Ureasil–polyether hybrid film-forming materials

Colloids and Surfaces B: Biointerfaces 101 (2013) 156–161

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Ureasil–polyether hybrid film-forming materials L.K. Souza a,b , C.H. Bruno a,c , L. Lopes d , S.H. Pulcinelli d , C.V. Santilli d , L.A. Chiavacci a,∗ a

Faculdade Ciências Farmacêuticas, UNESP, Araraquara, Sao Paulo, Brazil Johnson & Johnson Consumer Companies, São José dos Campos, Sao Paulo, Brazil c Department of Health Sciences, UFES, São Mateus, ES, Brazil d Institute of Chemistry, UNESP, Araraquara, Sao Paulo, Brazil b

a r t i c l e

i n f o

Article history: Received 4 January 2012 Received in revised form 5 June 2012 Accepted 11 June 2012 Available online 29 June 2012 Keywords: Film-forming material Hybrid materials Sol–gel

a b s t r a c t The objectives of this work were to study the suitability and highlight the advantages of the use of cross-linked ureasil–polyether hybrid matrices as film-forming systems. The results revealed that ureasil–polyethers are excellent film-forming systems due to specific properties, such as their biocompatibility, their cosmetic attractiveness for being able to form thin and transparent films, their short drying time to form films and their excellent bioadhesion compared to the commercial products known as strong adhesives. Rheological measurements have demonstrated the ability of these hybrid matrices to form a film in only a few seconds and Water Vapor Transmitting Rate (WVTR) showed adequate semiocclusive properties suggesting that these films could be used as skin and wound protectors. Both the high skin bioadhesion and non-cytotoxic character seems to be improved by the presence of multiple amine groups in the hybrid molecules. © 2012 Elsevier B.V. All rights reserved.

1. Introduction One of the greatest challenges for pharmaceutical research is the development of new technology platforms for drug delivery that overcome the therapeutic limitations of conventional pharmaceutical forms. New technology platforms for drug delivery aim for usage flexibility, which includes the ability to be loaded with different drugs at varying dosages and to have tunable release profiles [1,2]. The most extensively used pharmaceutical form for a systemic effect is an oral one, which has some therapeutic inconveniences, including patient compliance, gastrointestinal side effects and first pass effect. Additionally, the skin is very important for the administration for many pharmaceuticals [3], and the Dermal Therapeutic Systems (DST) and Transdermal Therapeutic Systems (TTS) represent alternative routes for drug administration. These drug delivery platforms typically employ a patch, which has a backing layer, an adhesive, a drug reservoir and a removable protection liner. An assortment of patches is available on the market that have varying levels of structural complexity. Specifically, the most complex ones have liquid-filled reservoir devices, and the least complex ones have a drug-in-adhesive matrix [4–6]. The film-forming materials technology is a novel approach for drug delivery, and although further research is needed, it is an

∗ Corresponding author at: Faculdade de Ciências Farmacêuticas, UNESP, 14801902 Araraquara, Sao Paulo, Brazil. Tel.: +55 16 33016966; fax: +55 16 3306960. E-mail address: [email protected] (L.A. Chiavacci). 0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2012.06.009

innovative and promising breakthrough option for the substitution of DTS and TTS [7]. All the complexity related to product manufacturing and usage could be simplified by a film-forming solution that is able to build a thin and transparent film on the skin. Only a few studies in the literature report these systems and their applications as skin and wound protectors or as drug-delivery devices [7,8]. Usually, film-forming materials are formulations that contain an organic polymer dissolved in ethanol, polyvinyl alcohol or other appropriate organic solvents [7]. Most film-timing formation relies on the evaporation rate of the solvent and is subject to change pursuant to the functioning of some parameters, such as room temperature and skin moisture. In addition, the relationship between the structure of the polymeric matrices and the final properties of film-forming systems has been poorly explored in the literature. The use versatility of organic polymeric solutions as films-forming substance is also restrict once the ability of control of the drugdelivery behavior is strongly dependent on the chemical nature of the drug and the polymer, being necessary adjustments in the formulation for each drug. Presently, some commercial formulations of film-forming materials are used only as tissue glue in the surgical field or as a wound or skin protector [9–11]. In this context, organic–inorganic hybrid matrix materials that are formed by ureasil cross-linked polyethers (Scheme 1) have high transparency, mechanical resistance and flexibility, which are properties that are highly attractive for cosmetic applications. Previous studies showed the ability of ureasil–polyether hybrid materials to host a variety of drugs molecules. These hybrid systems have a high drug loading capacity and have the possibility

