Tailor-made antioxidative nanocrystals: production and in vitro efficacy Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) submitted to the Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin by Run Chen from Hunan, China July 2013 The enclosed doctoral research work was performed during the period from October 2010 to July 2013 under the supervision of Prof. Dr. Cornelia M. Keck and co-supervision of Prof. Dr. Rainer H. Müller in the Institute of Pharmacy, Freie Universität Berlin, De- partment of Pharmaceutical Technology, Biopharmaceutics & Nu- triCosmetics. 1st reviewer: Prof. Dr. Rainer H. Müller 2nd reviewer: Prof. Dr. Cornelia M. Keck Date of defence: 28. August 2013 To my parents and my wife Table of Contents Table of contents Table of contents ......................................................................................................... 1 1. Introduction ......................................................................................................... 4 1.1. Introduction: delivery problems in dermal application ...................................................... 5 1.2. Submicroncrystals & nanocrystals - definitions & properties ............................................ 7 1.3. Overview of production methods of submicroncrystals & nanocrystals ............................ 9 1.4. The commercial use of submicroncrystals & nanocrystals ............................................... 12 1.5. Incorporation into dermal products ................................................................................. 13 1.6. Mechanism of action on the skin...................................................................................... 13 1.7. Neuroprotective effect ..................................................................................................... 15 1.8. In vitro and in vivo performance ...................................................................................... 16 1.9. First commercial products ................................................................................................ 19 1.10. Summary ........................................................................................................................... 20 2. Aims of thesis ..................................................................................................... 22 3. Materials and method ......................................................................................... 26 3.1. Materials ........................................................................................................................... 27 3.1.1. Active pharmaceutical ingredients .......................................................................... 27 3.1.1.1. Apigenin ............................................................................................................... 28 3.1.1.2. Hesperetin ........................................................................................................... 29 3.1.1.3. Quercetin ............................................................................................................. 29 3.1.1.4. Rutin .................................................................................................................... 30 3.1.1.5. Hesperidin ........................................................................................................... 31 3.1.2. Surfactants ............................................................................................................... 31 3.1.2.1. Plantacare® 2000 UP ........................................................................................... 32 3.1.2.2. Poloxamer 188 ..................................................................................................... 32 3.1.2.3. Tween® 80 ........................................................................................................... 32 3.1.2.4. Sodium dodecyl sulfate ....................................................................................... 33 3.1.3. Reagents and tested substances in cell culture test ................................................ 34 3.1.4. Other materials ........................................................................................................ 34 3.1.4.1. Cellulose acetate membrane ............................................................................... 34 3.1.4.2. Dimethyl sulfoxide ............................................................................................... 34 3.1.4.3. Glycerin 87% ........................................................................................................ 34 3.1.4.4. Preservative ......................................................................................................... 34 3.1.4.5. Water ................................................................................................................... 35 3.2. Methods ........................................................................................................................... 35 1 Table of Contents 3.2.1. High speed rotor/stator shear force dispersion ...................................................... 35 3.2.2. High pressure homogenization ................................................................................ 35 3.2.3. Wet bead milling ...................................................................................................... 36 3.2.4. Photon correlation spectroscopy ............................................................................. 36 3.2.5. Laser diffractometry ................................................................................................ 37 3.2.6. Optical microscopy .................................................................................................. 37 3.2.7. Zeta potential ........................................................................................................... 37 3.2.8. High performance liquid chromatography .............................................................. 38 3.2.9. Powder X-ray diffraction .......................................................................................... 38 3.2.10. Cell culture test ........................................................................................................ 39 3.2.11. KRL Test .................................................................................................................... 39 3.2.12. pH measurement ..................................................................................................... 40 3.2.13. Dry residue test ........................................................................................................ 40 3.2.14. Sterilization .............................................................................................................. 40 4. Flavonoid nanocrystals - influencing factors ......................................................... 41 4.1. Preparation of flavonoid nanocrystals .............................................................................. 42 4.1.1. Formulation ............................................................................................................. 42 4.1.2. Production method .................................................................................................. 43 4.2. Result and discussion ........................................................................................................ 