Modified low molecular weight poly-vinyl alcohol as viscosity enhancer

Materials Today Communications 21 (2019) 100634

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Modified low molecular weight poly-vinyl alcohol as viscosity enhancer a,b,⁎

a,b

a

a

T

a,c

Gemma Leone , Marco Consumi , Simone Pepi , Alessio Pardini , Claudia Bonechi , ⁎ Gabriella Tamasia,c, Alessandro Donatia,c, Claudio Rossia,c, Agnese Magnania,b, a b c

Department of Biotechnology, Chemistry and Pharmacy, University of Siena, via A. Moro 2, Siena 53100, Italy INSTM, via G. Giusti 9, 50121 Firenze, Italy CSGI, via della Lastruccia 3, 50019 Sesto Fiorentino, Italy

A R T I C LE I N FO

A B S T R A C T

Keywords: PVA STMP Viscosity enhancer Eye drop Human tears

Dry Eye Disease (DED) is a multifactorial disease that provokes a reduced vision quality and eye fatigue. It is often treated with artificial tears that must have enhanced viscosity in comparison with human tears. Phosphorylated low molecular weight PVA was synthesized to obtain a viscosity enhancer for the production of artificial tears. Three different concentrations were analyzed, verifying that, thanks to the combined action of low molecular weight and presence of anionic groups inserted along the chains, formulations can be sterilized by filtration without losing their viscosity properties, stability and transparency (light trasmittance > 85%). Formulations were characterized by Infrared and UV spectroscopy, thermal analyses and rheology. The developed viscosity enhancer shows adequate viscosity values for the foreseen application. Indeed, its viscosity is higher than natural tears (1.5 mPa.s) but lower than 30 mPa.s (sticky sensation) at the shear stress values the eye is subjected. It shows a mean viscosity value of 20 mPa.s at shear rate 1 s−1 (interblinking phase: open eye), and 10.5 mPa.s at 10,000 s-1 (blinking phase).

1. Introduction Tear film (TF) guarantees the integrity and health of the ocular surface, which is a complex structure comprised of cornea and conjunctival tissues [1]. TF protects the eye against external attacks, such as infections caused by different microorganisms and guarantees the cornea receives sufficient nutrients and oxygen [2]. However, various factors as aging, hormonal changes, pathologies, habits as contact lens wear, computer use or alcohol consumption [3,4] can led to tear evaporation or a decrease in tear quantity, causing a multifactorial syndrome called Dry Eye. Dry Eye Disease (DED) is defined as “a multifactorial disease of the ocular surface characterized by a loss of homeostasis of the tear film, and accompanied by ocular symptoms, in which tear film instability and hyperosmolarity, ocular surface inflammation and damage, and neurosensory abnormalities play etiological roles” [5]. DED affects over 20% people worldwide with a high impact on the quality of life. It is estimated that a total cost of $3.8 billion is spent each year for the treatment of DED only in the United States [6]. Despite the etiology, dry eye main symptoms are reduced vision quality, eye fatigue, redness [2] and it is managed with various types of artificial tears [7]. Eye drops must be as comparable as possible with human tears that are a shear-thinning solution, consisting of a

⁎

mucous layer, an aqueous layer, and a lipid layer which covers the surface at the aqueous–air interface [8,9]. Artificial eye drops must fulfill several requirements, lubricate the surface of the eye, remain on the surface of the eye as long as possible and delay the appearance of dry spots to not be used too often [10]. They should possess sufficient viscosity and an adequate rheological behavior, to lubricate the surface and remain as long as possible. This is generally obtained using lipid based empty or medicated nanocarriers [11,12] or, more frequently, polymeric solutions. Sodium hyaluronate (NaHA) and sodium carboxymethylcellulose (CMCNa), being able to increase tear film stability by virtue of their viscoelastic and anionic properties, are the most utilized viscosity enhancer [13,14], even if other polymers, such as poly vinylalcohol (PVA) or poly-vinyl pyrrolidone (PVP), whose compatibility with eye compartment is known [15], are widely used. However, the majority of eye drops formulations have been made using low concentrations of high molecular weight polymers. These high molecular weight polymer solutions cannot be sterilized by filtration and they can go under fragmentation during steam sterilization. This treatment affected not only their physical properties but generated fragments that can act as endogenous danger signals [16]. This study seeks to develop an artificial drop formulation based on low molecular weight modified PVA. The optimal behavior played by

