#copolymerization

Abstract

In the rapidly developing world a great concern regarding the protection and wellbeing of living beings and environment has led to the necessity for development of accurate monitoring processes through accurate and sensitive sensors. For these sensor applications conducting polymers nanocomposites have been widely used. In this article we will discuss about the conducting polymers, their mechanism of conduction, the sensing mechanism and different types of conducting polymers-based gas sensors. The prospects of conducting polymers-based gas sensor nanocomposites are also discussed in this article.

Keywords: Conducting polymers; nanocomposites; gas sensors; polymers; sensors

Abbreviations: CPs: Conducting Polymers; PANI: Polyaniline; PPy: Poly Pyrrole; PTs: Polythiophene; PEDOT: Poly 3,4-ethylenedioxythiophene

Introduction

Conducting Polymers

Recently the conducting polymers (CPs) have gained attention of researchers because of their tunable conducting properties, easy preparation, light weight and ease of process. Due to their higher conductivity values the CPs are referred as “synthetic metals”. CPs are being used for variety of different application including energy storage devices, solar cells, capacitors, EMI shielding, pressure sensors and gas sensors. Although most of their potential is still unexplored. CPs are intrinsically conductive in nature due to their conjugated system coupled with atoms or conjugated structure with alternate single and double bonds. The mechanism of conductivity of CPs is based on the transmission of polarons and bipolarons and there are different factors that influence the conductivity of CPs, some of them are the conjugation length, chain length, polaron length, the charge transfer of adjacent molecules and type and concentration of doping [1-3]. Most common and important CPs include, polyaniline (PANI), poly pyrrole (PPy), polythiophene (PTs) and poly 3,4-ethylenedioxythiophene (PEDOT) [4,5].

Sensing Principle of CPs and the factors that influence the performance of the conducting polymer-based gas sensors

The mechanism of gas sensing in the case of CPs based sensors, involves the measuring of resistance variation with the interaction of gas and the sensing material. The interaction between gas and the sensing material changes the physical properties of the CPs sensors, such as resistance, absorbance and current [6]. Following are types of CPs based gas sensors reported in the literature based on their operational mechanisms

a. Chemiresistors

b. Semiconductor Sensors

c. Optical Gas Sensors

d. Piezoelectric Crystal Sensors

e. Amperometric Sensors

There are many factors that influence the performance of the gas sensing performance of CPs based sensors, including nature of the sensing material, design of the sensor, type and concentration of doping, chemical interaction of gas and CPs, physical interaction of gas and CPs etc. [7].

Prospects of CPs based gas sensors

Despite scientific advancement in the field of gas sensors there are some areas in which researchers are facing challenges some of them are the selectivity for a target gas, in depth understanding of response time and the mechanism of sensitivity. So, in future we will see that much more attention will be given for the development of flexible and wearable sensor devices developed through the advanced synthesis methods to improve dispersion and to get uniform properties of the nanocomposites [8,9].

Conclusion

In this article we have discussed the CPs structure, properties and conducting mechanism. We have also discussed the factors influencing the gas sensing of CPs based sensors, like design of the sensor, nature of the material etc. different types of gas sensors based on the sensing principles are also enlisted in the article. In future we will be seeing advanced synthesis and fabrication techniques in order to improve the repeatability, sensitivity and reliability of the CPs based gas sensors.

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Abstract

This study aims to design some medicinally important biopolymer based Nano carriers. The study undertakes synthesis, characterization of Chitosan-co-lactic acid nanoparticles and optimization of swelling ratio. The Chitosan-co-lactic acid nanoparticles were prepared by the shaking of lactic acid emulsion in Chitosan solution by emulsion crosslinking method and characterised by transmission electron microscope (TEM) analysis (size), and Fourier transform infrared spectra (FTIR) (structural) and SEM (morphology). The particles were dispersed in a phosphate buffer saline (PBS) and the swelling response of these nanoparticles was studied as a function of chemical composition, pH and temperature of swelling medium. To determine the encapsulation efficiency, a centrifugation method was applied. Also, the assessment of biocompatibility has been made on the basis of two in-vitro test which are BSA adsorption test and haemolysis assay. Our result revealed that equilibrium swelling was influenced by the composition of nanoparticles and the swelling medium. These observations and assessment indicate that these nanoparticles have good capability for use in drug delivery and suitable for biomedical application.

Keywords:Chitosan; Nanoparticles; Swelling Behaviour; Drug Delivery Devices; Lactic Acid

Introduction

Cancer is a disease which refers to a group of tumour which is formed by an excessive growth of abnormal cells [1]. Cancer is characterized by uncontrolled cell growth, and the potential to get larger into neighbouring tissues and to metastasize. Surgery may not be always more effective due to the involvement of the tumour with neighbouring tissues [2]. In such conditions, Chemotherapy (a destructive agent) is a conventional approach with the major potential to cure localized and metastasized cancers. Chemotherapy is a general term used to describe using several drugs that are destructive to malignant cells and tissues [3]. Anticancer drugs are effective against a wide range of tumours, formulated to treat wide range of tumours but its clinical applications are inhibited due to its poor solubility [4]. The main drawback of these drugs is that they not have enough of selectivity towards tumour cells. Because of the weak selectivity, they kill both normal and cancer cells and result in toxicity to normal tissues [5,6]. The target site-specific delivery can be attained by a drug delivery system. Drug delivery systems can reduce the side effects of anticancer drugs via encapsulation methods, which decrease the scrape of toxic drugs, protect the chemotherapeutic drug prior to attaining the target cells. Encapsulation is one of the effective procedures, as this not only enhances water solubility of the drug but also reduces gastrointestinal impatience and provides a long-lasting release rate [7,8]. The purpose of chemotherapy is the sustained, controlled, and targeted delivery of anticancer drugs for a long period which can be achieved by polymeric nanoparticles [9].

A drug delivery system is generally associated with particulate carriers, such as polymeric micelles [10], emulsions, liposomes [11] and nanoparticles (NPs) [12], which are devised to localize drugs at the target sites. The nanoparticles are submicron-sized, colloidal particles that carry drugs to the target or release drugs in a controlled manner in the body. Controlled drug delivery offers advantages like reduced dosing frequency, better therapeutic control, reduced side effects, and, consequently, better patient compliance. After preparation, nanoparticles are usually dispersed in liquid [13-15]. Recently, polymer nanoparticles have attracted significant interest in chemistry, pharmaceutics and biomaterial science for biomedical application and controlled release [16]. Both synthetic and natural polymers were studied with the purpose of making nanoparticles [17]. Nanoscale drug delivery systems are made from biocompatible and biodegradable material constitute an extend to drug delivery and tumor targeting [18].

However, among the variety of polymers that were used for drug-loaded nanoparticles, chitosan has received great attention in both the medical and pharmaceutical fields [19]. Chitosan, a biodegradable and biocompatible polymer, is a modified natural carbohydrate and the second most abundant polysaccharide in nature [20]. It can be synthesized by the partial N-deacetylation of chitin, chitosan has important applications in various areas and has been the topic of much research in recent years. Since the amine group −NH2 can be protonated to NH3+, which may provide chitosan with a favourable gel forming property, it has been all inclusive examined in the medical industry for its potential in the development of pH-sensitive drug release systems [21]. It consists of repeating units of glucosamine and N-acetyl-glucosamine, the proportions of which determine the degree of deacetylation of the polymer. Chitosan is available in a wide range of molecular weights and deacetylation degrees. Due to its characteristics, chitosan has attained increasing attention in the pharmaceutical field [22]. As one of the well-recognized organic acids, lactic acid has established its potential for use in several biomedical applications due to its antibacterial and excellent biocompatible properties with low toxicity and bio-absorbability in vivo [23, 24]. It has been widely used in biomedical applications, such as sustained drug delivery systems, implants for orthopaedic devices and absorbable fibres [25]. Chitosan and Lactic acid have highly pH-dependent swelling properties, which could be used to control the release of drugs and may be used to treat cancers.

Chemotherapy is a conventional approach with the major potential to cure localized and metastasized cancers [26], in which drugs that are commonly used in cancer therapy are infused through the blood vessels [27] (Figure 1).

