Combining Dietary Phenolic Antioxidants with Polyvinylpyrrolidone
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1 Combining Dietary Phenolic Antioxidants with 2 Polyvinylpyrrolidone: Transparent Biopolymer Films based on 3 p-Coumaric Acid for Controlled Release 4 Marco Contardia,b*, José A. Heredia-Guerreroa*, Susana Guzman-Puyola, Maria Summac, José J. 5 Benítezd, Luca Goldonie, Gianvito Caputoa,, Giovanni Cusimanof, Pasquale Piconef, Marta Di Carlof, 6 Rosalia Bertorellic, Athanassia Athanassioua, Ilker S. Bayera*. 7 a Smart Materials, Istituto Italiano di Tecnologia, Via Morego, 30, Genova 16163, Italy 8 b DIBRIS, University of Genoa, via Opera Pia 13, 16145, Genoa, Italy 9 c In Vivo Pharmacology Facility, Istituto Italiano di Tecnologia, Via Morego, 30, Genova 16163, Italy 10 d Instituto de Ciencia de Materiales de Sevilla, Centro mixto CSIC-Universidad de Sevilla, Americo 11 Vespucio 49, Isla de la Cartuja, Sevilla 41092, Spain 12 e Analytical Chemistry Facility, Istituto Italiano di Tecnologia, Via Morego, 30, Genova 16163, Italy 13 f Istituto di Biomedicina ed Immunologia Molecolare "A. Monroy", CNR, via Ugo La Malfa 153, 14 Palermo 90147, Italy 15 16 * Corresponding authors 17 Email address: [email protected] (Marco Contardi); [email protected] (José A. Heredia- 18 Guerrero); [email protected] (Ilker S. Bayer). 19 1 20 Abstract 21 Polyvinylpyrrolidone (PVP) is probably one of the most utilized pharmaceutical polymers with 22 applications ranging from blood plasma substitute to nanoparticle drug delivery, since its synthesis in 23 1939. It is highly biocompatible, non-toxic and transparent film forming polymer. Although excessive 24 solubility of PVP in aqueous environment is advantageous, it still poses several problems for some 25 applications in which sustained targeting and release are needed or hydrophobic drug inclusion and 26 delivery systems are to be designed. In this study, we demonstrate that a common dietary phenolic 27 antioxidant, p-Coumaric acid (PCA), can be combined with PVP covering a wide range of molar ratios 28 by solution blending in ethanol, forming new transparent biomaterial films with antiseptic and 29 antioxidant properties. PCA not only acts as an effective natural plasticizer but also establishes H- 30 bonds with PVP increasing its resistance to water dissolution. PCA could be released in a sustained 31 manner up to a period of 3 days depending on PVP/PCA molar ratio. Sustained drug delivery potential 32 of the films was studied using methylene blue and carminic acid as model drugs, indicating that the 33 release can be controlled. Antioxidant and remodeling properties of the films were evaluated in vitro by 34 free radical cation scavenging assay and in vivo on murine model, respectively. Furthermore, the 35 material resorption of films was slower as PCA concentration increased, as observed by full-thickness 36 excision in vivo model. Finally, the antibacterial activity of the films against common pathogens such 37 as Escherichia coli and Staphylococcus aureus and the effective reduction of inflammatory agents such 38 as matrix metallopeptidases were demonstrated. All these properties suggest that these new transparent 39 PVP/PCA films can find a plethora of applications both in food and pharmaceutical sciences including 40 skin and wound care. 41 42 Keywords: Polyvinylpyrrolidone, p-coumaric acid, biomaterial, antioxidant properties, antibacterial 43 activity, wound dressing. 2 44 1.- Introduction 45 Most commercially available medical formulations derive from natural products1. For instance, there 46 are many drug delivery systems and therapeutic approaches based on the use of biopolymers as well as 47 alternative natural bio-active agents such as essential oils2-4 and propolis5, with polyphenols as their 48 main active components. These organic chemicals are characterized by the presence of phenol 49 structural units and have notable antioxidant properties6. Among this large class of compounds, 50 hydroxycinnamic acid derivatives, also known as dietary phenolic compounds, such as ferulic, caffeic, 51 and p-coumaric (4-hydroxycinnamic acid, PCA) acids have gained a growing interest due to their 52 remarkable biological activity: they can reduce the inflammatory response7, 8, show protective effect 53 against cancer9-11, affect the angiogenesis process12-14, and interact with the β-amyloid (i.e., the main 54 protein involved in Alzheimer disease), affecting the formation of toxic protein fibrils15, 16. For 55 instance, Abdel-Wahab et al.17 demonstrated that PCA protects rats’ hearts against doxorubicin- 56 induced oxidative stress, while Ferguson et al.18 showed that ferulic and p-coumaric acids have a better 57 activity in comparison to curcumin, other common polyphenol, protecting cultured mammalian cells 58 against oxidative stress and genotoxicity. On the other hand, Luo et al.19 described a double 59 antibacterial action mechanism for PCA. Recently, several studies have demonstrated that these 60 medically beneficial compounds