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cross-linked nodes. The hydrolysis of –(SiOEt)3 was initialized by adding 1 mL of a water/ethanol mixture (0.06 v/v) that contained 110 mg kg−1 of a HCl catalyst (oral toxicity limit 900 mg kg−1 ). Unsupported films were obtained after drying under vacuum at 25 ◦ C for 24 h. These films were submitted for cytotoxicity assay testing and water vapor permeability measurements. 2.2. Visual determination of gel time To evaluate the gel time during the film formation, the hydrolysis and condensation reactions of the hybrid precursor were induced by the addition of a solution that contained water, ethanol and a catalyst in different proportions. Shortly thereafter, the mixed film-forming solution was spread in an acrylic plate covered by Teflon® using a film extensor (slot cavity of 0.254 mm). After a given time period, a glass slide was placed on the film without pressure. The film was considered to be a gel if no liquid was visible on the glass slide after removal. If any remaining liquid was visible on the glass slide, the experiment was repeated with a longer drying time until reaching closer film time [15]. 2.3. Rheological determination of gel time

Scheme 1. Representation of ureasil cross-linked polyether hybrids.

to tuning finely the delivery profiles [12,13]. Moreover, the use of polyethylene oxide (PEO) as the organic polymer makes the hybrid materials more hydrophilic compared to the hybrid materials formed by using polypropylene oxide (PPO). The drug release from these non-water soluble hybrids network is controlled by its hydrophobic or hydrophilic character. Thus, the choice of PEO or PPO for the hybrid synthesis determines the nanostructure of the hybrids and the molecular weight of polyether [12,13]. In this work, we have assessed the possibility of using ureasil–polyether organic inorganic hybrids as film-forming materials that have optimized properties, such as optical, mechanical and biocompatible characteristics. Furthermore, we evaluated their application viability and highlighted the advantages of employing these hybrids as skin and wound protectors that are able to simultaneously delivery drugs with modulated release profiles. 2. Experimental 2.1. Synthesis of the film-forming hybrid materials The organic–inorganic hybrid matrixes were prepared by a sol–gel route. Ureasil, the cross-linking agent, was covalently bonded to both ends of the macromer polyether [14] by reacting the aminopropyl terminal groups of the functionalized PEO (O,O -bis(2-aminopropyl)-poly(ethylene oxide) with 3-(isocyanatopropyl)-triethoxysilane) in a molar ratio of 1:2. These commercially available reagents (Fluka, Aldrich) were stirred together in tetrahydrofuran (THF) under reflux for 24 h. Then, the THF solvent was removed by evaporation at 60 ◦ C, which led to the hybrid precursor (EtO)3 Si(CH2 )3 NH(C O)NHCHCH3 CH2 (PEO)-CH2 CH3 CHNH(O C)NH(CH2 )3 Si(OEt)3 . This synthesis [14] was used for PEO with average molecular weights (Mw) of 500 and 1900 g mol−1 , and these are labeled hereafter as PEO500 and PEO1900 hybrids. The same synthetic process was used for PPO with average molecular weights (Mw) of 400 and 2000 g mol−1 , and the products are labeled hereafter as PPO400 and PPO2000 hybrids. The film formation was induced by the hydrolysis of –(SiOEt3 )3 followed by a condensation reaction to form the ureasil