The intravenous injections cause drug decomposition in the blood after an initial rapid increase in concentration [28]. Drug delivery system can reduce these inefficiencies via encapsulation methods, which decrease the lesions of drugs. Even though anticancer drug proves to be remarkably nontoxic and has promising medicinal properties, its relevance in anticancer therapies is inadequate owing to its low aqueous solubility and poor bioavailability. These drug formulations are higher to conventional medicines with deference to control release, targeted delivery and therapeutic impact. Polymeric nanoparticles have concerned noteworthy consideration in the study of drug delivery systems as they offer a means for localized or targeted delivery systems of a drug to specific tissue/organ sites of interest with an optimal release rate [29]. Polymeric nanoparticles act as nanocarriers with many advantages, such as low toxicity and high stability. In this study, the Chitosan-co-lactic acid nanoparticles were prepared by emulsion crosslinking method. The Synthesized nanoparticles were characterized by Fourier Transform Infrared (structural), Scanning electron microscopy (morphology of nanoparticle surface), Transmission electron microscopy (size range) and their water imbibition capacity has been evaluated under varying experimental conditions. The present work aims to preliminary study will be determined for nanoparticles drug loading capacity, encapsulation efficiency and in vitro bioavailability of nanoparticles using UV-VIS spectrophotometer.

Materials and Methods

Chitosan was obtained from Hi Media Pvt. Ltd, (Mumbai, India) and Lactic acid, paraffin oil and Glutaraldehyde were purchased from Loba Chemie (Mumbai, India). By using disodium hydrogen orthophosphate, Phosphate buffer saline (PBS) was prepared. Throughout the experiments all other chemicals were of analytical grade and used as received without further purification.

A. Preparation of Chitosan-co-lactic acid nanoparticles

Chitosan – Co- Lactic acid nanoparticles were prepared using an emulsion crosslinking technique with minor alteration in protocol [30]. Briefly, aqueous phase of chitosan was prepared by dissolving the appropriate amount of chitosan in acetic acid solution (2.5%) and then poured in paraffin oil i.e. oil phase with vigorous shaking for 30 min (shaking speed 300 rpm) at the same time emulsion of lactic acid was added to the mixture with uniform shaking. After some time, emulsion of gluteraldehyde was added to the mixture with uniform shaking. The reaction mixture was kept for predetermined time period to attain crosslinking reaction. After that synthesized nanoparticles were separated from the medium by washing thoroughly with toluene, acetone and dried. The dried and powder nanoparticles were kept in air tight polythene bags and were subjected to further analysis. The advantage of this method was credited to its mild conditions achieved without applying harmful organic solvent, heat or vigorous agitation that are damaging to sensitive proteins.

Characterization of Chitosan-Co-Lactic Acid Nanoparticles

Nanoparticles were characterized at two levels which one is physiochemical and the second one is biopharmaceutical characterization.

Physiochemical characterization – Under these level of characterization nanoparticles was characterized by the following methods:

FTIR spectral analysis: FTIR was used to characterize the structure of chitosan-co- lactic acid nanoparticles. The IR spectra of chitosan-co- lactic acid nanoparticles were recorded by a Shimadzu, Japan. Model- 8400S FTIR spectrophotometer and the samples were prepared by KBr pellet technique.

SEM analysis: The morphology of chitosan-co-lactic acid nanoparticle was observed using a scanning electron microscope (SEM) (HITACHI S-3700N) and the samples were coated by gold.

TEM analysis: Transmission electron microscope (TEM) was used to examine the size of nanoparticles using JEOL (JEM-2100) Japan with an acceleration voltage of 200 kV

Particle Size analysis: The distribution of Chitosan-co- lactic acid nanoparticles were characterized by Malvern instrument Ltd. for mean particle size (mean intensity weighted diameter, Zaverage) and poly dispersity index (width of the size distribution, PDI).

The swelling behaviour of Nanoparticles influence their usability as a biomaterial in medicine pharmaceutical.

The mechanism of water diffusion into the nanoparticles is important for assessing the suitability of nanoparticles as drug delivery, as the amount of drug released is dependent on the rate and transport mechanism of water diffusing into polymer matrix. The swelling response of Chitosan-co- lactic acid nanoparticles was determined by an accepted gravimetric method [31]. In a typical experiment appropriate amount of nanoparticles were dispersed in an applicable volume of Phosphate Buffer Saline (10 ml) until equilibrium was reached. The representation of polymer relaxation during water sorption has been shown in Figure 2. The swelling ratio of nanoparticles was calculated from the following equation-

Swelling ratio= Wt / W0

Where, Wt is the weight of nanoparticles (time t)

W0 is the weight of nanoparticles (time 0)

Drug Loading and Encapsulation Efficiency

Drug loading capacity and encapsulation efficiency was dogged by centrifugation method with slight modification in the protocol [32]. The mixture of nanoparticles in drug solution was centrifuged at 10,000 rpm for 2 hr and the amount of free drug in the supernatant was determined by UV spectrophotometer. The drug loading and drug entrapment efficiency were defined by the following equations respectively: % loading = Wd – Wo/ Wo (Wd – weight of drug loaded NPs, Wo – weight of dry NPs)

% EE = (A – B/ A )* 100

Where A - Total amount of drug loaded B- free drug in supernatant

Invitro Cytotoxicity

The MTT assay was performed to measure the metabolic activity of cells to reduce yellow colored tetrazolium salt 3-(4,5- Dimethyl thiazol- 2-yl)-2,5- diphenyltetrazolium bromide to purple colored formazan. Prior to extraction test materials was sterilized with acetone under aseptic conditions. Extract was prepared by incubating test material chitosan- lactic acid in physiological saline at 37+1O C for 72 + 2h at an extraction ratio of 0.2g/ml. The extract was mixed with MEM2X medium to get 50% extract. This was further diluted with culture medium to 25%, 12.5% and 6.25%. Physiological saline without test material processed similar to test material extract was considered as reagent control. Cells cultured in normal medium was considered as cell control. Equal volume (100μl) of various dilution of test samples, extract of negative control, cell control and Positive control were placed on sub confluent monolayer of L-929 cells. After incubation of cells with various concentration of test sample and controls at 37+1 C for 24+2h, extract and control medium were replaced with 50μl MTT solution, wrapped with aluminum foil and were incubated at 37+2 C for 2h. After discarding the MTT solution, 100 μl of Isopropanol was added to all wells and swayed the plates. The color developed was quantified by measuring absorbance at 570 nm using a spectrophotometer. The data obtained for test samples were compared with cell control.

Evaluation of In Vitro Blood Compatibility

In the present investigation compatibility of nanoparticles has been judged by monitoring the amount of protein adsorbed by the nanoparticles. When biomaterials come in contact with biological fluid such as blood, protein within millisecond begin to adhere to the surface through a process known as protein adsorption. Protein adsorption is the first process that occurs after implantation of a biomaterial in the human body [33,34]. The foremost event occurring at the interface of the blood- material contact is the adsorption of plasma proteins (bovine serum albumin, fibrinogen, etc.) which subsequently influences the adhesion of leukocytes, macrophages or platelets and ultimately leads to fibrous encapsulation. Thus, the adsorption of proteins could be one of the determinants of biocompatible nature of the material [35]. The adsorption of proteins was studied as below: A known volume of protein solution of definite concentration was mildly shaken with the known quantity of nanoparticles for a definite time period and the remaining concentration of protein was monitored in the supernatant solution spetrophotometrically. The amount of the adsorbed protein was calculated with the help of the following mass balance equation. Adsorbed amount (mg m-2) = (Co-Ca) V / A

Where, Co and Ca are the concentrations of protein solution (mg per mL) before and after adsorption, respectively. V is the volume of the protein solution and A is the surface area of the adsorbent.

Haemolysis experiments were performed on the surfaces of the prepared nanoparticles as reported elsewhere [36]. In this experiment, dry nanoparticles were equilibrated in normal saline water (0.9% NaCl solution) for 24 h at room temperature and human blood was added. After 20 min, 20 mL of saline was added to stop haemolysis and the sample is incubated for 60 min at 37 0C. Positive and negative controls were obtained by adding 0.025 mL of human ACD blood and saline solution, respectively to 2.0 mL of distilled water. Incubated samples were centrifuged for 45 min, the supernatant was taken out and its absorbance was recorded on a spectrophotometer at 545 nm. The percent haemolysis may be calculated using the following relationship.

% Hemolysis = A test sample – A (-) control / A (+) control – A (-) control Where A = Absorbance.

Results and Discussion

The Chitosan-Co-lactic acid nanoparticles were synthesized by emulsion crosslinking method. Chitosan Co-lactic acid nanoparticles were formed instantaneously due to the protonated amino groups of chitosan and carboxyl groups of lactic acid electrostatically interact. The representation of nanoparticles synthesis polymer has been shown in Figure 3.