are also suitable for the treatment of skin issues due to their strong 61 antioxidant properties2, 4. Indeed, several types of skin damages and diseases are characterized by local 62 and/or protract inflammatory and oxidant events that drastically increase the levels of interleukin and 63 free radicals, causing cellular damages, slowing down the healing process, and potentially driving into 64 chronic state20. Ghaisas et al.21 evaluated the administration of ferulic acid as an oral solution and in the 65 form of ointment for topical delivery in an excision wound model on diabetic rats that produced the 66 reduction of inflammation and the acceleration of the healing process. In addition, PCA and other 67 phenolic acids have been reported as efficient photoprotective agents for skin against dangerous solar 68 ultraviolet radiation (UVB)22-24. 69 Although PCA has antioxidant properties, as previously demonstrated in cultured cells7, 10, 25 and 70 animal models25-29, its activity on cutaneous wound healing is yet to be investigated in detail. In this 71 sense, incorporation of PCA in pharmaceutical polymeric matrices, like PVP, can yield alternative 72 antibiotic-free biomaterials that can be used in the treatment of infected wounds. PCA integrated skin 73 dressing systems can potentially regulate the levels of free radicals and pro-inflammatory molecules for 74 healing skin along with other important parameters, such as antibacterial activity, good adhesion, 3 75 exudate absorption, transparency and resorbing from the skin30. Recently, we demonstrated the 76 possibility of using polyvinylpyrrolidone (PVP) as a biocompatible polymer matrix for fabrication of 77 skin wound dressing films with high level of transparency, adhesion to the skin, with controlled 78 ciprofloxacin release31. 79 Here, we present the fabrication of new biomaterials in the form of transparent, free-standing films 80 composed of different proportions of PCA and PVP blended in ethanol solutions. We demonstrate that 81 both components strongly interact by H-bonds and this allows fine-tuning of thermal, mechanical, and 82 hydrodynamic properties. The incorporation of PCA into PVP not only confers antioxidant and 83 antibacterial properties to the polymer, but also controlled release of model drugs due to increased 84 water dissolution resistance. Furthermore, PVP/PCA 2:1 films demonstrated promising activity in 85 reducing matrix metallopeptidase 9 (MMP-9) levels in a wound mice model that indicates future 86 potential for chronic wound treatment. 87 88 2.- Materials and Methods 89 2.1.- Materials 90 Polyvinylpyrrolidone (MW = 1300000 Da), p-Coumaric acid (≥98.0% HPLC; MW = 164.16 g/mol), 91 carminic acid (CA; MW = 492.39 g/mol), methylene blue (MB; MW = 319.85 g/mol), ethanol, and 92 phosphate buffered saline (PBS) solution (pH 7.4) were purchased from Sigma-Aldrich and used 93 without further purification. 94 2.2.- Preparation of the films 95 PVP/PCA solutions with different molar ratios were prepared by dissolving PCA and PVP powders in 96 15 mL of ethanol. The solutions were first shaken (standard lab shaker) overnight, casted onto plastic 97 Petri dishes (5 cm diameter), and dried for 2 days under an aspirated hood ambient conditions (16-20°C 98 and 40-50% R.H.). Films with PVP/PCA molar ratio of 10:1, 7.5:1, 5:1, 3.5:1, and 2:1 were prepared, 99 Table 1. The films had an average thickness of (170 ± 30) µm. Samples with higher PCA contents were 100 not prepared due to the segregation of both components and macroscopic crystallization of p-coumaric 101 acid. A control film of only PVP at 3% (w/v), labeled as PVP/PCA 1:0, was also fabricated. PCA 102 powder was the sample PVP/PCA 0:1. 4 103 To investigate the dispersion and delivery of model drugs in the PVP/PCA formulation, samples loaded 104 with methylene blue (MB) and carminic acid (CA), were prepared. In particular, 10 mg of MB and 15 105 mg of CA, respectively, were added to 15 mL solutions of PVP/PCA 1:0, 10:1 and 2:1. 106 107 Table 1. Sample labeling, molar ratio between PVP and PCA, molar fraction of PCA, weight 108 percentage of both components, and concentration of drugs used. Weight % of Weight % of Molar ratio Molar fraction Weight % Sample Label MB respect CA respect PVP/PCA of PCA of PCA to PVP to PVP PVP/PCA 1:0 1:0 0.00 0 - - PVP/PCA 10:1 10:1 0.09 12.8 - - PVP/PCA 7.5:1 7.5:1 0.12 16.3 - - PVP/PCA 5:1 5:1 0.17 22.6 - - PVP/PCA 3.5:1 3.5:1 0.22 29.5 - - PVP/PCA 2:1 2:1 0.33 42.2 - - PVP/PCA 0:1 0:1 1.00 100 - - PVP/PCA 1:0 + 1:0 0.00 0 1.0 1.6 MB PVP/PCA 10:1 + 10:1 0.09 12.8 1.0 1.6 MB PVP/PCA 2:1 + 2:1 0.33 42.2 1.0 1.6 MB PVP/PCA 1:0 + 1:0 0.00 0 1.0 1.6 CA PVP/PCA 10:1 + 10:1 0.09 12.8 1.0 1.6 CA PVP/PCA 2:1 + 2:1 0.33 42.2 1.0 1.6 CA 109 110 2.3.- Scanning Electron Microscopy (SEM) 111 Morphology of the films was analyzed by Scanning Electron Microscopy (SEM), using a variable 112 pressure JOEL JSM-649LA microscope equipped with a tungsten thermionic electron source working 113 in high vacuum mode, with an acceleration voltage of 10 kV.