Rheological measurements (n = 3 replicates) were carried out using the oscillatory shear mode with the Rheometer, Haake Rheostress, RS-1 model, by using cone-plate geometry (32 mm in diameter). Measurements were performed “in situ” during film formation (sol–gel transition) at a constant temperature of 25 ± 0.1 ◦ C. Storage (G ) and loss (G ) time lapse moduli as a function of time were measured at a fixed frequency (0.5 Hz) and tension (40 Pa) using a gap of 0.105 mm during the isothermal sol–gel transition to determine the time of film-formation [16]. 2.4. Bioadhesion analysis A standard test method used to verify in vitro bioadhesion is the Strength Test [17]. In the first phase, the material to be analyzed was fixed in a biological substrate. Then, one probe was applied with a known force for a known period of time. In the second phase, the probe was withdrawn in a controlled way from the substrate. The bioadhesion using this technique was evaluated through a measurement of the maximum force required to separate the film formed from the biological surface (pig ear skin). Then, the work adhesion was calculated based on the area under the force–distance curve. The TA.XT Plus Texture Analyzer Systems and Stable Micro Systems were used to measure the withdrawn resistance. The Texture Analyzer was equipped with Mucoadhesion Test Rig tool, which has a cylindrical probe of 10 mm in diameter. The pig ear skin used in the test was obtained from fresh, sacrificed pig, and it was cleaned and carefully dissected to keep uniform thickness using a dermatome of 500 ␮m in thickness. The skin was then stored at 0 ◦ C until use. The pig ear skin was placed between two acrylic plates in the Mucoadhesion Test Rig tool, and the system was placed in a beaker with water at 32 ◦ C, which is a skin-like temperature. Enough of the film forming solution to make a thin layer over the skin (36 ␮L) was applied to the pig ear skin substrate, and then the probe was lowered to the substrate at a constant speed of 0.5 mm/s. Thereafter, the probe was kept in touch with the substrate with a force of 0.98 N for 330 s, which is considered the maximum time desired for a polymeric solution to form a film on the skin [7]. Afterward, the probe was withdrawn at the speed of 0.5 mm/s, and the resistance to separate the probe from the substrate and the maximum force required for detachment were measured. The PPO400, PPO2000, PEO500 and PEO1900 hybrid polymer systems were tested. Some commercial film-forming products were also tested, including Band-Aid®

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Liquid Bandage and New Skin® . We also tested bandages with acrylic adhesives that are known to have a strong resistance for removal, including Band-Aid® Tough Strip Bandage, and bandages with hot-melting pressure sensitive adhesives that are known to have good resistance for removal, such as Band-Aid® Flexible Fabric Bandage. All data were expressed as the mean ± S.D. from eight (n = 8) independent experiments. 2.5. Cytotoxicity assays A material in contact with human tissues should not liberate any agent that may be toxic or have an adverse effect on healing process. The cytotoxicity procedure described in this work follows the ISO International Standards Organization (ISO/EN 10993) [18] guidelines for testing biomedical materials. The cell viability was assessed using trypan blue dye exclusion at the beginning of each experiment and was always higher than 80%. For MTT assays, human fibroblast cells (2 × 105 ) were cultured in DMEM (Dubelcco’s Medium Eagle Modified) containing 5% FBS (Fetal Serum Bovine) in 96-well plates and exposed to a conditioned medium. The conditioned media were obtained by immersion of the hybrid materials in DMEM for 48 h. Thereafter, the plates were centrifuged, the supernatants were replaced with MTT dissolved in PBS and the cells were incubated in the dark for 4 h. Finally, the medium was then aspirated and the MTT reduction product, formazan, was dissolved in isopropyl alcohol and quantified spectrophotometrically at 540 nm. The cell concentration was measured using a calibration curve made for the cell line tested using the MTT-staining method, and the results were expressed as a percentage of the control (untreated cells). All data were expressed as the mean ± S.D. from five independent experiments. 2.6. Statistical analysis The data are presented as the mean ± S.D. of five independent series of experiments with six repetitions in each. Statistical analysis was performed using Student’s t-test. The Tukey test with 95% confidence was applied to compare the means. Statistical analyses were performed using the InStat software program for Windows (GraphPads software, San Diego, USA). 2.7. Water–vapor permeability