FTIR spectral analysis is a common method for investigating the intermolecular and intramolecular interaction in the nanoparticles. The observed spectra of chitosan, chitosan-colactic acid are shown in (Figures 4a & 4b). The peak assignment of chitosan was as follows (cm−1): 3468 cm-1 (O-H stretch overlapped with N-H stretch), 2922 cm-1 (C-H stretch), 1699 cm-1 (amide I band, C=O stretch of acetyl group), 1597 cm-1 (the bending vibrations of the N–H), 902 cm-1 and 1157 cm-1 (saccharide structure), 1367 cm-1 (CH3 symmetrical deformation). After the conjugation reaction, the absorption band of C-H stretch at 2922 cm-1 observably shifted to 2955 cm-1 and the absorption band of C-N stretch at 2141 cm-1 shifted to 2154 cm-1, the absorption band of O-H stretch overlapped with N-H stretch at 3468 cm-1 and shifted to 3458 cm-1. All these differences pointed out that carboxyl group of lactic acid was successfully conjugated to the amino group of chitosan. Moreover, the crosslinking of chitosan & lactic acid by gluteraldehyde also occurs via electrostatic forces.

The SEM image of nanoparticles is shown in Figure 5. A Figure 5 is clearly suggests that the chitosan-lactic acid nanoparticles has non smooth morphology, fracture surface and dispersed with aggregates.

The nanoparticles are eventually convenient for any pharmaceutical purpose or not, is decided by one of important factor i.e. the size of nanoparticles. It has been reported that the smaller the size of nanoparticles is, the larger their circulation time in blood vessel. In this study, size of nanoparticles which is characterized by TEM is shown in Figure 6, which clearly shows that an average size range of about 100- 180 nm with nearly spherical shaped nanoparticles were synthesized.

The distribution of particles was evaluated by the polydispersity index (PDI). When the amounts of PDI varied between 0.01-0.7, the dimensional distribution is narrow & in high values of PDI> 0.7 particle have a wide dimensional distribution. According to the PDI of sample (PDI 1), it could be deducted that the nanoparticles had a wide dimensional distribution with Z (Figure 7). av 334.5d-nm. The colloidal stability of nanoparticles and nanoparticle surface charge was observed by means of zeta potential measurement. The nanoparticles have a susceptibility to accumulate, which may dangerous if those particles are injected intravenously because it leads to precipitation. The value of zeta potential for nanoparticles is -14.4 mV, which indicate that nanoparticles have charged surface.

The swelling behavior of the nanoparticles are mostly affected by the amount of components, functional groups (hydrophobic and hydrophilic) of the polymer. We have studied in this part the influence of functional groups of the chitosan and lactic acid on the swelling behavior by varying the quantities of their concentrations.

The effect of variation of pH on the swelling behaviour has been examined by performing water sorption experiments at different pH 2.6, 7.4 and 9.0. The observed results are shown in Figure 8, which reveal that the swelling behaviour at different pH values gets significantly affected. It was observed that the swelling behaviour of nanoparticles was maximum at pH 2.6 and above this pH, it started to decrease, which may be explained as below. Due to low pH, nanoparticles were swelled immediately as a result of osmotic pressure and charge repulsion between the ions present in nanoparticles. The swelling behaviour of nanoparticles was more in acidic medium than the neutral and basic medium. The chitosan has pKa value of about 6.5 due to amino group which leads to a protonation and increased charge density of the nanoparticles may enhance the osmotic pressure inside the particles due to electrostatic repulsion between NH2 +- NH2 + group. This osmotic pressure difference between the internal and external solutions of the particles is balanced by the water sorption. While at higher pH, swelling ratio is decreased due to the deprotonation of the amino units of chitosan. In addition, the hydrophobic sidechain aggregation and hydrogen bonds in the copolymers are much stronger, which will also lead to lower swell ability of the samples. The results revealed that the nanoparticles changed their swelling property when the environmental pH is changed. The water permeability can be converted in response to a change in the environmental pH, it would be a desirable characteristic for a pH sensitive controlled release system with controllable swelling ability (Figure 9).

The effect of variation of temperature on the swelling ratio has been examined at varying temperatures ranging between 15 to 45 0C. The observed results are presented in Figure 10, which reveal that the value of swelling ratio significantly rises at the particular level and then start over to decrease. It may be due to the large quantity of water which was penetrated into internal matrix of nanoparticles as a result of the enhanced rate of water diffusion. The resultant initial increase in the swelling of nanoparticles may be due to the large quantity of water which was penetrated into internal matrix of nanoparticles as a result of enhance rates of water diffusion and segmental mobility of macromolecular chains [37]. However, beyond 35 0C the swelling ratio was reduced, it may be due to the reason that at high temperature the hydrogen bonds get weaken between the solvent and polymer chains.

The swelling behaviour of nanoparticles in biological fluid was investigated by using urea, D-glucose, potassium iodide (KI), Phosphate Buffer Saline and the physiological fluids such as saline water. The observed results are presented in Figure 11, which reveal that the swelling behaviour was more in PBS than the other fluids. The possible reason for the swelling behaviour of nanoparticles in these solvents may be due to the presence of ions in the swelling medium which leading to an osmotic pressure differences between the biopolymer and the external solution. The swelling behaviour is influenced by the forces of interaction between solvent molecules and polymer [38].

The swelling extent of nanoparticles on chitosan concentration is shown in Figure 12. When the quantity of chitosan is varied in the range of 0.1 g to 0.45 g in the synthesis of the nanoparticles, the swelling behaviour was significantly affected. The results are show that with an increase in the chitosan concentration their swelling behaviour increased and reaching the maximum value of swelling capacity. Which may be due to the fact that with increasing amount of chitosan, hydrophilicity of nanoparticles also increases. In addition, higher chitosan content enhances the hydrophilicity of the nanoparticles in chitosan-co-lactic acid causes a stronger affinity for more absorption of water. However, a further increase in monomer concentration (beyond 0.35 g), however results in decreased swelling. This is probably due to the reason that at much higher content of chitosan, the Nano spheres become morphologically and structurally dense that the decrease the diffusion of water molecules into the particles gets slowed down [39].

The dependence of the swelling extent of nanoparticles on amount of lactic acid was investigated by varying the amount of lactic acid in the range of 1 ml to 4 ml in the feed mixture of the nanoparticles, the swelling ratio is significantly affected. The observed results are presented in Figure 13, which indicate that the swelling behaviour was decreases with increasing amount of lactic acid. The decreasing swelling ratio is due to the fact that with increasing amount of lactic acid hydrophobicity of nanoparticles also increases. In addition, higher lactic acid content enhances the hydrophobicity of the chitosan-co-lactic acid nanoparticles causes a weaker affinity for absorption of water. The amount of free water molecules is dependent on the nanoparticle structure that ultimately influences the swelling ratio. The results may be explained by the other fact that as the content of lactic acid increases in the nanoparticles, the polymeric nanoparticles becomes dense and compact, a compact nanoparticle structure contains lower quantity of free water [40].

Loading of Drug

To determine the carrier capacity of nanoparticles with respect to drug, various batches of nanoparticles containing 0.50, 1.0, 1.5, 2.0, 2.5, 3.0 mg/ml of drug were prepared keeping the amount of nanoparticles as constant. It was observed that as the concentration of drug increases, loading of the drug also increases but after certain concentration the loading efficacy decreases due to saturation of the polymer matrix. The obtained results of cisplatin and 5-Fluoro uracil drug are shown in Tables 1 & 2.

Encapsulation Efficiency

Various batches of nanoparticles containing different composition of biopolymer were prepared keeping the concentration of drug as constant. The initial concentration of drugs plays an important role in the encapsulation efficiency of chitosan-co-lactic acid nanoparticles. The obtained results are shown in Tables 3 & 4, which shows that the increase the concentration of chitosan in nanoparticles encapsulation efficiency also increases. It was found that the encapsulation and loading efficiencies of chitosan-co-lactic acid combination nanoparticle could be attributed to the fact that chitosan-co-lactic acid has a more electrostatic attraction with drugs [41]. % EE = (A – B/ A)* 100

Invitro Cytotoxicity

The MTT Assay of L929 cells after contact with 50%, 25%, 12.5% and 6.25% extracts of Chitosan- lactic acid showed 9.51%, 9.87%, 27.79%, 43.28% metabolic activity respectively. Positive control showed 25.17% and negative control showed 93.17% metabolic activity.