Corresponding author at: Department of Biotechnology, Chemistry and Pharmacy, University of Siena, via A. Moro 2, Siena 53100, Italy. E-mail addresses: [email protected] (G. Leone), [email protected] (A. Magnani).

https://doi.org/10.1016/j.mtcomm.2019.100634 Received 19 June 2019; Received in revised form 5 September 2019; Accepted 6 September 2019 Available online 09 September 2019 2352-4928/ © 2019 Elsevier Ltd. All rights reserved.

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Scheme 1. Schematic representation polymer phosphorylation.

of

Fig. 1. A: IR spectra of pristine PVA 31 kDa polymer (P31: grey) and its phosphorylated derivative (P31P: black); B: magnification of the 1800–1000 cm−1 region of the spectra, where it is possible to appreciate major differences between P31 and P31 P polymers.

2. Materials and methods

polysaccharides as viscosity enhancer is due to their anionic nature, PVA has been modified adding, along its chains, phosphate moieties which can both stabilize the pH without altering the osmolality and give to the polymer an adequate shear thinning behavior thanks to its anionic structure. Moreover, since phosphate moieties are chemically bound to the chains, the risk associated with the use of phosphate based buffering systems is reduced. Phosphate buffer systems are often used to regulate osmolality. Recent studies have shown that a high concentration of phosphate buffer may cause irreversible corneal calcification with visual loss. Bernauer et al. [17] analyzed fifty-nine samples of commercially eye drops finding that 44% of commercial products had a phosphate concentration above physiological levels (> 1.45 mmol/L) and, even if the definition of a critical phosphate concentration is difficult, particular attention should be paid in using soluble phosphate salts. Therefore, in this work, PVA 31 kDa was phosphorylated and 4%, 10% and 20% solutions in NaCl 0.9% tested as viscosity enhancer. The effect of phosphorylation on water interaction capability, thermal stability and transparency of pristine PVA was also evaluated.

2.1. Materials PVA (MW 31 kDa; hydrolysis degree: 89%), Trisodium trimetaphosphate (STMP) (MW 305.9 Da) and all the other reagents were purchased from Fluka Sigma-Aldrich (Switzerland). All solvents were of analytical or HPLC grade. 2.2. PVA phosphorylation A 5% w/v solution of poly(vinyl alcohol) (PVA) was prepared in basified water adding NaOH 2 M drop by drop until pH 12 was reached to deprotonate hydroxyl moieties. Trisodium trimetaphosphate (STMP) in a molar ratio of 1:1 with respect to PVA was added. After addition, pH = 10 was regulated. Solution was magnetically stirred for 2 h and then lyophilized. The freeze-dried polymer was dissolved in water and dialyzed against water for 48 h and, once purified, lyophilized again. The phosphorylation degree was determined by a spectrophotometric assay using a commercial kit (Test Spectroquant Merck 2

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Table 1 Main wavenumbers observed in the IR spectra of P31 and P31 P together with their assignments. Sample

wavenumbers (cm-1)

assignments

P31

3328

Intra and inter molecular H-bonded OH stretching CH2 asymmetric and symmetric stretching COOR stretching CH2 bending Crystallinitya CeO + Alcoholic OH bending Intra and inter molecular H-bonded OH stretching CH2 asymmetric and symmetric stretching COOR stretching CH2 bending eOeP]O stretching CeO + Alcoholic OH bending PeO bending PO3 +PeOeC stretching

2945-2913-2850 1730 1439 1147 1098 3339

P31P

2945-2916-2856 1736 1436 1287 1101 1016 921

a stretching mode of CeO and CeC of a portion of the chain where an intramolecular hydrogen bond is formed between two neighboring OH groups that are on the same side of the plane of the carbon chain [26].