Table 1 Visual film-forming time determined as function of catalyst amounts. Hybrid precursor/ catalyst ratio (w/v)

Visual gel time (s)

PEO1900

41.6 20.8 10.4

900 240 60

PEO500

41.6 20.8 10.4

840 240 60

PPO2000

41.6 20.8 10.4

900 240 60

PPO400

41.6 20.8 10.4

840 300 60

reactions to obtain a gel point in less than 5 min, which is a convenient work-time for film-forming applications [7]. The experimental values for the gel times are listed in Table 1 and indicate that the higher the hybrid/catalyst ratio in the film-forming solution, the higher the gel time is. Specifically, a hybrid/catalyst ratio equal to 41.6 (m/v) yielded an average gel time of 14–15 min. This length of time is regarded as lengthy for a solution to dry onto films and often results in films with irregular shape. A long drying time may impact the patient acceptance and compliance once used in pharmaceutical preparation. For the 10.4 (m/v) hybrid catalyst ratio, the average gel time was 1 min. The intermediate 20.8 (m/v) hybrid/catalyst ratio showed an average gel time of 4–5 min. In the second phase of this development, we performed rheological measurements to determine the gel time by a conventional methodology adopted in different technological fields, such as food industries, colloidal and polymeric systems processing, cure time of adhesives and so forth [16]. The sol–gel transition that was induced by the addition of an acid hydrolysis solution (proportion hybrid/catalyst of 10.4 (m/v)) to PEO500, PEO1900, PPO400 and PPO2000 hybrid precursors was monitored. Fig. 1 shows the storage (G ) and loss (G ) moduli time lapse throughout the isothermal film formation for the PEO1900 hybrid. The shapes of these curves, which have two distinct regions, are essentially the same for the films prepared with the PEO500, PPO400 and PPO2000 precursors. The first one, which occurred in the initial time period (t < 53 s),

Measurements of the Moisture Vapor Transmitting Rate (MVTR) using PERMATRAN-W Model 101K were performed for the unsupported film prepared by spreading the solution in an acrylic mold. The samples were conditioned using standard conditions of 23 ± 1 ◦ C and 50 ± 2% relative humidity for a minimum of 4 h prior to testing. The samples were placed on a 2.5 cm2 plate with a grill. A positioning adhesive was used to attach the sample onto the plate, and a tape was also added to ensure a seal around the sample. The test control parameters were set to convergence by the cycle mode and the duration/length/time-span of the test lasted 5 min. The sample parameters were set to an area/cell of 2.5 cm2 and a thickness of 0.0254 mm. Each cell was analyzed over 5 min on a seriatim basis until the difference in the last five results was less than 1% for each cell. The sample results were calculated by the software and were given in g/m2 /24 h. 3. Results and discussion In the first phase of this development, the film timing was visually evaluated in the hybrid material. The objective was to determine the best formulation between the hybrid precursor and the catalyst used to promote the hydrolysis and condensation

Fig. 1. Temporal evolution of G and G for a representative sample, the PEO1900 hybrid.

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Table 2 Time of gel/film formation obtained by rheological measurements. Results are expressed as the mean ± S.D. for n = 3 (replicates). Hybrid matrix

Gel time (s)

PEO1900 PEO500 PPO2000 PPO400

111 (±2) 35 (±1) 130 (±3) 67 (±2)

Table 3 Maximum value of adhesion force (Fmax ) and of work of adhesion (Wad ) measured for organic–inorganic matrixes and commercial film-forming materials or adhesive products. Results are expressed as the mean ± S.D. for n = 8 (replicates). Polymer

Fmax (N)

Wad (mJ)

PEO1900 PEO500 PPO2000 PPO400 New Skin® Band-Aid® Liquid Bandage Band-Aid® Tough Strips Band-Aid® Flexible Fabric

3.10 (±0.06) 3.11 (±0.09) 3.15 (±0.08) 3.13 (±0.09) 0.11 (±0.02) 0.21 (±0.03) 2.12 (±0.08) 1.13 (±0.07)

