a commonly used synthetic polymer with excellent mechanical properties and extensively used in the preparation of films (Chifiriuc et al., 2012; Grumezescu et al., 2013). PVA based hydrogels and membranes have excellent characteristics like non-carcinogenicity, biocompatibility, nontoxicity, high swelling in aqueous solutions and desirable physical and thermal properties (McNeely & Kovacs, 1975; Torres et al., 2013). PVA is also used for the preparation of skin replacement materials, contact lenses, reconstruction of vocal cord (Gao et al., 2012), replacement of artificial cartilage, and hydrogels for biomedical applications (Liang et al.,
2009) such as wound healing/wound dressing (Rasool et al., 2010), drug delivery, membranes form for reverse osmosis/waste water treatment, removal of heavy metals, controlled release of agricultural fertilizers and coating materials for industrial applications (Islam et al., 2011). To develop the coordination among natural and synthetic polymers, different crosslinking agents are available like: formaldehyde, acetaldehyde, glutaraldehyde, TEOS, etc. But TEOS is a preferred crosslinker because silane crosslinkers are mainly suitable to form inorganic crosslinks in the amorphous zone of polymers in order to deliver the covalent bonding between inorganic content and polymer chains, usually used in biomaterials and other applications. It is easy to bind via condensation reactions and nontoxic in comparison with formerly reported crosslinkers (glutaraldehyde, epichlorhydrine, borate and tripolyphosphate) (Jena & Raju, 2008; Liang et al., 2009; Islam et al., 2011). The main issue in the conventional drug release systems is that mostly they are not site oriented or targeted and release the drug in bulk amount, unable to maintain its concentration. So, a diminute amount is absorbed by the body and the rest is excreted without any change. Thus, to deal with such drugs which are not easily absorbed by the body, it is necessary to use alternative ways to maintain drug dosage in actual range otherwise the given prescription becomes useless for the patients who is unable to achieve the desired results. For this issue, the injectable hydrogels are more attractive for releasing drug in a controlled way with optimum/required delivery periods. These are also more suitable for biomedical applications as they can lessen patient distress, infection risk and reduce operation cost of hydrogel implantation (Hou et al., 2004; Lee & Tae, 2007). The most commonly used polymers for the fabrication of injectable hydrogel are cellulose, carboxymethyl cellulose, poly(vinyl pyrollidone), PVA, chitosan, poly(ethylene glycol), etc. (Hoare & Kohane, 2008; Kadajji & Betageri, 2011; James et al., 2014).
Neomycin sulfate (NMS), is a water soluble aminoglycoside. It is an antibiotic obtained through fermentation of Streptomyces fradiae. It is basic in nature and thermally stable compound. It retains its effectiveness in aqueous solution and other vehicles compared to other antibiotics. It also retards the growth of Gram-positive and Gram-negative bacteria (Livingood et al., 1952; Adams et al., 1996; Clarot et al., 2005). In this study, cationic and anionic polymers are blended with PVA and crosslinked via TEOS to develop a novel pH-responsive hydrogel for controlled drug release. The swelling trend of the prepared hydrogels is evaluated in different solvent media. All the hydrogels represented the highest swelling at acidic pH while low at basic and at pH 7. This specific pH-sensitive behavior at pH 4 recommended them as injectable controlled drug delivery system. Hydrogel with the best response is preferred to attain the objective of this research. NMS is an aminoglycoside antibiotic, chosen as a model drug and its released mechanism is evaluated in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). The prepared NMS loaded hydrogel showed release in a controlled way in SIF solution. The amount of NMS release is examined through UV-Vis spectroscopy. 2.