KqaA– Darmstadt – Germany – ISO6878/1 and US-standard Methods 4500-P-E) following the producer instruction. Calibration curve was constructed using five non zero points in the range of 0.5 mg/L-10 mg/L (R2 = 0.9999), using a standard phosphate solution. Each sample (15 mg) was heated up to 900 °C and the obtained residue was dissolved in 10 mL of bi-distilled water. The absorbance of the resulting solutions was quantitatively quantified at λ = 713 nm, after dilution. The absorbance was recorded trough a dual-beam (against water) UV-Perkin Elmer Lamda 25(optical pathway, 10 mm; cuvettes, PMMA/UV grade). The phosphorylation degree was expressed as mg of P per mg of polymer. 2.3. Infrared analysis FTIR spectra of dried pristine (P31) and modified polymer (P31 P) were recorded with a Thermo Nicolet IS20 apparatus equipped with an attenuated total reflection accessory (ATR) and a 45° Germanium crystal as internal reflection element, purging the instrument with nitrogen. The spectra were acquired between 4000 and 750 cm−1 and 64 scans at a resolution of 2.0 cm−1 were averaged. FTIR spectra were analyzed using the OMNIC™ software [18]. 2.4. Thermogravimetry (TGA)

Fig. 2. A: thermograph of pristine PVA 31 kDa polymer (P31); B: thermograph of phosphorylated PVA (P31 P); derivatives of weight as a function of temperature of P31 and P31P.

Thermogravimetric Analysis (TA) was performed using a Q600 thermogravimetric analyzer (TA Instruments-Waters, USA). Sample, 15 mg in weight, was inserted in a platinum pan and heated from 30 °C to 900 °C with a heating ramp of 10 °C/min under nitrogen. Sample was set up in three replicates [19].

“blinking shear rate” (shear rate: D = 10,000 s−1) can be calculated using the following formula [21]:

2.5. Rheological analysis

where σ is stress (Pa) and K = σ for D = 1 s−1 Then viscosity can be quantified:

σ = K•DN

η = σ/D

P31 P was dissolved in NaCl 0.9% w/v to prepare three solutions with increasing concentration: 4% w/v (P31P4), 10% w/v (P31P10) and 20% w/v (P31P20). The obtained solutions were filtered through a 0.22 μm filter and subsequently subjected to flow-test and creep-test. The apparent viscosity of the samples (22 mL in volume) was determined by a flow test performed in a shear rate range 1–100 s−1, at 37 °C, using a controlled strain rheometer equipped with a concentric cylinders system (Discovery HR2, hybrid rheometer, TA Instruments, Leatherhead, United Kingdom) [20]. Applying the Power law model to the formulation viscosity curves, the rate index N, also called fluidity index, can be obtained and subsequently the apparent viscosity for

The zero-shear viscosity (η0), i.e. the viscosity of the material when no shear stress is applied, was obtained by a creep test. Creep test was performed subjecting the material at a fixed stress value of 0.1 Pa for 500 s. To confirm that the stress value 0.1 Pa falls within the range of linear responses of the material, a creep test was also performed at 0.05 and 0.5 Pa, to verify that the response of the material is the same despite the applied stress [22].