3.31 (±0.05) 12.31 (±0.06) 1.13 (±0.04) 8.21 (±0.06) 0.82 (±0.03) 0.94 (±0.02) 2.02 (±0.05) 0.92 (±0.03)

shows the loss modulus G increasing continuously; however, the storage modulus G was too low to be measured by the rheometer. This property is typical of a Newtonian fluid presenting the molecular weight of the hybrid molecules increasing continually by formation of a growing amount of the siloxane cross-linking nodes. In the second region, which occurs at an advanced time period (t > 53 s), G increases faster and exceeds G , which is evidence of a transformation from a Newtonian fluid to a viscoelastic solid that is associated with the formation of a three-dimensional cross-linked network. The intersection point between the G and G curves observed at 111 s characterizes the film formation time. These values determined for the different hybrids obtained with hybrid/catalyst ratio of 10.4 m/v are listed in Table 2. The observed increase in the film formation time with the polyether molecular weight can be explained by the expected increase of the steric hindrance effect and by the decrease in the total amount of ureasil groups. The differences observed among the film formation time measurements that were obtained by visual and rheological analyses for the samples prepared with hybrid/catalyst proportion of 10.4 m/v are not meaningful and can be attributed to experimental errors due to the subjectivity of visual examinations. Additionally, in the rheological measurements, a tension was applied that changed the cross-linking time of the polymer. In a future use on the skin as a film-forming material, a tension will also be applied during its application for spreading; therefore, we believe that the gel time obtained by rheology is quite realistic. One of the required aspects for a film-forming material to be applied to protect wounds from further injury or to be used as a transdermal drug delivery device is to remain adherent to the skin during the suitable therapeutic time period. The bioadhesion of the hybrid film to the skin can be evaluated both by the maximum value of adhesion force (Fmax ) and by the adhesion work (Wad ). In the literature, we have not found any data related to Fmax and Wad for films used on the skin. For comparison, the same bioadhesion tests were performed with the follow commercial film-forming materials and bandage products approved by the FDA: “New Skin® ”,“Band-Aid® Liquid Bandage, “Band-Aid® Tough Strip Bandage” and “Band-Aid® Flexible Fabric Bandage”. The Fmax and Wad results were obtained for all hybrid films and all commercial products, and they are listed in Table 3. When we compare the maximum adhesion force and the adhesion work of the commercial film-forming materials and the bandage products to the results calculated for the hybrid matrices, the last ones present higher Fmax

Fig. 2. Water vapor permeability values for ureasil–PEO and ureasil–PPO hybrids. Results are expressed as the mean ± S.D. for n = 3 (replicates).

and Wad values, which suggest superior bioadhesion properties. All commercial products that were compared to the hybrid matrices claim to have high adhesion and remain on the skin for at least 24 h, which allows us to reinforce the proposition of using hybrid matrices as semi-occlusive dressings and/or transdermal systems due to their bioadhesion properties. From the hybrid films analyzed in this work, we observed that the bioadhesivity is affected by the molecular weight and the chemical nature of the polymer used to obtain the hybrid matrices (Table 3). In fact, some materials traits, such as the presence of chemical groups, the ability to form hydrogen bonds with the biological substrate, ionic charges on the surface of both the polymer and the biological substrate, the high flexibility of the high molecular weight polymer chains and the appropriate values of surface tension, may lead to diffusion through the layer of epithelial tissue increasing bioadhesivity. The structural formulas of the ureasil–PEO and ureasil–PPO hybrid materials, which are shown in the Scheme 1, reveal that both hybrids present urea groups that are able to form hydrogen bonds with the biological substrate [19]. As seen in Table 3, the adhesion work (Wad ) is higher for the ureasil–PPO and ureasil–PEO hybrids prepared with polyether of lower molecular weight, which confirms the key role of urea groups on the bioadhesion of the hybrid films. Furthermore, the work of adhesion (Wad ) of ureasil–PEO hybrids are higher than those observed for ureasil–PPO hybrids of similar molecular weight. This feature indicated that the hydrophilic character and the higher flexibility of ureasil–PEO provided greater bioadhesion to these matrixes. The human body is constantly losing water to the environment by evaporation through the skin. Trans Epidermal Water Loss (TEWL) is a passive diffusion process that is very important for skin functions, such as body temperature control. Occlusion, meaning impairment of the TEWL, influences several properties of the skin, such as hydration of the stratum corneum, skin temperature and blood flow, and can therefore increase the percutaneous absorption of certain drug substances depending on the anatomic site and the drug vehicle [20–22]. Various skin parameters, such as pH and bacterial flora, are also influenced by an occlusive treatment and result in an increased risk of infection and skin irritation [23]. Accordingly, the degree of occlusion is an important feature of a drug-delivery system that is supposed to be worn on the skin for a prolonged period of time. Fig. 2 shows the Moisture Vapor Transmitting Rate (MVTR) for the different hybrid films. The literature is divergent on the concept of occlusion. According to the