Materials and methods
2.1
Materials
Chitosan (Mw = 17103.41 g/mol, degree of deacetylation (DDA) = 90.28%; viscosity = 200 cp) was received from Biolog (GmbH) Trademark. Alginate (Mw = 60275 g/mol) extra pure food grade, poly (vinyl alcohol) (Mw = 72,000 g/mol; D-85662; degree of hydrolyzation ≥ 98 %), acetic acid (Mw = 60.05 g/mol (100%)) extra pure, absolute ethanol and glacial acetic acid were purchased from Merck, Germany. TEOS (98.5 %) was purchased from DAEJUNG chemical & metals Co. Ltd., Korea. Hydrochloric acid (37%) was purchased from Labscan Asia, co. Ltd.,
Thialand. NaCl and CaCl2 were purchased from sigma Aldrich St. Louis, Missouri, United States. NMS (ATTC B05CA09) was obtained locally and used as a model drug. E-coli gram negative bacteria (ATTC 25922), yeast extract and tryptone were also used. Fluorescein diacetate, dimethyl sulphoxide, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium chloride and potassium chloride were obtained from sigma Aldrich. HeLa cell lines were purchased from ATTC. All chemicals were of analytical grade and used without any purification. 2.2
Fabrication of hydrogels
Chitosan (0.60 g) was dissolved in 50 mL of 2 % acetic acid solution with continuous stirring on a hot plate at 50 ˚C. Alginate (0.20 g) was soaked overnight in 25 mL distilled water in a separate beaker and magnetically stirred for 1 h at room temperature. The prepared chitosan and alginate solutions were blended and stirred for 1 h at 50 ˚C. Preheated distilled water (25 mL) at 80 ˚C was taken and 0.2 g of PVA was added in it and stirred until it dissolved. This PVA solution was added in previously made chitosan-alginate solution and stirred for 1 h at 50 ˚C (referred as CPA). 25 µL (0.5 drop) TEOS was dissolved in 5 mL ethanol having one drop of 0.1 M HCl solution and added drop wise in CPA solution at 50 ˚C during continuous stirring on a hot plate for 2 h. This blended solution was poured on a glass petri dish and dried at room temperature. After complete drying, it was peeled off to get dried hydrogel and finally it was vacuum dried at 60 ˚C for 6 h in drying oven (LVO- 2040, Lab Tech, Korea). The dried hydrogel was stored in polythene bags in a desiccator. CPA was the control hydrogel contained chitosan-PVA-alginate without crosslinker while CPA1, CPA2 and CPA3 hydrogels have 25 µL (0.5 drop), 50 µL (1 drop), and 75 µL (1.5 drop) TEOS (silanol) along with the same amount of chitosan, PVA and alginate, respectively. 3.
Characterization
3.1
Fourier transform infrared (FTIR) analysis
FTIR spectra of chitosan, PVA, alginate and all CPA hydrogels were taken on Shimadzu IRPrestige-21, Kyoto, Kyoto Prefecture, Japan, with attenuated total reflectance (ATR) mode at a resolution of 4 cm-1 with programmed range of 4000-400 cm-1. 3.2
Thermogravimetric analysis (TGA)
TGA was carried on TA instrument SDT Model Q600; TA Instrument, New Castle, DE, USA, with nitrogen flow of (15 mL/min). The temperature ramp was kept at 20 ˚C/min from ambient temperature to 600 °C. 3.3
Scanning electron microscopy (SEM)
The surface morphology of hydrogels was determined through SEM Model JEOL/EO JSM 6480 (LA) Akishima, Tokyo, Japan. The images were collected at different magnifications. 3.4
Swelling experiment
The swelling experiment was conducted in distilled water, buffer and ionic solutions by following procedure; vacuum dried hydrogels were cut into tiny fragments (25 mg), weighed and dipped in a glass vial contained 40 mL of respective solvent. After a specific time interval (20 min), the excess of external solution was discarded from vial and cleaned the vial with tissue paper and swollen weight of hydrogel sample was measured. The experiment was revised at the same time interval until the equilibrium was attained. This experiment was repeated thrice for each hydrogel to obtain mean value for more accurate results. Degree of swelling was determined by equation 1. Swelling (g/g) =
(1)
Where, Wd is the weight of dried hydrogel and Ws is the weight of swollen hydrogel at time t. Buffer solutions of different pH (2, 4, 6, 7, 8 and 10) were used to study the pH response. Ionic solutions of NaCl and CaCl2 of different concentrations (0.1, 0.3, 0.5, 0.7, 0.9 and 1M) were prepared and swelling of hydrogel samples was determined in each solution. To find the crosslinking degree of the hydrogels, the samples were cut into small pieces and known weight of hydrogel samples (0.1g) was put into a stainless steel cloth according to the ASTM 2765 (Mir et al., 2014). Soxhlet extractor was used to extract the crosslinked material in the presence of distilled water at 100 ˚C for 10 h. After extraction, the samples were dried in a drying oven until the weight become constant. The degree of crosslinking was calculated by weighing the dried crosslinked samples using equation 2. (2) Where, Wg is the weight of dried crosslinked sample and Wo is the initial weight of the sample. 3.5
Antimicrobial test
Antimicrobial activity of the hydrogels was investigated using liquid diffusion method. P.lice strains of E.Coli (gram -) were used as bacterial modal. The method involves the following steps; LB-media was prepared by taking 10 g NaCl, 5 g yeast extract and 10 g Tryptone in 800 mL distilled water and the solution was maintained at pH 7. After this, the volume of the solution was raised up to 1000 mL and autoclaved it for 1 h. To prepare the control solution, 20 mL of above prepared LB-media and 20 µL P.lice strain of E.Coli (for the growth of bacteria) were taken in a 50 mL flask and incubated at 38 ˚C for overnight. To prepare the test sample solution, 3 g of hydrogel sample was added in above mixture (20 mL LB-media and 20 µL P.lice strain of E.Coli) and same
procedure was repeated. On the next day, the absorbance of control and test sample solutions at 600 nm was taken from UV visible spectrophotometer, Labomed, Inc. UVD-3500, USA.