Evaluation of Biocompatibility

The selection of a material to be employed as a biomaterial for a specific end use must meet several criteria such as favourable physicochemical properties, function desired, nature of the physiological environments, adverse effects in case of failure, expected durability and considerations relating to cost and ease of production. Whatever being the type of materials the biocompatibility is the foremost requirement for all biomaterials. In the present study, the assessment of in vitro biocompatibility has been made on the basis of two in vitro tests viz. BSA (bovine serum albumin) adsorption test, and heamolysis assay as discussed below:

In the present investigation, the biocompatibility of prepared nanoparticles has been judged by monitoring the amount of protein (BSA) adsorbed by the nanoparticles. The results are shown in Table 5, which indicate that the amount of adsorbed BSA decreases with increasing amount of chitosan, in the feed mixture of nanoparticles. The observed results may be explained on the basis of fact that chitosan, is hydrophilic in nature and do not provoke either any damage to blood cells or the surface of plasma proteins which are the main requirement for biocompatibility. Furthermore, it is also known that protein adsorption is more favourable onto hydrophobic surface rather than hydrophilic one. Thus, an enhanced hydrophilic nature of nanoparticles with increasing content of chitosan results in lower protein adsorption.

Heamolysis studies were performed on nanoparticles of various composition and the results are summarized in Table V, which indicate that with increasing chitosan content, the extent of heamolysis decreases. The observed results may be attributed to the reason that with change in chitosan concentration in the feed mixture of the nanoparticles, the surface composition favourably changes, which improves the blood compatible quality of the material.

Conclusion

Nanoparticles of chitosan and lactic acid were prepared by emulsion crosslinking method and characterized by various analytical techniques like FTIR spectroscopy, SEM and TEM, which confirmed the functional groups of nanoparticles, surface and size of nanoparticles. The swelling behavior of Nanoparticles was investigated in this study. It was noticed that the water sorption property was dependent on the chemical composition of nanoparticles of chitosan-co-lactic acid. With the increase in amount of chitosan up to 0.35 g, the adsorption of PBS increased, whereas, a further increase in chitosan above 0.35 g, resulted in a fall in swelling ratio. On increasing the amount of lactic acid content, the degree of swelling ratio was decreased. The extent of water sorption by these nanoparticles was found to increase from neutral to acidic pH and decrease in alkaline pH at room temperature (30 0C). A lower value of degree of swelling was observed in simulated biological fluids like saline water, KI solution and D-glucose solution. The nanoparticles were characterized by various analytical techniques like FTIR, SEM and TEM, which clearly showed the presence of various functional groups present on the nanoparticles of chitosan-co-lactic acid, morphology and the size of nanoparticles. It was observed that the water imbibition capacity was dependent on the chemical composition of the nanoparticles. The current approach is mainly based on the entrapment of an anticancer drugs into the nanoparticles to be protected and transported by the bloodstream. Polymeric nanoparticles do not provide any benefit in the treatment of disease, but rather they act as carrier for the drugs. In the present studies the addition of model drug, cisplatin and 5-fluoro uracil to polymeric nanoparticles results in 46% and 73.6% drug entrapment respectively. Also, the assessment of biocompatibility has been made on the basis of two in-vitro test viz BSA adsorption test and haemolysis assay. These assessment is indicating that these nanoparticles are suitable for biomedical application.

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Abstract

This article will give complete overview on recent development in co-process excipient technology and approaches involved in its development. The co-processed excipients help to overcome deficiencies occurring with use of single general grade excipients. Formulation scientist recognized that the single component excipient cannot always provide the essential performance in manufacturing or formulation to certain active pharmaceutical ingredient (API). Hence attention on the production of multifunctional excipient with improve performance was seen to meet the need of formulation. In order to justify rise in new drug development and also high industrial output demand new combinations of existing excipient were tested for improving excipient functionality. Now days the particle engineering of individual excipient and their combinations by using co-processing technique has provided an attribute tool for developing high functionality excipients to modern drug delivery system. The co-processing is a process with interaction of two or more excipients at sub particle level which turn to provide a synergy of functionality improvement as well as masking the undesirable properties of individual excipients. It is synergistic outcome of the combination of excipients as material property into consideration .The specific excipients are best suited for particular dosage form as a selection criterion for excipients and various excipient interaction. The excipient as like of API need to be stabilized and to be standardized along with safety evaluation parameters. These co-excipients have high functionalities as compared to the individual excipients like better flow property, reduced lubricant sensitivity and compressibility.

Keywords: Excipients; Co-Processing; Particle Engineering; Novel Drug Delivery System

Introduction

In recent year scientists have found that a single excipient cannot always provide the requisite performance to allow the certain API to be manufactured or formulated. The co-processed excipients are combination of two or more compendial or non-compendial excipients which are designed to physically modify their properties in manner that are not achievable by simple physical mixing and without a significant chemical change. Development of the co-processed excipients starts with selection of excipients to be combined, preparation method, their targeted proportion to get optimized product with desired physico-chemical parameters and minimizing avoidance with batch to batch variations. Excipient of a reasonable price has to be combined with optimal amount of functional material in order to obtain the integrated product, with a superior functionality than simple mixture of components. The co-processing is interesting because products are physically modified in special way without altering chemical structure. The fixed and homogenous distribution of components is achieved by embedding then within mini granules. Segregation is diminished by the adhesion of actives on the porous particles making process validation and in process reliable and easy control. Co-processed excipient enhances performance of established excipient that possess performance and improvements: in flow, increased surface area, compaction etc [1-3].

Types of Excipients [2,4-6]

Generally, types of excipients are classified into four types enlisted below:

i. Single chemical entity

ii. Physical mixture or blends of excipients

iii. New chemical entities

iv. Co-processed excipients

i. Single chemical entity

The single entity excipient is nothing, but the primary component used as excipient. It may contain other components like:

a. Concomitant component: is a balance between excipient composition and its functionality. These components should be considered as part of composition profile and hence not be construed as being a undesirable, nor confused with presence of added substance [2].

b. Processing Aids: are chemical substances which are used for specific processing need and benefit in excipient manufacturing process, like to improve chemical and physical process ability, to enhance a chemical synthesis reaction, to provide stabilization during the manufacturing process etc. The processing aids can be removed during the excipient manufacturing process or, depending on process clearance capability; it may remain as low level residual in final excipient. [4,5].

c. Additives: It is defined as any substance not normally consumed as a food by itself and one which is intentionally added to food, or for technical purpose in manufacture, processing, preparation or storage of such food to improve their physico-chemical properties. Normally additives are added by simple mixing process and they are incorporated in amount required to produce intended effect. Additives added not be of official grade, it should be of appropriate quality with safety been evaluated for their proposed use. Hence, the additive does not detrimental impact on excipient function and the final drug product safety/efficacy [5].

ii. Physical mixture or blends of excipients

It is a simple physical mixture or blends of two or more excipients to maintain shear processes where, individual components are mixed together without significant change. For solid mixture the individual excipients remain physically separate at particulate level. Excipient blends can be either solid or liquid after simple mixing typically for short duration [3,4].

iii. Novel excipients or new chemical entities

An excipient used for first time in formulation or by a new route of administration is classified as novel or new according to the ICH Guideline. On the other hand, this guideline defines known excipients that are commonly used and well-established usually included in pharmacopoeias and used in registered drug products. When an excipient has not previously been used in a pharmaceutical formulation then there are a number of conditions set out by the US and European regulatory authorities to allow for its use. They are not fully qualified by existing safety data with respect to currently proposed level of exposure, route of administration or duration of exposure [4,5].

iv. Co processed excipients

It is a combination of two or more compendial or noncompendial excipients that are designed to physically modify properties in such manner not achievable by simple physical mixing without significant chemical change. Various methods are available for co-processing like milling, granulation, melt extrusion, spray drying etc. The choice for the specific method/ technique will depend on the material used and desired physical properties to be achieved. Likewise, ratios of the components may vary depending on target performance to attain [2,5].

History

The key objective for co-processing was to achieve a product with additional value associated to the ratio of its functionality and cost. The aspect that occurs during the co-processing method is not understood absolutely but appears to achieve product components in intimate association with each other as single entity. This association cannot be achieved by simple dry blending but requires appropriate co-processing technique.

