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Designing of biodegradable and biocompatible release and delivery systems of selected antioxidants used in cosmetology Magdalena Maksymiak, Renata Debowska, Karolina Bazela, Agata Dzwigalowska, Arkadiusz Orchel, Katarzyna Jelonek, Barbara Dolegowska, Marek M. Kowalczuk, and Grazyna Adamus Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01065 • Publication Date (Web): 07 Oct 2015 Downloaded from http://pubs.acs.org on October 9, 2015
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Designing of biodegradable and biocompatible release and delivery systems of selected antioxidants used in cosmetology Magdalena Maksymiak1, Renata Debowska2, Karolina Bazela2, Agata Dzwigalowska2, Arkadiusz Orchel3, Katarzyna Jelonek1, Barbara Dolegowska4, Marek Kowalczuk1, 5and Grazyna Adamus1* 1
Polish Academy of Sciences, Centre of Polymer and Carbon Materials, 34 M. CurieSklodowskiej St., 41-819 Zabrze, Poland
2
Dr Irena Eris Centre for Science and Research, 107A Pulawska St., 02-595 Warszawa, Poland 3
School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical
University of Silesia, Katowice, Poland, Chair and Department of Biopharmacy, 8 Jednosci St., 41-208 Sosnowiec, Poland 4
Department of Laboratory Diagnostics and Molecular Medicine, Pomeranian Medical University, 72 Powstancow Wielkopolskich St., 70-111 Szczecin, Poland
5
School of Biology, Chemistry and Forensic Science, Faculty of Science and Engineering, University of Wolverhampton, Wulfruna Street, Wolverhampton, WV1 1SB, UK
ABSTRACT Conjugates of antioxidants: p-anisic and vanillic acids with non-toxic, biocompatible and biodegradedable oligo-(R,S)-(3-hydoxybutyrate) carrier were synthesized and their structural and biological characterization was performed. The molecular structure of the bioconjugates, in which antioxidants are covalently bonded with oligo(3-hydroxybutyrate) chains, has been proven by mass spectrometry supported by NMR. The bioconjugates hydrolytic degradation studies allowed to gain thorough insight into the hydrolysis process and confirmed the release of p-anisic acid and vanillic acid. In vitro studies demonstrated
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that all of the conjugates studied were well tolerated by KB and HaCaT cell lines as they had no marked cytotoxicity, while conjugates with a relatively short OHB carrier are optimal to support keratinocyte function. The preliminary study of the biological activity confirmed the protective effect of VA-OHB conjugates against H2O2-induced lipid peroxidation in human keratinocytes (HaCaT). It was also demonstrated that the selected bioconjugates can penetrate all layers of the skin, which shows their functionality and opens up their potential application in cosmetology. Keywords: cosmetic delivery system, antioxidants, biodegradable polyesters, ESI-MS analysis, tandem mass spectrometry *Corresponding author: Tel.: +48 32 271 60 77 /ext. 226/ E-mail address:
[email protected] (G. Adamus) 1. Introduction Antioxidants are compounds that can cause delay or inhibition of the lipids oxidation or other molecules by impeding the initiation or propagation of oxidizing chain reactions1. The antioxidant activity of phenolic acids arises mainly from their redox properties, which play a significant role in adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides2. Most of the antioxidants and free radical neutralizers, which are currently applied in cosmetics, are absorbed quickly into deeper layers of the skin and are then carried away by the bloodstream. Temporary binding of antioxidants and free radical neutralizers to specific polymeric carriers is a novel approach resulting in their retard penetration into deeper layers of the skin and consequently promoting intracellular antioxidant and free radical neutralizing activity3.