3.6
In Vitro Cytotoxicity/Cell Viability
HeLa cell lines were used for cell culturing and cytotoxity analysis. The culture was maintained under standard conditions (incubated at 37 ˚C under 5 % CO2 and 95 % air). The cell viability was assessed with fluorescein diacetate (FDA) staining. 10 μL of 2 mg/mL of FDA stock in dimethyl sulfoxide (DMSO) was diluted in 5 mL of phosphate buffer saline (PBS) and 1 mL of this solution was used to stain each sample in 12 well plates. The cells were incubated for 5-10 min and washed with PBS. Positively stained cells were observed under fluorescent microscope (Nikon TS 100) for green fluorescent filter. The surface of the plate was coated with 100 μL of each hydrogel solution, air dried and then UV sterilized. The cultured cells were seeded in 10,000 cells per cm2 density and cultured over night to ensure proper cell attachment and incubated again for 48 h. After incubation, the media (used for cell culturing) was removed, the cells were washed with PBS, and neutral red media was added and incubated again for 2 h. After two hours, neutral red media was removed and the cells were again washed either PBS. Freshly prepared de-staining solution (49 % deionized water, 50 % absolute ethanol and 1 % glacial acetic acid) was added and culture was incubated for 10 min and its optical density was taken at 570 nm on UV visible spectrophotometer, Labomed, Inc. UVD-3500, USA. 3.7
Controlled drug (NMS) delivery analysis
3.7.1
Preparation of simulated solutions
Simulated gastric fluid (SGF) of pH 1.2 was prepared by taking 1 g NaCl in 3.5 mL of HCl and diluted up to 500 mL with distilled water.
Simulated intestinal fluid (SIF) of pH 6.8 was prepared by adding 0.2 M solution of 250 mL of KH2PO4 and 0.2 M solution of 118 mL of NaOH (Islam et al., 2013). 3.7.2
Drug loading procedure
To prepare the drug loaded hydrogel samples, NMS (100 mg for each blend) in 25 mL of distilled water was loaded into two separate blends of chitosan (0.60 g in 50 mL of 2 % acetic acid solution), PVA (0.20 g in 25 mL of distilled water) and alginate (0.20 g in 25 mL of distilled water) and stirred for 1 h at 50 ˚C. 50 μL (1 drop) of TEOS was added in each of these solutions and stirred for 3 h. These drug loaded blend solution were poured on glass petri dishes and dried at room temperature. After complete drying, these were peeled off to get the dried hydrogels and finally vacuum dried at 60 ˚C for 3 h in drying oven (LVO- 2040, Lab Tech, Korea). The vacuum dried drug loaded hydrogels (1.245 g each) were placed separately in SGF and SIF (100 mL each) solutions at 37 ˚C. After every 10 min, 5 mL solution was collected using a syringe from each SGF and SIF solution having drug loaded hydrogel. Fresh solutions (5 mL) of SGF and SIF were added back every time to each beaker of SGF and SIF solution to keep the volume up to 100 mL. Subsequently, the samples were collected for next 2 h from SGF solution and 3 h from SIF solution. The amount of released NMS by drug loaded hydrogels was estimated spectrophotometrically at 550 nm by UV visible spectrophotometer, Labomed, Inc. UVD-3500, USA. The standard drug solution (100 ppm of NMS drug in SGF and SIF) was taken as reference for UV visible analysis. 4.