Primarily, co-processing concept was implemented in food industry to improve stability, dispersibility, gelling properties of food additives. Few co-processed combinations used were Micro crystalline cellulose (MCC) and galactomanan with glucomannan, MCC with Carboxy methyl cellulose (CMC) and sodium alginate complex.

In pharmaceutical industry, the co-processing concept was established in early 1990 with the introduction of co-processed combinations like MCC and calcium carbonate. Cellactose is a co-processed combination of cellulose and lactose, ProSolv is a combination of MCC and colloidal silicon dioxide [7,8].

Excipients Selection Criteria in Co-Processing

The combination of excipient selected for co-processing should compatible and non-reactive to each other. For example, two hexitol group excipient Mannitol: Sorbitol are co-processed to achieve good compressibility as tablet excipient where first is poorly compressible and less hygroscopic and later is good compressible but highly hygroscopic to create balance between plasticity and brittleness. A few other examples of co-processed excipients studied are combination of Lactose: PVP K30: Starch as a ready to use excipient for tablet with diluents, binder and disintegrant property, MCC: Sodium starch glycolate for direct compression immediate release formulations, Dibasic Calcium Phosphate dihydrate co-processed with Pre gelatinised starch for direct compression with binder and diluents property [9-12].

Regulatory Aspects in Co-Processed Excipient

Excipients combinations through co-processing do not produce any chemical modification in the incorporated excipients. Individual properties of excipient are retaining in the combination, the only novelty is the improved functionality with same physical form. Hence, the co-processed combination does not require any toxicological study and can be considered safe if the parent excipients are declared safe by the regulatory agencies GRAS. Very few co-excipients are described in official monograph like Dispersible Cellulose in B.P., Compressible Sugar in U.S.P/NF. Their non-compendial status is the major hurdle in the success of coprocessed excipient in the market. This obstacle will be overcome in the coming future as with New Excipient Safety Evaluation Procedure (NESEP), and excipients now could be reviewed outside the FDA drug approval process. Affirmative opinion from experts of IPEC team will limit the risk of FDA dismissal of drug based on excipient and could encourage innovation in the pharmaceutical industry [13,14].

Current & Future Status of Co-process excipients

At present co-processed excipients have less applicability in the pharmaceutical industry but surely these will be utilized with enhanced functional properties at low concentration in future. Very few new chemical entities are being introduced in market due to stringent rules and regulations by considering safety, efficacy, and cost. Also, very few improvements in existing excipients will help in increased use of co-processed excipients in coming future. Uprising technologies and many more modification are implementing daily in Pharmaceutical industry to achieve high production rate. A growing popularity of high functionality excipients counterpart tablet machine increasing speed capabilities, to modulate the permeability, solubility, or stability of drug, increasing performance expectations related to disintegration, dissolution, bioavailability etc. makes a fantastic opportunity for the development of novel drug delivery system. Forthcoming novel excipient combinations and new co-processing techniques without any doubt going to attract both in research and pharmaceutical industry [15,16].

Fundamental solid-state properties of excipient

The solid-state properties are characterized by three levels as molecular, particle and bulk. These levels are closely linked with each other, reflecting change in one level with another level shown in (Figure 1). The first molecular level is crystal lattice and include phenomenon such as polymorphism, pseudo polymorphism and amorphous state. The second level consist of individual particle size, shape, porosity, and surface area. The third is bulk level comprises of a group of particles and their physical properties. The individual levels depend over each other which provides framework for the development of novel combination of existing and new grade of excipients. The essential solid-state properties of particles like morphology, size, shape, porosity, density, surface area influence excipient functions such as flowability, dilution, lubrication and disintegrating potential, compressibility and Compatability.

The primary characteristic associated with excipient is that no chemical change occur during co-processing and also the reflected changes showed in the physical properties have to prove that coexcipient do not show any chemical change. There should not be covalent bond formation when these individual ingredients are combined to form co-excipient. Also, stereochemical environment, reorientation, bond breaking and the intermolecular forces responsible for new shapes confirms the formation of a new composition. Analytical techniques for characterization used like particle size distribution, specific surface area, SEM images and X-ray diffractogram (XRD) etc. However, the techniques NMR, HPLC, FTIR, DSC, are used in order to aid control their structural properties [17-19].

Methods Of Coprocessing [20-25]

i. Spray Drying.

ii. Solvent Evaporation.

iii. Crystallization.

iv. Melt Extrusion.

v. Granulation/Agglomeration

i. Spray Drying

This technique is applicable for transformation of feed from fluid state into dried particulate form by spraying the feed into the hot drying chamber. It involves continuous drying operation processing. The feed may be in the form of solution or suspension. The dried product can be obtain in powders, granules or agglomerates depending upon it’s physical and chemical properties, dryer design and final powder properties desired. The spray drying combination of ingredient under solid or dry form during drying, by atomizing active compound in suspension or solution form (Figure 2).

ii. Solvent Evaporation

It involves use of liquid as a vehicle to dissolve excipient in organic solvent in which it is immiscible. A core excipient material are also microencapsulated in dissolved or dispersed liquid phase to obtain appropriate size microparticles. The mixture is then heated if necessary, to evaporate the solvent. Once all the solvent gets evaporated, the mixture temperature is reduced to ambient temperature (if required) with continuous agitation. Also, the particles can be used in isolated form as powders or coated sub states. The obtained material can be either water soluble or insoluble depending on material property. After complete drying of obtained mixture is in the form of new single entity co-excipient [26,27].

iii. Crystallization

Crystallization is a solid crystals formation process of precipitating naturally or artificially from the solution and rarely deposited directly from a gas. It is a chemical solid-liquid separation technique, in which mass transfer of solute from the liquid to crystalline solid is obtained and it must be supersaturated. It means that solution has to contain more solute dissolved, unless equilibrium i.e. Supersaturation is achieved. It can be achieved by various methods such as, solution cooling, addition of second solvent to reduce solubility of the solute or by chemical reaction. Change in pH is one of the most common method used in industry for crystallization.

iv. Hot Melt Extrusion

The technique involves mixing of co-processed excipients with meltable binder on a water bath, and then cooling agglomerates to solidify the mass which is subsequently pass through sieve for size reduction. It is a solvent free technique and requires a less processing time. The concentration of selected excipient ratio is a critical variables in the process. The process is a formation of small pellets, beads from the molten mass which is an extruded through extruder. Schematic presentation shown below (Figure 3).

v. Granulation /Agglomeration

The granulation is a process of forming or crystallizing into grains. The granules typically have size range between 0.2 to 4.0mm depending on their use. The Agglomeration is synonymous to granulation process involves particle size enlargement technique to alter product properties. The Agglomeration process is widely used to enhance physical properties like flowability, bulk density, wettability, and product appearance. In pharma industry two types of granulation technologies are employed, mainly dry and wet granulation. Wet granulation is a more preferred method for co-processing (Table 1).

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JUNIPER PUBLISHERS- ACADEMIC JOURNAL OF POLYMER SCIENCE

Abstract

Glutathione may prevent age-related, oxidative damage to ocular tissues but has poor corneal penetration. Hydrogel formulations were investigated to determine an optimized ocular delivery system. The rheology and texture profile of formulations were investigated at 25°C. The 1% w/v glutathione was incorporated into systems demonstrating desirable characteristics for ocular vehicles and physical characteristics re-determined. In vitro cumulative glutathione delivery across the bovine cornea was measured over 8 hours at 32ºC in Franz diffusion cells. Carbopol-containing Poloxamer systems exhibited shear-thinning behavior desirable for ocular formulations whilst Polyvinyl alcohol (PVA) and Polyacrylic acid (PAA) systems exhibited Newtonian behavior. Of the glutathione-containing systems, 0.2% w/v Carbopol 1342 was the most viscous with a viscosity of 960cP at a shear rate of 100sec-1. All formulations significantly increased the amount of glutathione delivered across the cornea (relative to an aqueous solution of glutathione) with the exception of 0.1% w/v Carbopol 940 (p=0.12). Formulations containing 0.1% w/v Carbopol 934, 0.1% w/v Carbopol 1342 and 0.2% w/v Carbopol 940 improved penetration dramatically (ca. 30%); but were not significantly different from each other. Therefore, Carbopol and Poloxamer formulations demonstrated enhanced penetration of glutathione across the cornea. The 0.2% w/v C940-containing-Poloxamer formulation was determined to be the most promising for ocular delivery of glutathione.