3

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2.7. Statistical analysis The quantitative data are presented as the mean value ± standard deviation of three replicates. OriginPro 8.0 was used for statistical analysis. Multiple comparisons were performed by one-way ANOVA and individual differences tested by Tukey's test after the demonstration of significant intergroup differences by ANOVA. Differences with p < 0.05 were considered significant [24]. 3. Results and discussion 3.1. PVA phosphorylation PVA phosphorylation by STMP is schematized in Scheme 1. Phosphorylation conditions were chosen basing on authors previous observations [15,25]. PVA molecular weight deeply affects the phosphorylation process. Low molecular weight PVA (i.e. 27 kDa and 31 kDa) can be crosslinked by STMP only working with concentration higher than a threshold of 5% w/v. Polymer solution was basified to pH 12 to guarantee the deprotonation of hydroxyl groups but the reaction was conducted at pH 10 since this pH value favors a high phosphorylation degree whereas counteracts the crosslinking process. Reaction was ended after 2 h since previous studies demonstrated that longer times did not affect the phosphorylation degree on PVA polymers [26] differently from what observed by Benjaurau et al. [27] for polysaccharides. 3.2. Infrared analysis Infrared analysis was performed to verify the introduction of phosphate moieties along the chains. P31 and P31 P spectra are depicted in Fig. 1A. The main wavenumbers observed in the IR spectra are summarized in Table 1 together with their assignments. Both spectra show intermolecular and intramolecular hydrogen bonded OH band that is slightly shifted towards higher wavenumbers in P31 P spectrum, indicating a lower involvement in hydrogen bond network. Both spectra show asymmetric and symmetric stretching of CH2 band in 2945-2850 cm−1 range [28] and a band at 1730 cm−1 due to C]O stretching of acetyl groups, according with P31 relatively low hydrolysis degree (89%). Nevertheless, its intensity is significantly higher in P31 spectrum in comparison with IR spectrum of P31 P. The C]O band intensity significantly decreases after phosphorylation because of the high pH value used to deprotonate hydroxyl moieties (P31 P). A first significant difference is found in the band at 1147 cm− 1 that is generally used to evaluate the crystallinity degree. As explained by Mansur et al. [29], it is related to the stretching of CeO and CeC involved in an intramolecular hydrogen bond between two OH moieties on the same side of the plane, and its intensity is affected by the crystalline portion of the polymeric chains [29]. The lower intensity of 1147 cm−1 band, together with the OH band shift towards higher wavenumbers in P31 P spectrum, confirmed a partial disruption of the hydrogen bond network that is imputable to the involvement of part of hydroxyl groups in phosphorylation of the polymer, being deprotonated hydroxyl groups the attaching points of phosphate moieties. The most evident difference between P31 and P31 P spectra is the presence in the latter of very intense bands due to phosphate moieties, at 1287 cm−1 and at 1016 cm−1 (O]PeO stretching and PeO bending, respectively) [28,29], thus confirming the phosphorylation reaction (Fig. 1B). UV quantification indicates a degree of phosphorylation of 2.8·10−3 ± 0.3·10-3 mg of P per mg of polymer.

Fig. 3. A: Viscosity curve of 4% aqueous solution of P31 P (P31P4); B: Viscosity curve of 10% aqueous solution of P31 P (P31P10); C: Viscosity curve of 20% aqueous solution of P31 P (P31P20).

2.6. Trasmittance Light transmittance through the P31P4, P31P10 and P31P20 solutions was measured using a UV–vis Spectrophotomer (Perkin Elmer Lamda 25; optical pathway, 10 mm; cuvettes, PMMA/UV grade). Three scans from 700 to 380 nm were averaged. Transmittance was expressed as light trasmittance percentage [23].

3.3. Thermal analysis Thermographs of native (P31) and phosphorylated PVA (P31 P) are depicted in Fig. 2. The total weight loss of the two polymers, registered 4

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Table 2 Fluidity index (N) and viscosity values as a function of shear rate of P31 P solutions compared with a hyaluronan based commercial tear drop formulationa and human tearsb. ηA (mPa.s)

Sample

P31P4 P31P10 P31P20 Human tears Commercial tearsa

N

D=0 s-1

D=1 s-1

D=100 s-1

D=10000 s-1

1.037 0.9877 0.9932 ———————— 0.9686

2.63 ± 0.04 20.8 ± 0.4 48.1 ± 0.3 7 ± 2c

4.11 ± 0.04 19.42 ± 0.09 47.8 ± 0.9 —————— 24.4 ± 0.3

2.53 ± 0.04 18.5 ± 0.2 45.2 ± 0.4 ————————— 18.3 ± 0.7

5.76 ± 0.04 10.5 ± 0.2 38.7 ± 0.6 1.5b 13.9 ± 0.6

All data are significantly different (p < 0.05). a [30]. b [34]. c [33].

stretching are also increased, thus permitting an increase of chains hydration as already observed for other polymers [30]. In 200–400 °C range, we can observe thermal decomposition of free aliphatic chains and elimination of volatile products. The relative weight loss for P31 and P31 P are 74.6 ± 0.4% and 49.7 ± 0.3%, respectively (p < 0.05). The third weight change, occurring between 400–600 °C, is related to structured portions of the polymer chains, and P31 and P31 P lose 17.6 ± 0.9% and 30.7 ± 0.6%, respectively (p < 0.05). The relative weight loss recorded in second and third temperature ranges permitted us to further confirm PVA phosphorylation. Indeed, the presence of phosphate groups along the chains drastically affects the residue and decomposition fragments composition, increasing the heavier component percentage. Since phosphorylation significantly affects thermal behavior of the polymer, weight derivative was also plotted versus temperature to evaluate if a qualitative change in temperature, at which the main weight losses are recorded, occurs (Fig. 2C). In 200–400 °C range, where a degradation of free aliphatic chains occurs, peaks at 317 °C and at 296 °C are observed for P31 and P31 P, respectively. This shift can be attributed mainly to the difference in chemical composition of the systems, with a large contribution played by the hydrogen bond network destabilization due to the introduction of phosphate groups, as evidenced also by infrared analysis. Thanks to the presence of phosphate moieties the temperatures at which the weight loss of heavier chains is detected (400–600 °C) shifted towards higher values (423–440 °C and 436–450 °C for P31 and P31 P, respectively).

