Dry eye disease is a relatively common ocular problem, which causes eye discomfort and visual disorders leading to a decrease in the quality of life. The aim of this study was to find a possible excipient for eye drop formulations, which is able to stabilize the tear film. A cationic thiolated polyaspartamide polymer, poly[(
Dry eye disease (DED) has been reported to afflict 7–33% of the population, thereby reducing their quality of life. For normal vision, continuous moistening of the ocular surface is needed. Important roles are played in this by a sufficient quality of tears, maintenance of the normal composition of the tear film, normal lid closure, and regular blinking [
DED is accompanied by changes in mucin distribution and glycosylation, a dysfunction of MUC4 and MUC5AC and a high calcium level [
One way to stabilize the tear film in cases of DED is to use liquid thiolated polymer formulations, whose structures are similar to those of ocular mucins, as they are protein-like polymers bearing a considerable number of thiol groups. The solutions of such polymers are therefore able to mimic the physiological properties of mucins, such as tear film stabilization. The formation of disulphide bonds with the mucus layer leads to strong mucoadhesion, which may be further strengthened by the formation of ionic bonds between the cationic groups of the excipient and the anionic groups of the mucins. The strong adhesion promotes a prolonged residence time and a protective effect for the corneal/conjunctival epithelium. Liquid formulations also serve as lubricants, prolonging the breakup time of the tear film. Moreover, thiolated polymers have antioxidant and radical scavenging properties and can therefore be useful excipients in artificial tear formulations for the therapy of DED [
We earlier described thiolated poly(aspartic acid) (ThioPASP) polymers, which are biocompatible [
For the synthesis of the polymers, L-aspartic acid (Merck, extra pure), phosphoric acid (Sigma Aldrich, 99%), cysteamine (Acros Organics, 95%),
The precursor polymer of cationic ThioPASP, polysuccinimide (PSI), was synthesized by the thermal polycondensation of L-aspartic acid in a solvent-free reaction at high temperature and reduced pressure. PSI and cysteamine were dissolved in DMF under a nitrogen atmosphere and the solution was stirred for 72 h at room temperature. An excess of
Synthesis of cationic ThioPASP-DME polymers.
Osmolality and pH were measured in 10% w/w aqueous solutions of ThioPASP-DME. Osmolality measurements based on the freezing point depression of a solution were carried out with an automatic osmometer (Knauer Semimicro Osmometer, Germany) in 3 parallels. 150
The pH of ThioPASP-DME solutions prepared with distilled water was determined with a pH meter (Testo 206-pH2, UK) [
Optical tests were performed by the measurement of transmittance with a UV-spectrophotometer (Thermo Scientific Evolution 201 UV-Visible Spectrophotometer, Thermo Fischer Scientific, Shanghai, China) in the wavelength range 200–800 nm. In our investigations, the thickness of the samples was 10 mm. The transmittance in aqueous solutions of ThioPASP-DME was determined at 10% w/w.
The
The wettability of ocular surfaces with cationic ThioPASP formulations (10% w/w ThioPASP-DME polymers in PBS) was studied with an OCA Contact Angle System (Dataphysics OCA 20, Dataphysics Inc., GmbH, Germany). Microscopic slides were covered with 20
The effect of the oxidative agent on the polymer solutions and the interaction between the polymer solution and the ocular mucin were investigated by rheology. The rheological properties were studied with a Physica MCR101 rheometer (Anton Paar, Austria). The measuring device was cone and plate type (the diameter was 25 mm, the gap height in the middle of the cone was 0.046 mm, and the cone angle was 1°). ThioPASP-DME was dissolved in PBS and the gelation test was initiated by the addition of model oxidant. For the investigation of the interaction between the polymer and the ocular mucin, the polymer was mixed with a mucin dispersion in PBS and in the presence of 20% w/w model oxidant (the final mucin concentration was 5% w/w, while the final polymer concentration was 10% w/w). As blank measurement, the polymer solution without mucin was measured. The structural changes in the formulation were characterized by frequency sweep tests. The oxidative effect on the eye can induce gelation, and the interactions between ThioPASP-DME and mucin can also result in structural changes; the storage modulus (
Tensile test also provides information on the interfacial interaction of the polymer and the ocular surface. Measurements were performed with a TA-XT Plus (Texture analyser (ENCO, Spinea, I)) instrument equipped with a 1 kg load cell and a cylinder probe with a diameter of 1 cm. The force and work needed to separate the polymer solution from the ocular surface are measured, which can characterize the strength of the interaction. Three different test conditions were used: the ocular surface was modelled (1) with 50
The porcine conjunctiva was obtained from a slaughterhouse, freshly detached from the connective tissue and stored at −20°C until the measurement. 10 parallel measurements were carried out. Test conditions were as follows: 20
The results were evaluated and analysed statistically with GraphPad Prism software (version 5). One-way and two-way ANOVA (with Bonferroni posttests) analysis were applied [
During ocular drug delivery formulation, several excipients are used which can change the physical and physiological properties of the ocular surface and the stability of the tear film [
Osmolality and pH of aqueous ThioPASP-DME solutions (10% w/w).