Keywords: Glutathione; Hydrogel; Ocular delivery; Rheology; Newtonian behavior; Sustained release; Permeation

Introduction

Glutathione (γ-glutamyl-cysteinyl-glycine; GSH) is a highly hydrophilic thiol tri-peptide with a low-molecular weight of 307.4Da [1,2] The reduced form of GSH serves as a strong intracellular antioxidant [1,3] and plays an important role in the protection of the eye from oxidative stress [4]. In addition to its antioxidant activity, glutathione serves as a cofactor of enzymes involved in the degradation of peroxides, such as glutathione peroxidises. Moreover, as a component of NADPH pathway, it prevents cell components from being oxidized thus promoting DNA regulation and protein synthesis [3]. Franco et al [5] also reported glutathione’s role in the regulation of cell-cycle, signal transduction and immune response [5]. Given that all ocular tissues contain GSH as a primary antioxidant, replenishing the amounts of GSH in the eye could reduce the incidence of ocular diseases related to oxidative stress and aging, such as cataracts. The cornea is particularly susceptible to chemical insults and damage because it is the outer-most layer of the eye and therefore exposed to the harsh environment. GSH maintains normal hydration, protects the cellular membrane and degrades xenobiotic agents [4] in the cornea. Therefore, GSH may play a role in the treatment of keratitis and other corneal diseases [4].

Due to anatomical and physiological characteristics, the eye is uniquely shielded and a highly protected organ that presents many barriers to effective ocular drug delivery [6]. The main physical barriers shielding the eye are tear film and cornea, which protect the anterior of the eye, Tear film is a protective layer that prevents the entry of foreign molecules into the eye. The blood-retina barrier, which protects the back of the eye [7]. The cornea is the main barrier against drug penetration to the inner tissues of the eye due to its small surface area and relative impermeability [8]. In addition, many ocular enzymes in the cornea could degrade glutathione and prevent its therapeutic effect. These enzymes include endopeptidases (plasmin, collagenase) and exopeptidases, such as the hydrolytic aminopeptidase, which degrade amino acids [9].

The main advantages of stopical administration are its ease and convenience of use and localized drug effects, thus, reducing systemic absorption and avoiding enzymatic degradation through hepatic first pass metabolism [10]. However, the natural ocular defense mechanisms lead to poor bioavailability. Blinking increases the production of the tear film’s aqueous layer which in turn increases tear turnover to approximately 1μL/min, which is equivalent to 16% of the tear film turnover [11]. Both the removal of excess fluid and tear turnover reduce the extent of drug absorption. Conventional eye drops require frequent administration, with each drop being more than 30μl, which is estimated to be the maximum volume the eye can accommodate without overflow. Drainage of instilled solution occurs within 15–30 seconds after administration [12], therefore the majority of the drugs administered are removed before absorption. Although ocular ointments have the viscosity to increase pre-ocular drug retention time, they often cause irritability to the eye, resulting in lower compliance. In addition, their oily medium is not compatible with water soluble drugs such as GSH. Consequently, an ocular formulation with desirable characteristics that shows promising potential would be required to effectively deliver glutathione. This delivery system should prevent enzymatic degradation of glutathione, enhance its pre-corneal retention and penetration.

Hydrogels can be formed by dispersing polymers in an aqueous medium where they undergo swelling to produce a viscous gel capable of increasing pre-corneal retention time, prolonging contact time by increasing mucoadhesion and controlling drug release [12]. These properties increase trans-corneal absorption and hence the amount of drug reaching the anterior chamber of the eye. Poloxamers are non-ionic triblock copolymers comprised of ethylene oxide and propylene oxide, which are non-irritating to the ocular surface. With favorable characteristics such as strong hydrogen-bonding, high MW and sufficient flexibility to interact with the mucus network contribute to excellent mucoadhesion. Another defining characteristic include their ability to increased stability of drugs, Carbopol (CP) is a synthetic high-MW polymer comprised of acrylic acid that are cross-linked with either allyl ether of pentaerythritol or allyl sucrose [3]. They are anionic and therefore offer increased mucoadhesion hence contact time [13]. In addition, CP is reported to open the cellular tight junctions and could promote trans-corneal penetration of glutathione [14]. PVA has been used extensively in ocular formulations due to its ability to maintain ocular osmotic pressure while PAA has been widely used as a viscosity enhancer [2]. In this project, we developed a novel hydrogel formulation which composed of CP, PAA and PVA, and used as a carrier system for ocular delivery of GSH. This delivery system is aimed to enhance the permeation of the GSH over the cornea, thus, to maximize the bioavailability of GSH via ocular administration.

Materials and Methods

L-glutathione reduced≥99% was purchased from Sigma-Aldrich (USA). P407 and P188 were purchased from BASF (Germany). C934NF were from Noveon (USA) while C940NF and C1342NF polymer were purchased from Lubrizol (USA). PVA came from Applichem (Germany) and PAA was from Polyscience Inc (USA). Sodium hydroxide (NaOH) was from Scharlau (Spain). All other chemicals and solvents were of analytical grade. Bovine eyes were obtained from Auckland Meat Processors (New Zealand) and stored at20°C until required.

Methods

Preparation of Formulations

The CP formulations were prepared by adding the required amount of CP to 35mL of milli-Q water and were continuously stirred until the CP was completely dissolved. The solution was cooled to 4°C and the required amount of Poloxamer 407 (P407) and Poloxamer 188 (P188) were added with gentle stirring. The formulation was stored in the refrigerator and stirred every 30 minutes until the Poloxamers were completely dissolved. Milli-Q water was added to make up to a final volume of 50mL. The pH of the formulations was then adjusted to 7.4 by the addition of a small amount of 1M of NaOH. (Table 1) lists the samples prepared using the method mentioned above.

PVA formulations (Table 2) were prepared by adding the required amount (w/v) to 35mL of milli-Q water, which was then heated to 50°C under constant stirring until all PVA was dissolved and made up to 50mL with milli-Q water. The pH was adjusted to 7.4 by adding a small amount of 5M NaOH. The corresponding concentration of PAA samples were added according to (Table 2) and dilutions were made with milli-Q water. The control used in the screening of the formulations was milli-Q water.

The polymer combinations were shown in (Table 3). The CP formulations containing glutathione were prepared by dissolving 0.5g of glutathione in milli-Q water. Once glutathione was dissolved, the required quantity of CP was added. The solutions were continuously stirred until CP was completely dispersed and the Poloxamers were prepared according to the method described above. The control for samples mentioned in (Table 3) was aqueous solution of glutathione. Simulated tear fluid (STF) was prepared based on the method used by Hagerstrom et al [15]. Based on the physical parameters obtained, several potential formulations with glutathione will be investigated in this study.

Characterization

Rheological Properties

The rheological properties were examined using the Brookfield DV-III+ rheometer (Brookfield Engineering Laboratories, Inc. USA) with spindle 40 for PVA and PAA samples and spindle 52 for the CP formulations. This was carried out in a temperature- controlled environment at 25±1°C to simulate average room conditions. Each sample went through 20–40 loops of shearing, in between which there was a 10 second delay. The speed of shearing increased by 5rpm at each loop and the torque was kept between 9 to 90% by manipulating the starting rpm to ensure accuracy of data. The range of rotational speed was between 1 and 394 rpm corresponding to a shear rate range between 2 and 788s-1.

Mechanical and Mucoadhesive Properties

TA-XT plus texture analyzer (Stable Micro Systems, UK) with a 5kg load cell at 25±1°C was used. Texture analyzer was calibrated. For the measurement of the mechanical properties, a 10mm diameter delrin cylinder probe was twice compressed into each formulation at the rate of 2 mms-1 to a depth of 5mm with a trigger force of 0.1g. A delay of 15 seconds was allowed between the compressions. Data collection and calculations were gathered from the Texture Exponent 3.0.5.0. The force-time plot measured mechanical properties such as hardness, compressibility, adhesiveness and cohesiveness. This experiment was conducted in triplicate.

Hardness is measured by the peak force of the first compression cycle. Compressibility is measured as the positive force area during the first compression of the probe [3,16]. Adhesiveness is the negative force area of the first compression [16]. Cohesive force of each sample is the ratio of the first and second positive force area of the two consecutive compressions.