Results and discussion
The proposed interactions (covalent and hydrogen bonding) of silane crosslinked blends of CS, PVA and alginate having hydrogel properties are presented in scheme 1. These properties especially the pH sensitivity could be used for the controlled/targeted release of drugs. 4.1
FTIR analysis
FTIR spectroscopy was conducted to investigate the functional groups of pure chitosan, PVA, alginate, CPA (control), and crosslinked CPA1, CPA2 and CPA3 hydrogels as shown in Figure 1. These spectra confirmed the presence of incorporated components. The bands of amide I and amide III were observed at 1642 and 1245 cm-1, respectively while the band of amide II was observed at 1556 cm-1 (Islam et al., 2012). Amide IV and amide V bands were appeared at 600 cm-1 and 660 cm,-1 respectively. A band due to pyranose ring was observed at 898 cm-1 which confirmed the existence of chitosan and alginate in the samples (Islam et al., 2011). Sodium alginate showed the characteristic absorption bands at 1414 and 1543 cm-1 due to carboxyl anions. The C–O stretching bands of C–O–C bridge were observed at 1030 cm-1 in chitosan and alginate. The absorption bands at 1424 and 1090 cm-1 were ascribed to the bending (–OH) and stretching (C–O) of pure PVA, respectively. The asymmetric stretching bands in the range of 1130-1000 cm-1 indicated the presence of both –Si–O–Si and –Si–O–C which confirmed the crosslinking via silanol groups. A broad band between 3570-3100 cm-1 showed -OH stretching of intra- and inter-molecular hydrogen bonds in the prepared hydrogels (Ma et al., 2008). A vibrational band of alkyl group (–CH stretching) was observed at 2921 cm-1. The silanol (–Si–OH) stretching vibration was observed at 3725 cm-1 (Kamoun et al., 2015). 4.2
Thermogravimetric analysis
TGA was carried out to study the effect of crosslinker i.e. TEOS content on the thermal stability of the hydrogels. The weight loss (%) behavior of prepared hydrogel samples are shown in Figure 2.
TGA of pure chitosan, alginate and prepared hydrogels showed that the degradation was occurred in two differentiated steps whereas in PVA it took place in three well differentiated steps. The thermogram of PVA exhibited three regions of mass loss. After the water loss step, the decomposition occurred mainly in the second and third degradation steps that occurred at 281 and 420˚C, respectively and reflected the decomposition of pendant groups and backbone chains of PVA (83 % weight loss). At 600 ˚C, a residue of 4.0 % could be found (Santos et al., 2014). For chitosan, CPA, CPA1, CPA2 and CPA3, the first weight loss was observed at 260 °C while for alginate it was observed at 285 °C due to dehydration. For chitosan and alginate, the onset degradation was observed at 260 and 303 °C and offset was found at 349 and 362 °C, respectively. All hydrogel thermograms showed two steps degradation; first degradation was attributed to the breakdown of side chains and pendent groups while second phase was corresponded to the scission of backbone polymer chains (Hench, 1998, Buranachai et al., 2010; Honary et al., 2010). In the controlled hydrogel (CPA) having two stages for degradation, the onset of degradation started earlier having no crosslinker as CPA showed 30 % weight loss at 280 ˚C while second step was due to main degradation and offset occurred at 400 ˚C. The 30 % weight loss was increased up to 289 °C in CPA3. Whereas, 60 % weight loss was observed at 350 ˚C in CPA but with the addition of crosslinker, the same weight loss (60 %) was observed at 370 °C for CPA1 and for CPA3 it was at 410 °C. This increase in temperature (at 30 % and 60 % weight loss) confirmed the increase in thermal stability with the addition of TEOS. The higher degree of crosslinking with increase in the amount of TEOS resulted in hindrance in the chains movement. 4.3
Scanning electron microscopy
The morphology of the prepared chitosan/PVA/alginate hydrogels are shown in Figure 3. The hydrogels without crosslinker showed roughness and surface irregularities (Pieróg et al., 2012;
Kamoun et al., 2015) with the formation of visible depressions (Figure 3a and 3b) (Eldin et al., 2015) while with the addition of TEOS (CPA1), uniform and smoother surface was observed. SEM images clearly showed that the prepared hydrogels have defined porous structure (Straccia et al., 2015) as the addition of crosslinker imparted a key role in the development of pores having size ranged from 2-30 µm in CPA2. These micrographs showed that by increasing the amount of crosslinker (50 μL), number of pores with good distribution on the surface was observed (Fig. 3e and 3f). In fact, the number of –OH groups were increased with increase in the crosslinker amount instigated the development and more expansion of hydrogel network among the polymer chains. This might be the reason of more water absorption in CPA2 and make it suitable for gaseous exchange and encapsulation of biochemical agents (Rashidzade et al., 2014; Rasool et al., 2010). 4.4