Fig. 4. Compliance as a function of time of P31P4, P31P10 and P31P20 solutions.

in the 30–900 °C range, is 100 ± 1% for P31 (Fig. 2A) and 93 ± 2% for P31 P (Fig. 2B). This first result confirms the presence of inorganic component in the second one that cannot be volatilized in the analyzed temperature range. For both P31 and P31 P three main weight loss steps are present. The first one, 30–120 °C, is related to the loss of free bulk water and hydration water. The former is dependent on the humidity state of the sample whereas the latter is related to polymer chemistry being the water strictly bound to the polymer chain. P31 showed a weight loss of 0.88 ± 0.02% whereas P31 P showed a significantly higher weight loss of 3.79 ± 0.04% (p < 0.05). Phosphate moieties along the chain provoke an increase of polymer hydrophilicity. Moreover, being the polymer negatively charged, the chain repulsion and

3.4. Rheological analysis The obtained polymer was dissolved in physiological solution (0.9% NaCl) to obtain three different polymer concentrations, 4%, 10% and 20% w/v, with an osmolality of 305 ± 2 mOsm/kg, close to human tear osmolality (304 ± 23 mOsm/kg [31]). The presence of phosphate

Fig. 5. Light Trasmittance (%) along visible spectrum of P31P4, P31P10 and P31P20 solutions. 5

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must be higher than 7 mPa.s (natural tears) without exceeding 30 mPa s beyond which there is blurred vision and irritation. Viscosity values of the P31 P solutions are significantly higher than that of natural tears, reaching the threshold value of 10 mPa.s, which has been demonstrated to be the critical value from which retention began. Rheological analyses indicate that, among the different concentration solutions, P31P10 is the only one still valuable as potential component of eye drops formulation. Furthermore, it shows increased rheological performance with respect to hyaluronan based commercial tears drops.