Mucoadhesive force measurements were performed on freshly prepared bovine cornea using TA-XTplus texture analyzer (Stable Micro Systems, UK) with a 5kg load cell at 25±1°C. The cornea was stabilised on the mucoadhesion test rig. A small amount of sample was applied to the 10mm delrin cylinder probe surface, after which the probe was lowered at a constant speed of 2mms-1 and a trigger force of 5g. After 10 seconds of contact, the probe was moved away at a speed of 0.5mms-1, generating a curve. The area under the curve (AUC) was calculated by the Texture Exponent 3.0.5.0 program. The work of mucoadhesion was calculated through Equation 1[3]. Each measurement was conducted in triplicates.

where πr² is the surface area of the cornea.

Permeability Studies

Ex-vivo drug permeation studies were performed by Franz diffusion cell (VTC 200, Logan Instrument Corp). The thermostat of the Franz diffusion cells was set at 32±1°C to mimic the ocular surface temperature. Bovine cornea is placed between the donor and the receptor chamber. Twelve mL of STF served as the dissolution medium in the receptor chamber which functioned as the reservoir. One mL of formulation was placed in the donor chamber and the cap was covered with parafilm to prevent evaporation. Samples (0.4mL) were collected at predeterminate time intervals and replaced by 0.4mL of STF for the first four hours, after which a greater amount of sample was withdrawn and replaced in order to maintain sink condition. UV spectrometer was blanked with STF at the wavelength of 215 nm and the absorbance of each sample was obtained. The absorbance for samples presented in Table 3 was measured and concentrations were calculated. In order to evaluate the penetration rate, the apparent permeability coefficient (Papp) was calculated, using the following equations.

where ΔQ / Δt is the steady-state of the linear portion of the graph which presents the amount of drug in the receptor chamber versus time, A represents corneal area available for diffusion (1.766cm2), Co is the initial glutathione concentration in the donor chamber and 60 is the conversion factor from minute to second [2,17]. The linear branch of the permeation data was determined using correlation analysis. A minimum of six data points in the linear branch were taken to calculate the flux, J, (μg.cm-2s-1) by linear regression. The flux was then divided by the concentration in the donor (μg.cm-3) in order to calculate Papp (cm.s-1) [2,18].

Cryo-Scanning Electron Microscopy (Cryo-SEM)

Cryo-SEM was used to evaluate the hydrogel structure at swollen state. Images were obtained through Philips XL30S FEG (Field Emission Gun) SEM (Netherlands). Cryo unit, Gattan Alto 2500 was employed, including a fracture stage and a sputter coater, with a coating temperature of less than -120ºC. Samples were placed on a brass specimen holder and heated to allow thermogellation. The sample were frozen by using liquid nitrogen (-200ºC) and then cut and allowed to heat to -90ºC under vacuum. This allowed the frozen water in between gel pores to evaporate, generating the clear structure of polymer hydrogel. The surface of the sample was sputtered with gold for 4 minutes at -120ºC, to minimize any charge builds up, after which the samples were viewed under the cryo-SEM.

Statistical Analysis

Statistical data was analyzed using Microsoft Excel 2007, with two-way variance analysis (two-way ANOVA) and pair wise comparisons were performed using t-tests. p<0.05 indicates a significant difference.

Results

Characterization of Rheological Properties

Hydrogel ocular formulations should ideally have a viscosity of around 1000–5000 cP in order to maximize pre-ocular retention time and the delivery of glutathione. Formulations with similar viscosities were plotted together for clear and apparent analysis. The 0.3% C1342 formulation was not included as it was too viscous and the method that was used to evaluate rheological properties was not suitable [19]. All CP dispersions exhibited non-Newtonian shear-thinning (pseudoplastic) behavior (Figure 1) i.e. decreasing viscosity with increasing shear rate.

It was observed that the increase in viscosity with increasing CP concentration was proportionally similar at 0.1% and 0.2% for C934 and C940 at a shear rate of 40s-1. Conversely, C934 exhibited a much greater increase in viscosity at 0.3% compared to that of C940 (Figure 2). The viscosity difference appears to be proportional between 0.1% and 0.2% of C1342 and 0.2% and 0.3% C934 at the shear rate of 40s-1. Moreover, the 0.1% and 0.2% C940-containing formulations were the least viscous, while the respective C934 formulations appeared to be three-times more viscous than C940-containing formulations (Figure 2). The difference in viscosity between all formulations at different concentrations appears to be significant apart from that between 0.1% C934 and 0.2% C940 and between 0.1% C1342 and 0.3% C940.

The difference in viscosity at different concentrations for each CP was statistically significant (p<0.05) (Figure 3A, 3B & 3C). The C940 exhibited the most proportional increase in viscosity with increasing concentration (Figure 3B); while the viscosities of C934 and C1342 appeared to have been more dramatically affected by the change in concentration. The rheological characteristics of PVA and PAA formulations are shown in (Figure 4). PVA and PAA results were analyzed separately from that of the CP formulations because their viscosity range was vastly different. The PVA and PAA formulations demonstrated Newtonian flow, where a linear relationship between shear rate and shear stress was evident (Figure 4) [7]. Furthermore, the Newtonian flow properties showed that the viscosity of these formulations remained constant despite increasing shear rate. The 25% PAA is approximately eight-times more viscous than the diluted 12.5% PAA and has a much higher shear stress than all the other three formulations. Similar to the CP systems, the viscosity of PAA and PVA also increased with increasing concentration. Again, there was a significant difference between the viscosities of these simple chain polymers where PAA appears to be more viscous than PVA. The 25% PAA displays a viscosity which is comparable to 0.1% C940-Poloxamer formulation after shearing (Figure 2).

Based on the optimal ocular formulation characteristic described by existing literatures available, the formulation demonstrating the most desirable rheological properties contains the 0.2% C940, it exhibited the lowest viscosity at the lowest shear rate investigated.

Characterization of mechanical and mucoadhesive properties

The hardness and compressibility of the hydrogel system during formulation screening increased with an increase in CP concentration. This trend can be observed for all CP-containing formulations (Figure 5A & 5B). Significant increase in hardness for C940 and C1342-containing systems was observed at concentration of 0.2% compared to concentration of 0.1% and there was significant difference compared to control (p<0.05). However, no significant differences between the CP systems and control at 0.1% (p>0.05) were observed. At 0.3% and 0.5%, all CP systems were significantly different, compared to control (p < 0.05). Conversely, PVA showed no distinctive change compared to control and no significant differences between the different concentrations was observed (p>0.05). PAA exhibited a significantly lower hardness compared to control (p<0.05), however, there was no difference when concentration was doubled. The hardness of the hydrogel in the presence of PVA was no different when it was compared to all 0.1% CP formulations (p>0.05). The results obtained were comparable to the previous literature data [20]. The compressibility of the hydrogel in the presence of C1342 at 0.3% and 0.5%, as well as C940 0.5%, was significantly different when compared against control (p<0.05). Similar to hardness, PAA showed a significantly lower compressibility in comparison to control (p<0.05).

A relationship exists between the increase in adhesion and a decrease in cohesion of formulations (Figure 6A & 6B), with a concentration increase. At 0.1%, C1342-containing formulations were significantly higher compared to control (p<0.05). However, adhesion of all CP-containing formulations increased with an increase in concentration. Conversely, for PVA and PAA formulation, no such trend can be observed with increase in concentration, and statistical analysis showed no significant difference when compared to control (p > 0.05). At 0.1%, 0.2% and 0.3% of C934 and C94 containing systems, the cohesive forces were significantly higher when compared to control (p<0.05), but this was not demonstrated at a concentration of 0.5% (p>0.05). Cohesion of 0.1% and 0.2% of C1342-containing formulations were significantly higher to control (p < 0.05). The cohesion forces of 0.1%, 0.2% and 0.3% of C934-containing formulations were significantly different compared to 0.5% of C1342, C940 and 0.1% of C940 respectively (p<0.05). The PVA and PAA formulations demonstrated higher cohesive forces than control (p<0.05) and showed a concentration dependent relationship similar to the CP formulations. There was no significant difference in cohesion between PVA and control (p>0.05), but both PAA concentrations had significantly lower cohesion compared to control (p<0.05).

The measured apparent mucoadhesion showed a difference between the control and all formulations screened (Figure 7). It also showed a consistent increase in mucoadhesive force with increase in concentration, however, there was no significant difference between different CPs at concentrations 0.1% and 0.3%. At 0.2%, only apparent mucoadhesion of C940 was significantly different to that of other CPs. At 0.5% all CP formulations were significantly different from each other. PVA and PAA showed a similar trend, PAA at 25% obtained similar results to 0.5% C934 containing formulation.