Study of swelling behavior in different media
4.4.1
Swelling in distilled water
The hydrogels swell by the absorption of water through diffusion process. This diffusion occurred due to the movement between external solvent and polymer chains. The swelling behavior of the developed CPA hydrogels in distilled water with respect to time is shown in Figure 4a. The swelling performance of all the hydrogels was different, a linear and continuous increase in the swelling was seen which increased with increase in time (Islam et al., 2012). The equilibrium time for CPA, CPA1 and CPA2 was observed at 80 min and for CAP3 it was 60 min. Maximum swelling was observed in CPA2 (68 g/g) while minimum swelling was noticed for CPA3 (38 g/g). Initially, an increase in swelling with the addition of TEOS (silanol) was observed in contrary to the normal trend of decreased swelling of hydrogels with the increase in amount of crosslinker. First trend of increase in swelling was due to the generation of pores and more diffusion of water molecules as discussed in section 4.3. The number of –OH groups were increased with increase in
the crosslinker amount caused the development of hydrogel network among the polymer chains which might be the reason of more water absorption at a higher crosslinker amount (50 µL) as the swelling analysis was repeated for three times and found the same results. The swelling was decreased with the maximum amount of TEOS (75 µL) which caused to shrink the spaces in the hydrogel network by the generation of more crosslinking points and made it more tough to be swollen by water. Also, the highest amount of crosslinker (75 µL) created more compact network resulted in the reduced swelling. This trend was in accordance with the normal trend of decrease in swelling at higher crosslinker amount (Rasool et al., 2010; Rashidzadeh et al., 2014). Generally, the swelling of hydrogels depends on the diffusion of solvent from extracellular matrix to its structure. The mechanism of swelling process was determined by equation 3. F=
(3)
Where ‘k’ is the swelling rate constant, n is the swelling exponent, F is the fractional swelling estimated through swelling ratio of Wt and Weq. Where, Wt and Weq are the swelling ratio at time t and at equilibrium time (min), respectively. The swelling data of CPA hydrogels in water was used to determine the values of n and k parameters. A plot of ln(F) vs ln(t) shows in Figure 4b and values of diffusion parameter are given in Table 1. For CPA, CPA1, CPA2 and CPA3, n ˂ 0.5; corresponded to the Fickian transport which showed that the rate of diffusion was lower than the rate of relaxation. The crosslinking degree of hydrogels represented the crosslinked portion or undissolved part of the CPA blends which was observed to be increased with increase in the concentration of crosslinker. The crosslinking degree of CPA hydrogels are given in Table 1. 4.4.2
Swelling in buffer solutions
The hydrogels are sensitive to change in pH therefore the swelling medium imparted a main role in the swelling of hydrogels. A slight change in pH of external solvent disturbed the charge balance in the complex which altered the interactions of polymer chains. The effect of pH on the swelling of CPA, CPA1, CPA2 and CPA3 hydrogels in buffer media is shown in Figure 5. The hydrogels expressed maximum swelling at acidic whereas least at neutral and basic pH. To study the swelling response at different pH buffers (2, 4, 6, 7, 8, and 10), all CPA hydrogels were soaked in specific buffer solutions up to their equilibrium time. The amino (-NH2+) group of chitosan and carboxyl (-COO-) group of alginate are the two basic groups existed in the prepared hydrogel. The amino group was stronger base and have greater tendency to accept the hydrogen ions first, therefore, the control hydrogel without TEOS and CPA1 with the least amount of TEOS exhibited highest swelling at pH 4. While in CPA2 and CPA3, the swelling was decreased with the increase in crosslinker amount. In acidic buffers, higher concentration of charged entities created charge repulsion and the entrance of solvent through diffusion resulted in the increased swelling ratio (Atta et al., 2015). CPA1 and CPA2 showed maximum swelling of 32 g/g at pH 2 while the lowest values of 4 and 6 g/g were observed at pH 7, respectively. All the hydrogels displayed a steady decrease in swelling from pH 4 to pH 6. At neutral pH, carboxyl group become unprotonated as it was strong acid than amino group, so, low swelling was observed. At basic pH, due to decrease in the degree of ionization of charged entities or deprotonation of ammonium ions (-NH3+), water was released from hydrogel resulted in the shrinkage of polymer chains and hence the reduction in swelling ratio while with further increase in pH up to 10, it was remained almost the same. 4.4.3