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of amine residues in the hybrid molecule, which could stimulate cell proliferation [19]. 4. Conclusions The ureasil–polyether hybrid matrices evaluated in this work will be a potent new technology platform for the drug-controlled release once they are able to produce non-occlusive transparent and thin films that are capable of drying quickly, do not stick onto outward surfaces and have no cytotoxic effects. These characteristics meet the requirements for cosmetic attractiveness for this novel approach to drug-delivery systems. Acknowledgements FAPESP, CNPq, PADC/FCF and Johnson & Johnson Consumer Companies. References Fig. 3. Cytotoxicity assay with human fibroblasts cell for ureasil–PEO and ureasil–PPO hybrids. Results are expressed as the mean ± S.D. for n = 5 (replicates).

British Pharmacopoeia, a material can be considered permeable to water vapor when the rate exceeds 500 g/m2 /24 h [24]. However, according to William Hart [25], water vapor permeability ranging between 200 and 6000 g/m2 /24 h in thin films can be considered non-occlusive because part of the water vapor is able to permeate the film in a high enough amount to maintain the hydration of the stratum corneum and to ensure appropriate amount of exudate/humidity. Accordingly, the MVTR values between 430 and 490 g/m2 /24 h observed for the ureasil–polyether hybrid films indicate that these materials present semi-occlusive properties and permit the permeation of a portion of the water vapor and retain the other part to hydrate the stratum corneum. The lower MVTR values of the ureasil–PEO films as respect to ureasil–PPO of similar molecular weight is in agreement with its higher hydrophilic character, which favor the absorption of water vapor into free volume of the hybrid network. Moreover, the free volume decreases and the amount of the siloxane nodes increases by decreasing the molecular weight of the polyether chain (Scheme 1) [12–14]. Consequently, the water vapor permeability is higher for the hybrid films prepared with polyether chains of lower molecular weight. Of note, the ratio between the MVTR values of PEO500 (470 g/m2 /24 h) and PEO1900 (430 g/m2 /24 h) is equal (1.09) to that calculated for the ratio between the MVTR values of PPO400 (490 g/m2 /24 h) and PPO2000 (450 g/m2 /24 h). This finding indicates that the effect of the molecular weight on the water vapor permeability through the film is not related to the chemical nature of the polyether used to synthesize the hybrids. These results suggest the dependence of the drug release mechanism on the swellability of the hybrid matrices evidenced previously [12] and suggest that this low flow of water through the films can be useful for the fine tuning of the release profile for incorporated drugs. The toxicity is another key decision criterion in the design of film-forming material to be applied for wound protection and for drug delivery. The assay based on MTT reduction is widely used to evaluate the cytotoxicity of biomaterials having a good correlation with in vivo assays [26,27]. Fig. 3 shows the viability of the cell calculated as described in Section 2.5. By observing viable cell count data, we can verify that the cross-linked ureasil–polyether hybrid matrices allow significant cell viability from 24 to 72 h. All of the hybrids presented cell viability higher than 80% and can be considered as safe for use as drug-delivery devices. The PPO400 proved to be least toxic hybrid. The increased survival of the cells cultured in medium conditioned by PPO400 may be due to the large presence

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