ThioPASP-DME degree of modification |
Osmolality (mOsm/L) in water |
pH |
---|---|---|
10 |
|
8.79 |
10 |
|
6.07 |
20 |
|
8.80 |
30 |
|
8.77 |
Reference |
|
6.65 |
Aqueous solutions of ThioPASP-DME polymers showed strong hypoosmolality (<100 mOsmol L−1), while the reference system was close to isotonic (301.4 mOsmol L−1). The solutions were alkaline (pH > 7). In order to modify the pH of the polymer solution close to that of the tear film (pH = 7.4), the synthesis was extended with a neutralization step. As a result, the pH of this polymer solution was approximately the physiological pH and that of the reference system (pH = 6.07), while the osmolality increased but remained hypoosmotic (<200 mOsmol L−1).
Transmittance spectra of 10% w/w ThioPASP-DME solutions were determined to study the effects of the solutions on the vision. The transmittance curves are depicted in Figure
Transmittance of ThioPASP-DME solutions.
The ThioPASP-DME solutions are not colourless but slightly yellow, though the transmittance is high over almost the whole range of the visible spectrum. There was no significant effect of the degree of modification (composition) of the ThioPASP-DME. Interestingly, the polymer solutions exhibited a noteworthy UV cut-off at 350 nm; this behaviour can be favourable in the event of eyes exposed to heavy UV radiation.
The refractive indices of the ThioPASP−DME 10, 20, and 30 and the reference solutions were 1.3483, 1.3491, 1.3499, and 1.3350, respectively.
As it is intended to use the ThioPASP-DME solutions in liquid eye drops, their spreading on the ocular surface is an important aspect. In our tests, the ocular surface was modelled with a microscope slide covered with a mucin dispersion. The measured contact angles are to be seen in Figure
Contact angles of ThioPASP-DME solutions.
The results indicate that the tested polymer compositions provide favourable wetting conditions on the model surface, because the contact angle is <90°.
The ThioPASP polymer solutions exhibited
No gelation was observed in the case of ThioPASP-DME solutions. The
Frequency sweep tests were performed with the aim of determining any synergetic interaction between the ThioPASP-DME and the mucin (Figure
Frequency sweep tests of (a) ThioPASP-DME 10, (b) ThioPASP-DME 20, and (c) ThioPASP-DME 30 with (filled symbols) or without (open symbols) mucin.
A minor increase in
Force was measured as a function of displacement during tensile tests. The adhesive force (the maximum in the curve) and the work of adhesion (the AUC) were calculated [
(a) Adhesive force and (b) work of adhesion of ThioPASP-DME solutions under (□) blank, (■)
Comparison of the blank with the
The mucoadhesivity of the new polymers was compared with that of hyaluronic acid solutions. HA as viscosity enhancing agent has been investigated for years as an active component of formulations applied in DED. Sodium hyaluronate increases the residence time and the precorneal tear film stability and the corneal wettability. It also decreases the evaporation rate of the tear film and improves the healing mechanisms of the cornea [
Under
Work of adhesion of polymer solutions under
DED is a multifunctional disease involving the tears and the ocular surface, associated with an increased osmolality of the tear film and inflammation of the ocular surface. The two most common causes of DED are insufficient tear production and excessive tear evaporation, both of which lead to hyperosmolality, ocular damage, or discomfort [
Because of the multifactorial pathology of DED, the therapy tends to be very varied. In the main treatments, artificial tears are used, especially preservative-free products, but unfortunately these provide only palliative therapy. In the event of inflammation, artificial tears are combined with oral omega-3 supplements, mucin secretagogues, short-term steroids, and daily cyclosporine A. When the DED is more severe, autologous serum, oral tetracyclines, prosthetic lenses, and systemic immune-suppressants are administered [
Osmolality has been deeply investigated in DED and is considered to be a very important factor. The osmolality of the tears in a normal eye is 310 to 334 mOsm L−1, but in DED the osmolality is higher. One aim of artificial tears is to counter this hyperosmolality, but the effect is generally only temporary. The osmolality of artificial tears is usually in the interval from 181 to 354 mOsmol L−1 [
In the treatment of DED, stabilization of the tear film is also very important. The tear film is stable for only a short time, because it ruptures in consequence of the concentration gradients and dispersion forces on the mucus layer. The rupture results in the loss of moisturization of the cornea, so that dry spots are formed, which irritate the corneal nerve endings and induce blinking. Thanks to the eyelid movements, a new tear film spreads over the eye surface. The dispersion forces, the interfacial tension, and the viscous resistance of the mucus layer affect the duration of rupture of the mucin layer and the breakup time of the tear film [
When all of these factors are taken into consideration, it appears clear that most of the physicochemical properties of the optimum eye drop formulation must be similar to those of the tear film and it must be hypoosmotic to balance the hyperosmotic tears in DED.