Swelling in ionic solutions
The nature of salt and its concentration have prominent impact on the swelling response of hydrogels. Therefore, NaCl and CaCl2 were used to prepare the ionic solutions to study the swelling behaviour. The two ionic solutions have same anion (Cl-1) but different cation charges on it. The swelling of control and crosslinked hydrogels was evaluated and shown in Figure 6. The swelling was reduced with enhanced concentration of both electrolytes. A high amount of salts in the external swelling solvent generated the charge screening effect due to excessive electrolytes which ultimately reduced the developed osmotic pressure between the external solvent and hydrogel. So, the diffusion was decreased and resulted in less swelling. Maximum swelling was observed at 0.1 M CaCl2 compared to the NaCl (0.1 M). The swelling value for CPA hydrogels in 0.1 M CaCl2 was 21 g/g, whereas, 18 g/g was determined in 0.1 M solution of NaCl. At lower concentration, Ca2+ ions formed complex with polymer so pore size was increased resulted in the increased swelling ratio (Islam & Yasin, 2012). 4.5
Antimicrobial results
Antimicrobial activity of the hydrogels was investigated using liquid diffusion method and results are given in Table 2. The absorbance value of the controlled solution was observed at 1.287 but on the other hand the absorbance values of test samples were below 1.287 which confirmed their good inhibition, because, if the absorbance values of test samples were same as the controlled solution, then these were not capable of inhibition of the bacterial growth. Particularly, CPA2 is -0.025 which showed excellent bacterial inhibition properties. The test samples contained chitosan having positive charged (cationic) and interact with negatively charged bacterial cell wall, which in turn caused the disruption onto the cell wall of bacterium, penetrated in bacterial cell and retarded its growth by avoiding DNA conversion into RNA.
4.6
In Vitro Cytotoxicity Analysis
The cell culture methods, also known as cytotoxicity analysis, were used to examine the toxic nature of the hydrogels. In vitro cytotoxicity of fabricated hydrogels was carried out using HeLa cell line (model cell line) and Neutral red assay by indirect method as shown in Figure 7. This figure shows that only few cells were attached to the CPA compared to standard and had round morphology. The cells were also appeared as clusters which showed that cell to cell adhesion forces were established before cell to matrix adhesion. In CPA1, the cells were uniformly distributed compared to CPA but unlike standard very few cells were attached. In CPA2, the cells were attached to the most of the surface area of hydrogel sample. The cell viability was good with round morphology and the cells were separated due to the nontoxic behaviour of material with higher cell count compared to CPA, CPA1 and CPA3 in which cells were not evenly distributed and few in numbers. Neutral Red is a supra-vital dye and all actively growing cells uptake the Neutral red from surrounding and bind it to lysosomes of cells. This test was widely used to check the cell viability and also for cytotoxicity assays (Repetto et al. 2008). The cell viability of the prepared hydrogel samples was compared with the standard sample (cell line without hydrogel sample) and found that CPA2 hydrogel containing 50 µL crosslinker have the highest cell viability i.e. the lowest cytotoxicity with cell survival rate of approximately 60 % compared to the other developed hydrogels (Figure 8). This maximum cell growth rate was attributed to the fact that 50 µL was the optimum concentration of crosslinker for the fabrication of CPA hydrogels used for the injectable drug delivery system. These results showed the nontoxic behaviour towards cells growth which performed their normal activities and undergone their normal proliferation and life cycle. This
outcome was strongly in accordance with the antimicrobial analysis of these hydrogels discussed in section 4.5. 4.7
Release Analysis of NMS
CPA2 was loaded by NMS drug and its release (%) mechanism as a function of time was studied in SGF and SIF. The entire drug was released in half an hour in SGF but according to the US pharmacopeia standard, the release should not be more than 10 % in SGF. This was the reason that this hydrogel could not be used for oral drug administration. NMS was released in a controlled manner in SIF (Figure 9) i.e. 83 % in 80 min, which was in accordance with the US pharmacopeia standard and the left amount could not be determined because hydrogel was broken down into fragments. The developed hydrogel possessed the property of controlled drug release and could be used as intravenous (IV) medication. These consequences specified that the hydrogels could be a suitable candidate for the injectable drug delivery and other biomedical applications. 5.