moieties covalently bound to the chains avoid their dispersion. Indeed, despite corneal calcification is dependent on several factors, high topical free phosphate ions concentrations can have an impact on the process of corneal calcification. In an animal model, rapid corneal calcification developed after treatment with 148 mmol/L solution [17], whereas a large area of calcification was found in humans after treatment with 50.9 mmol/L solution. Therefore, the definition of a critical phosphate concentration is difficult, even if it could be useful to maintain it as close as possible to that of human tears (1.45 mM). No phosphate ions release was quantified by P31P4, P31P10 and P31P20 solutions as a function of time. However, a quantification of the phosphate concentration after possible complete detachment from the chain, simulated using the heaviest conditions, i.e. heating, are measured and a maximum value of 2.5 mM, 6.3 mM and 12.6 mM was found for P31P4, P31P10 and P31P20, respectively. P31P4 and P31P10 show a phosphate concentration in the same order of magnitude of human tears whereas P31P20 one order higher. Tear drops should have specific rheological behavior to properly absolve their role. As widely reported, shear thinning fluids seem to have beneficial effect since exhibit low viscosity under high shear rates (blinking phase) avoiding excessive stresses for ocular surface and high viscosity at rest (interblinking phase), thus guaranteeing an increase in retention. Jossic et al. [10] demonstrated that shear thinning products are found to be more effective than Newtonian products, having a positive action with regard to maximum breakage time and maximum film homogeneity over the pupil to ensure visual comfort [10]. To verify that the developed preparations fulfil all these requirements, a flow step analysis was performed before and after the sterilization by the passage through a 0.22 μm filter. Viscosity curves were superimposable, thus confirming the applicability of such a sterilization process to low molecular weight PVA solutions. Viscosity curves obtained after the passage through the filter are depicted in Fig. 3. Power law model was applied to the formulation viscosity curves and the rate index N obtained. The fluidity index of P31P4 solutions is higher than 1 (1.037) indicating a weak shear thickening or dilatant behavior (Fig. 3A). Contrarily, P31P10 and P31P20 can be considered as shear-thinning solution (Fig. 3B-C). Beside the rheological behavior, adequate viscosity values must be shown. Indeed, an increase in tear volume steady value (i.e. 7 μL) occurs after eye drops instillation, but the excess volume is quickly cleared with a rate of 20%/min [32]. This results in a low effectiveness of eye drops. The best way to increase residence time is using systems with increased viscosity with respect to human tears. Since increasing fluid viscosity may also cause discomfort and damage to ocular epithelia due to an increase in the shear stresses during blinking, new vehicles should have specific viscosity values. Tiffany et al. [33] analyzed healthy human tears and found that human tears can be classified as shear thinning solution with a zero-shear viscosity of 7 ± 2 mPa.s (Table 2). Zero shear is the shear rate value the tears are subjected to when the eye is closed. Zhu et al. [34] indicated that artificial drops should have a viscosity higher than natural tears (1.5 mPa.s) but lower than 30 mPa.s to avoid a sticky sensation for the patient, even if they found that the retention began only after the fluid viscosity exceeded a threshold value of about 10 mPa.s. The shear rate on the cornea may differ considerably, it changes in the range 1–10,000 s−1. When the eyes are open, the shear rate depends on the gravitation only and it can be valuated approximately 1s−1. During blinking the shear rate is around 10,000 s−1 [30]. Fluidity index permitted to calculate the apparent viscosity for “blinking shear rate” (shear rate 10,000 s−1). The fluidity index and the apparent viscosity at a shear rate of 0, 1, 100 and 10,000 s−1 are summarized in Table 2. To determine the zero-shear viscosity it is necessary to identify the region in which the deformation is constant over time (dγ/dt = constant) and for which the viscosity corresponds to η0. The range was identified in the time interval from 120 s to 500 s. Compliance is plotted against time in Fig. 4. As stated above, the values of zero shear viscosity

3.5. Trasmittance The visible light transmission through P31 P solutions was higher than 85%. Thus, they can be considered a transparent solution and, consequently, suitable for the realization of eye drops. In Fig. 5, the UV spectrum in visible range is depicted. Among the three solution, only P31P20 shows values of trasmittance under the threshold percentage to be defined a transparent substance (i.e. 85%) along all the visible spectrum. 4. Conclusion Previous studies indicated that PVA modified with STMP did not have any toxic effect towards different cell lines, i.e. human anterior capsule cells, primary human microvascular endothelial cells adult (HMVECad) and normal human osteoblasts and articular chondrocytes [15,25,35,36]. So, phosphorylated low molecular weight PVA was synthesized to obtain a viscosity enhancer for the production of artificial tears or medicated eye drops. Phosphorylation process was verified by infrared spectroscopy and phosphorylation degree quantified by UV spectroscopy (of 2.8·10−3 ± 0.3·10-3 mg of P per mg of polymer). Three different concentration formulations were realized taking into account to have a phosphate ion concentrations not exceeding 10−3 M after possible complete degradation by the lyases present in the body, to avoid calcification and/or opacification. Thermal analysis indicated that the presence of phosphate groups increases the hydrophilicity of the material without altering the transparency of the polymer. Solutions at a concentration between 4% and 10% allow a light transmittance higher than the threshold value of 85%. This study has revealed that only P31P10 shows rheological properties adequate to the application as eye drops. Indeed, its viscosity in blinking phase (10,000 s-1) is higher than the threshold value of 10 mPa·s, but lower than the value which induces a sticky sensation and irritation (i.e. 30 mPa·s). Basing on these preliminary but promising results, next step will be to start the procedure to project and follow an in vivo model analysis (subjected to approval of Ethics Committee of our Institute) to confirm the capability of P31P10 polymer to correctly behave as viscosity enhancer in artificial drops formulations. Data availability Upon request to corresponding author. Declaration of Competing Interest None. Acknowledgements This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Authors would like to thank INSTM for the support. 6

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