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Phenolic acids can be easily absorbed in the human system and offer a host of anti-aging benefits. The most important anti-aging benefits of phenolic acids pertain to reducing antioxidant activity and preventing growth of abnormal cells as well as making the cells stronger and decayresistant. These acids are also useful in controlling inflammation, boosting the immune system, and improving blood circulation, all of which assembled benefit in anti-aging advantages to the body. They also reveal very good preservative properties, including antibacterial and antifungal effects4. p-Anisic (p-AA)
and vanillic acids (VA) (benzoic acid derivatives) possess antioxidant,
antitumor and anti-inflammatory properties, and it has also been found that both acids have biochemical properties, which makes them suitable as cosmetic ingredients5. p-Anisic acid has in recent years become increasingly significant as a multi-functional raw material in the cosmetics as well as food industry6. Moreover, it has been found that vanillic acid (natural phenolic acid) is a component of a natural origin cosmetic oil (argan oil) that is traditionally used as a cosmetic to treat light skin damage7. In recent years, increasing attention has been paid to polymeric controlled-release delivery systems of bioactive compounds. Polymer conjugation has the ability to modify the physical and functional properties of the bioactive unit, including its biocompatibility, solubility, stability, bioactivity as well as its biodistribution. A key area of recent and underway research focus is the design, synthesis and application of new polymer-based delivery vehicles capable of delivering therapeutics safely and efficiently to specific sites within the body. Among synthetic approaches for covalently bonded biodegradable polymer bioactive conjugates the four main strategies may be outlined. In the first one (i) bioactive compound may act as initiators of polymerization and as a result the polymer end caped conjugates may be obtained on this way 8-16. The second strategy
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(ii) is focused on incorporation of bioactive moiety into the polymer backbone17. The third strategy (iii) leads to polymer conjugates containing bioactive moieties attached as pendant groups and can be implemented either by grafting the bioactive compound into polymer backbone or end groups, via polycondensation, polyaddition or ring-opening polymerization of monomers containing such moieties18. Finally, the fourth strategy (iv) based on transesterification of high molecular weight polyester has been applied for the synthesis of delivery systems of selected bioactive species possessing either hydroxyl or carboxyl functionalities19. Recently, attaching cosmetics to specific polymer carriers has spurred particular interest. The literature provides some examples describing the use of poly(α-hydroxy acid) as polymeric carriers used for the delivery of biologically active compounds within the skin20. Also, all of these requirements are accomplished by polyhydroxyalkanoates (PHAs), which are represented by natural poly[(R)-3-hydroxybutyrate] (n-PHB)21. Lower molecular weight PHB was found in cell membranes of bacteria22 and was also detected in mitochondrial and microsomal membranes of animal cells23. Furthermore, the amphiphilic nature of PHBs results from the presence of hydrophilic ester carbonyl oxygen atoms and hydrophobic methyl groups additionally enables these polymers to penetrate hydrophobic regions such as cell membranes and hydrophobic pockets of proteins. Recent studies confirmed that PHB can be biodegraded in biological media such as blood and serum24. PHB generally degrades in vivo by hydrolytic breakdown, the degradation products are its oligomers and (R)-3-hydroxybutanoic acid which is a natural metabolite found in various mammalian body fluids25. The earlier studies carried out by some members of our team have shown that synthetic chemical analogue of n-PHB can be obtained via anionic ring-opening polymerization of (S)-β-
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butyrolactone26. The chemically synthesized atactic poly[(R,S)-3-hydroxybutyrate] (a-PHB) undergoes hydrolytic degradation faster than crystalline isotactic PHB27-28. The hydrolytic degradation product of atactic poly[(R,S)-3-hydroxybutyrate], i.e. (R,S)-3-hydroxybutanoic acid is non-toxic and its sodium salt was proposed as substitute for glucose as a brain nutrient in humans29 inspiring deeper interest in use of this polyester for medical applications. Synthetically
prepared
oligo-(R,S)-(3-hydroxybutyrate)s
(OHBs)
undergo
hydrolytic
degradation15, 27-28 and were found to be non-toxic30 . They have already been used as non-toxic carriers of selected drug precursors8-9,