In this work, we synthetized and characterized ThioPASP-DME, cationic thiolated polyaspartamide bearing both cationic tertiary amine and redox-responsive thiol pendant groups, as a potentially mucoadhesive and tear film-stabilizing excipient in the therapy of DED. The aim was the synthesis of a mucin analogue polymer which can interact with the ocular mucin via disulphide linkages and the ionic interactions between the positively charged polymer and the negatively charged mucosal surface. Thanks to these complex interactions, a continuous polymer network is formed on the surface, thereby preserving the tear film with maintenance of the hydration of the ocular surface. We assume that ThioPASP-DME polymers can function as ophthalmic drug demulcents, defined in US Food and Drug Administration (FDA) monograph 21 CFR 349 as water-soluble polymers applied topically to protect and lubricate mucous membrane surfaces and to temper dryness and irritation.
We first investigated the physiological acceptability of our formulations. Eye lubricants are recommended to be neutral or slightly alkaline. The pH of the ThioPASP-DME polymer solutions (pH = 8.7–8.8) was higher than that of normal tears (pH = 7.4) and could be therefore adjusted by using hydrochloric acid.
The polymer solutions (10% w/w) were hypoosmotic (87–90 mOsmol L−1) allowing the addition of other components, which is favourable in the therapy of DED. The neutralization process resulted in lower pH but higher osmolality (183.67 ± 1.25 mOsmol L−1), which is in the range of the osmolality of artificial tears (from 181 to 354 mOsmol L−1) [
Optical tests were performed in order to determine the degree of visual disturbance caused by these polymer solutions. The transmittance of the polymer solutions is slightly modified over a broad range of the visible spectrum and their refractive indices approximate to that of the tears. Thus, they do not greatly affect the quality of vision, while in addition they have a partial UV-filtering effect, which can be favourable in ophthalmic therapy.
The polymer solutions can readily spread on the simulated eye surface, as indicated by the low contact angles. This means that the formulations have the ability to establish strong interactions with the surface and to resist elimination immediately after administration.
The ThioPASP polymers are redox-sensitive and undergo gelling in response to oxidative stress or agents [
Tensile tests likewise verified the good adhesion of the polymer solution to the ocular surface. Besides hydrogen bonds, thiolated polymers are able to form covalent bonds with the cysteine-rich subdomains of mucin. We additionally immobilized other side groups with cationic, positively charged groups, so that ionic interactions can also occur [
ThioPASP-DME polymers showed better mucoadhesion compared with conventionally used HAs in DED, while the viscosity of their solution was not elevated.
We successfully adjusted the properties of ThioPASP-DME (pH and osmolality) to the desired physiological levels thereby resulting in a possibility to decrease side effects such as irritation and dehydration. In consequence of their similar structure to that of mucin, ThioPASP-DME solutions also have the ability to stabilize the tear film. They can interact with the ocular mucin and provide strong adhesion, ensuring an improved residence time and prolonged hydration of the ocular surface. Further beneficial properties of the polymer solutions, such as good spreading on the ocular surface, marked transmittance, and a partial UV-filtering effect, can provide new possibilities in the therapy of DED.
The authors declare that there are no competing interests regarding the publication of this paper.
This project was supported by the European Union and cofinanced by the European Social Fund (Grant TAMOP-4.2.2.A-11/1/KONV-2012-0035) and also by the New Széchenyi Plan (Grant TÁMOP-4.2.1/B-09/1-2010-0002).