Conclusions
Biopolymer based novel, pH-sensitive, crosslinked CPA hydrogels were formed by solution casting technique. CPA2 showed maximum swelling degree (68 g/g) in distilled water. The degree of swelling in distilled water was reduced with increase in the quantity of TEOS. CPA3 showed maximum thermal stability compared to the other specimens. The swelling behavior of the hydrogels for different pH showed high swelling in acidic and low in basic and neutral pH. This specific pH-responsive behavior at pH 7 recommended these hydrogels for the injectable controlled release of drug. In vitro cytotoxicity analysis exhibited the nontoxic behaviour toward cells growth executed their normal actions and experienced normal production and life cycle. The controlled release study of NMS (model drug) loaded CPA2 hydrogel exhibited that in SIF, 83% of drug was
released in a controlled manner within 80 min. This confirmed that these hydrogels could be a striking candidate for the injectable drug delivery and other biomedical applications. Acknowledgments The authors are highly thankful to Higher Education Commission of Pakistan for funding under the project (HEC-USAID) of “Development of Innovative Technical and Medical Textile Products” and Prof. Dr. Amtul Jamil Sami, Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore, Pakistan for cytotoxicity analysis.
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Figure 1. FTIR spectra of pure chitosan, PVA, alginate, CPA (control), CPA1, CPA2 and CPA3 hydrogels.
Figure 2. The thermograms of pure chitosan, PVA, alginate, CPA (control), CPA1, CPA2 and CPA3 hydrogels.
Figure 3. SEM micrographs of CPA (control) (a Å~ 250), (b Å~ 1000) CPA1 (c Å~ 90), (d Å~ 500) CPA2 (e Å~ 250) (f Å~ 1000) and CPA3 (d g~ 250) (d h~ 1000).
Figure 4(a). Time (min) vs swelling (g/g) of CPA (control), CPA1, CPA2 and CPA3 hydrogels in water at room temperature.
Figure 4(b). ln (F) vs ln (t) for CPA (control), CPA1, CPA2 and CPA3 hydrogels.
Figure 5. A plot of pH vs swelling (g/g) of CPA (control), CPA1, CPA2 and CPA3 hydrogels at different pH (2-10) of buffer solutions.
Figure 6. A plot of concentration (mol/L) vs swelling (g/g) of CPA (control), CPA1, CPA2 and CPA3 hydrogels in different molar concentration of NaCl (solid) and CaCl2 (hollow) solutions.
Figure 7. Optical micrographs obtained after 24 h incubation of prepared hydrogels and standard sample.
Figure 8. A comparison of cell viability of hydrogel samples with the standard sample.
Figure 9. Release analysis of NMS in SIF (PH 6.8) with time.
Scheme 1: The proposed interactions of chitosan, PVA and alginate via TEOS of prepared crosslinked hydrogels.
Table 1. Diffusion parameters and crosslinking degree of different CPA hydrogels. Parameters
CPA
CPA1
CPA2
CPA3
n
0.35
0.38
0.44
0.19
Intercept
-1.77
-1.82
-1.93
-1.22
k
0.170
0.162
0.145
0.295
Regression
98
97
93
87
45
71
80
87
(R %) Crosslinking degree (%)
Table 2. Absorbance data of controlled, CPA (control), CPA1, CPA2 and CPA3 hydrogel samples along with trends representing their antimicrobial behavior. Sample
Absorbance at
Trends
600 nm Controlled
1.287
----
CPA
0.211
Good
CPA1
0.004
Good
CPA2
-0.025
Excellent
CPA3
0.014
Good