31
. Furthermore, the biological studies of these drug
conjugates indicated that the conjugates of drug with OHB are more efficiently accumulated by cells comparing to unconjugated ones. It seems reasonable to assume that facilitating uptake of the drug conjugates by cells can be related to amphiphilic properties of OHBs. Additionally it was experimentally confirmed that the OHB oligomers containing more than four 3hydroxybutyrates (3-HB) repeating units could be taken up by cells30-31. The studies
presented herein are focused on the replacement of conventional forms of
antioxidants to the biodegradable polymeric release and delivery systems of antioxidants. Our hypothesis was that the conjugated form of the selected antioxidants shows modified biodistribution allowing control cosmetic release and improved penetration through the skin layers. The significance and the impact of our studies are related with the methods of coupling specific antioxidants with antiaging and free radical neutralizing properties with non-toxic, biocompatible and biodegradedable polymeric carriers, which are the most promising ways of obtaining cosmetic delivery system with retard cosmetic component release. The conjugates of oligo-3-hydroxybutyrate with p-anisic acid and vanillic acid were synthesized via ring-opening oligomerization (ROO) of racemic β-butyrolactone initiated by p-
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anisic acid potassium salt and vanillic acid sodium salt, respectively. To the best of our knowledge and according to relevant information, there are no earlier reports on the use of anionic ring opening oligomerization method for the synthesis of controlled delivery systems of p-anisic (p-AA) and vanillic acids (VA). Electrospray tandem mass spectrometry technique (ESI-MSn) supported by
1
H NMR
spectroscopy was used to determine the structure of individual macromolecules of the resulting conjugates (including the chemical structure of their end groups). The results of hydrolytic degradation studies of bioconjugates allowed to acquire comprehensive insight into the hydrolysis process, confirmed the release of p-anisic acid and vanillic acid and enabled to identify the degradation products of oligomeric carrier. Additionally, preliminary cytotoxicity, skin penetration and biological activity tests were conducted on the synthesized conjugates. 2. Experimental 2.1. Materials (R,S)-β-Butyrolactone (98%, Aldrich) was purified as described previously32. p-Anisic acid (≥99% (Aldrich), Vanillic Acid Sodium Salt (4-Hydroxy-3-methoxybenzoic Acid Sodium Salt, TCI Europe) and potassium hydroxide (pure pa, POCH SA) were used without additional purification. Potassium p-anisate (4-Methoxybenzoic Acid Potassium Salt) was obtained from a reaction between the corresponding acid and potassium hydroxide. Dimethylsulfoxide (DMSO, 99.8%, Aldrich) was stored over 4A molecular sieves prior to use. 2.2. Oligomerization of β-butyrolactone in the presence of p-anisic acid potassium salt and vanillic acid potassium salt p-Anisic acid-OHB and vanillic acid-OHB conjugates were synthesized via ROO of (R,S)-βbutyrolactone initiated by p-anisic acid potassium salt or vanillic acid potassium salt,
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respectively. The reaction was performed in dimethylsulfoxide (DMSO) at 18 °C in a glass reactor that was flamed and dry argon-purged prior to use. After 3 h of stirring, a β-butyrolactone monomer was added to the reactor containing the required amount of initiator in DMSO. The monomer concentration was equal to 1 mol dm-3 in each experiment. The average molar mass of the conjugates was controlled by the monomer-initiator ratio. The progress of polymerization was monitored by Fourier transform infrared spectroscopy (FTIR) based on the intensity of the signals arising from the carbonyl groups of the monomer and oligomer at 1815 and 1735 cm-1, respectively. When the reaction was completed, the resulting oligomers were protonated. The calculated amount of HCl in DMSO was added to the reactor, allowed to react for 10 min, and the mixture was freeze-dried. The resulting product was re-dissolved in CHCl3 and washed six times with distilled water in order to remove sodium chloride and potassium chloride, respectively, as well as DMSO residue. The obtained p-anisic acid-OHB and vanillic-OHB conjugates were precipitated in hexane and dried under vacuum for 48 h and analyzed using 1H NMR, ESI-MSn, and size exclusion chromatography (SEC). 2.3. Assessment of cytocompatibility of p-AA-OHB and VA-OHB conjugates 2.3.1. Statistical Analysis The data were analyzed using a one way ANOVA and -test. All the results are expressed as means ± SD. value of