Corneal permeability studies

The cumulative diffusion profiles of each various hydrogel formulations of glutathione over eight hours are shown in (Figure 8–10). All of the hydrogel systems showed a linear diffusion trend and showed a significantly higher diffusion when compared to the control (p<0.05). The permeation of 0.1% C940 formulation was significantly less than 0.1% C934, 0.1% and 0.2% C1342 (p<0.05). The remaining formulations did not show any differences when compared to each other. The ranking order of the different formulation groups in their ability to promote glutathione diffusion across bovine cornea is: 0.2% C940 and 0.1% C1342 > 0.1% C934, 0.2% C1342 and 0.2% C934 > 0.1% C940 > Control.

The apparent permeability coefficient (Papp) and Flux values were calculated and shown in (Figure 11). From the Papp of the different formulations measured, the permeability of glutathione was increased compared to water (p<0.01). The 0.2% C940 (p=0.03) and 0.1% C1342 (p=0.05) had an approximately 1.2-fold higher corneal permeability than 0.1% C940. All other hydrogel formulations did not show differences in their permeability (p > 0.05). The average Papp values of the hydrogels were in the range of 1.21–1.57 × 10–4 cm.s-1 compared to 0.64×10–4cm.s-1 produced by the control. This shows that the CP combinations enhance the permeability of drug by up to a 2.4-fold (p<0.05).

The Cryo-SEM images demonstrate the polymeric structure of the achieved CP hydrogel systems. Images of C934 and C1342 hydrogel systems with incorporated glutathione were obtained (Figure 12). Although, attempt was made to obtain images of C940 hydrogel structure, this could not be achieved due to experimental error. An air bubble was formed during preparation and, as it was cut, no formulation remained on the metallic rib for imaging; thus, the images of C940 hydrogel structure were not obtained and displayed.

Discussion

In order to increase the bioavailability of glutathione (and hence achieve post-corneal concentration), the pre-corneal residence time or penetration of the ocular formulation must be extended beyond that of the conventional formulations [21]. This may be achieved by increasing the viscosity, spreadability or mucoadhesion of the formulations; or by incorporating permeation enhancing materials into the formulation. The simplest approach is to enhance the viscosity because a two-fold increase in maximum bioavailability of a drug can be achieved. Therefore, viscosity is often considered as the major factor influencing ocular retention [12]. The increase in retention time of instilled formulations is apparent, once viscosity is greater than 10mPas but a very high viscosity results in ocular discomfort and epithelial damage can occur from blinking [22]. The difference in the effective number of polymers evaluated was likely the reason for the higher shear stress observed in this experiment. Upon the addition of NaOH solution to adjust pH to 7.4, carboxyl groups on the carbomers (pKa 3–5) are ionized, leading to electrostatic repulsion and dramatically increasing the rigidity of the formulation structure [23]. Thus, the ionized carboxylic groups could have also interacted with the small but very positive sodium ions, leading to a reduction in viscosity and shear stress. This theory could have been validated had the rheological characterization been carried out at both pH 5 and pH 7. Although Davies et al. [12] found the viscosity profile of PVA to be pseudoplastic in contrast to our findings; the conditions they used were extreme (85oC) and would not occur in the eye. Moreover, Davies et al. [12] prepared their PVA solution with PBS, introducing ions that possibly change the interactions between the molecules. Nevertheless, C934 was found to have greater viscosity-enhancing capability than PVA. Structurally, PAA are CPs without cross-link units and can be described as linear polymers (pKa~4.5) [24]. Although PAA has similar rigidity as the carbomers, it is less viscous because the strength provided by cross-links is much more robust.

The structures of hydrogels are highly dependent upon the interaction between the polymers and the swelling medium [25]. The mechanical properties of the hydrogel formulations were assessed through hardness, compressibility, adhesion and cohesion. The ability to prolong ocular contact time was measured through its strength of mucoadhesion [20,26,27]. The hardness is desired to be low in order for the formulation to be easily administered onto the ocular surface [27,28]. Therefore, lower concentrations of CP formulations (0.1% and 0.2%) are favoured. The “hardness” of anionic gels is dependent on the molecular network density and the degree of cross-linking, which is correlated to the polymer concentration, as well as viscosity [29–31]. C1342-containing hydrogels exerted the highest “hardness” out of all three CPs. This is likely to be due to increased cross-linking between its long alkyl methacrylate chain and allyl ethers of pentaerythritol and its high molecular weight compared to C934 and C940 [32]. Similarly, C934 with relatively lower molecular weight was the easiest formulation to deform due to its reduced rigidity. Additionally, surface molecules have a net inward force due to the cohesive forces being stronger than adhesive forces with the air, therefore a surface energy is created in parallel to the surface. This requires energy to break, which would have been measured as hardness [33–35]. The hardness of PAA was lower than the control. This may be explained through the accumulation of PAA molecules on the surface, which would push the liquid molecules into the bulk, leading to reduction in surface tension [33,34]. Compressibility claimed to measure the ease of spread of the formulations over the corneal surface; where a lower value was favorable. With increasing concentration, total resistance was greater hence more force was required as the parameter quantifies deformation under shear and compression [28]. The claimed “cohesion” is described as the spatial reformation of the hydrogel structure after successive compressions. It attempted to measure the relaxation of the polymers, which is dependent on time and the deformation [36]. The “cohesion” of the formulation hoped to examine the ability of the hydrogel to reform its structure after application to the ocular surface, where a high value was desired for maintenance of its structural integrity [27]. A general decline in cohesive force is shown with increase in concentration, which is due to the increase in mass of the polymers [37]. The adhesion is defined to be the interaction between different molecules within the hydrogel. This estimates the ability of the formulation to adhere to the target site [20]. At higher concentrations, there were more polymers present with greater capability to form chemical bonds [19]. The results showed a plateau at concentrations 0.3% and 0.5% for all CP-containing formulations. This may be explained by the high cohesive forces within the hydrogel structure to prevent adhesive forces forming with the probe [28]. The degree of mucoadhesion is dependent on the hydration, anionic charges, molecular weight and cross-linking of the polymer [32,38]. All CP-containing formulations were combinations of Poloxamers and CPs. This combination increased its mucoadhesive properties as the polyether components of Poloxamers were able to form tertiary carbon bonds with polyacrylic acid units of CPs, which may potentially expose different binding groups to increase mucoadhesive interactions [34,39]. Moreover, the CP-containing systems at 0.1% and 0.2% had a greater degree of swelling, therefore increased hydration. The polymers are flexible and free to diffuse to come in close contact with the corneal surface and interpenetrate with the mucus layer, to create a strong entangled network through non-covalent interactions [19,38].

The cumulative amount of glutathione that diffused across the cornea from the original administered dose (1mL), after eight hours ranged from 50.6% to 80.1% from the hydrogels compared to 45.0% from the control system. This is very high compared to the 5% or less, which is typically seen to reach the aqueous humour in living human eyes [40]. This is due to the absence of other ocular barriers such as tear turnover, tear dilution, lacrimation and nasal lacrimal drainage in the Franz diffusion cell apparatus [40]. The glutathione control group follows a linear trend across the corneal epithelial membrane and represents zero-order kinetics where diffusion was at a constant rate throughout the experiment. Conversely, the glutathione-loaded CP-containing hydrogels show a biphasic release profile. The initial phase was attributable to the release of glutathione from the outer pores of the gel surface and the second phase showed the sustained release of glutathione from the matrix of the polymer. This release profile shows benefits on application, rapid diffusion of glutathione through the corneal epithelial membrane will occur, followed by a sustained diffusion over time.

Conclusion

Glutathione is a potent anti-oxidant that is essential in the maintenance of tissue health. By replenishing the tissues with glutathione, it is hoped that the progression of ocular damages can be halted or even reversed. Hydrogels composed of CPPoloxamer, PAA and PVA were formulated and investigated as potential delivery systems for glutathione across the cornea. The CP-Poloxamer combinations exhibited favourable pseudoplastic behavior and cohesion/adhesion properties. These were further investigated for their ability to increase glutathione permeability across excised bovine corneas using a Franz diffusion cell. Penetration of glutathione across the cornea was significantly increased compared to the plain drug solution. The 0.2% C940- containing-Poloxamer formulation was determined to be the most promising for ocular delivery of glutathione. To study the efficacy of glutathione formulations on the reversibility of cataracts, in vivo tests can be performed in the future.

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