43 4.2.1. Influence of dispersion method on PCS measurement ........................................... 43 4.2.2. Size characterization ................................................................................................ 45 4.2.3. Zeta potential ........................................................................................................... 47 4.2.4. Short-term stability .................................................................................................. 48 4.2.5. Correlation with physicochemical properties .......................................................... 49 4.2.6. Influence of surfactant ............................................................................................. 52 4.2.7. Influence of concentration....................................................................................... 53 4.3. Summary ........................................................................................................................... 57 5. Tailor-made quercetin nanocrystals - size effect on in vitro efficacy ...................... 59 5.1. Preparation of quercetin nanosuspension for cell culture test ........................................ 60 5.2. Measurement of cellular ATP content .............................................................................. 61 5.3. Preparation of quercetin nanosuspension kinetic saturation solubility test .................... 62 5.4. Measurement of kinetic saturation solubility .................................................................. 62 5.5. Results and Discussion ...................................................................................................... 63 5.5.1. Size characterization of quercetin nanocrystals ...................................................... 63 5.5.2. Cellular ATP content with only toxins ...................................................................... 65 5.5.3. Cellular ATP content with only quercetin nanocrystals ........................................... 66 5.5.4. Cellular ATP content with toxins and quercetin ...................................................... 68 2 Table of Contents 5.5.5. Kinetic saturation solubility of quercetin powder and nanocrystals ....................... 74 5.6. Summary ........................................................................................................................... 75 6. ARTcrystals - a new method for the production of nanocrystals on industrial scale76 6.1. Preparation of rutin nanocrystals ..................................................................................... 77 6.1.1. Pre-treatment of rutin suspension .......................................................................... 77 6.1.2. Preparation of rutin nanosuspension with low energy/high energy HPH ............... 78 6.2. Result and discussion ........................................................................................................ 80 6.2.1. Pre-treatment .......................................................................................................... 80 6.2.2. Production of nanocrystals by conventional HPH .................................................... 80 6.2.3. Production of nanocrystals by low energy HPH ....................................................... 84 6.2.4. Production of nanocrystals with different surfactants by low energy HPH ............. 86 6.2.5. Comparison to conventional production methods .................................................. 91 6.2.6. Long-term stability ................................................................................................... 92 6.3. Summary ........................................................................................................................... 93 7. smartCrystals - large scale production and reproducibility of hesperidin nanocrystals ............................................................................................................... 94 7.1. Production procedure ....................................................................................................... 95 7.2. Wet bead milling step ....................................................................................................... 97 7.3. High pressure homogenization step ............................................................................... 101 7.4. Determination of dry residue test and pH value ............................................................ 103 7.5. Summary ......................................................................................................................... 104 8. Antioxidative capacity - KRL Test in hesperidin, rutin and quercetin nanocrystals 105 8.1. Hesperidin nanocrystals ................................................................................................. 107 8.2. Quercetin nanocrystals ................................................................................................... 107 8.3. Rutin nanocrystals .......................................................................................................... 109 8.4. Summary ......................................................................................................................... 112 9. Conclusion ........................................................................................................ 113 10. Summary ...................................................................................................... 116 11. Zusammenfassung ........................................................................................ 119 References ............................................................................................................... 123 Abbreviations........................................................................................................... 136 List of publications ................................................................................................... 139 Curriculum Vitae ...................................................................................................... 142 Acknowledgements .................................................................................................. 145 3 Introduction 1. Introduction (partly submitted to Household and Personal Care today - H&PC today, 2013) 4 Introduction 1.1. Introduction: delivery problems in dermal application Poorly soluble plant actives such as resveratrol, rutin, apigenin etc. are of high interest for delivery to the skin in consumer care and cosmetics, but their penetration into the skin is very low (Kitagawa, Tanaka et al. 2009). Therefore they have no sufficient dermal “bioactivity”. The problem is that these molecules are poorly soluble in water and simultaneously in oils. Therefore – in contrast to e.g. coenzyme Q10 – they can- not be dissolved in the oil phase of dermal creams. Application of a suspension of powdered active in a gel or the water phase of a lotion does not solve the problem. The saturation solubility Cs is too low, this results in a very low concentration gradi- ent between dermal formulation as “donor compartment” and the skin as “acceptor compartment” (Fig. 1 - 1 upper). The present regulatory trend is requiring more and more proofs of dermal effects, to substantiate claims for a dermal product. Therefore it is not sufficient anymore just to admix an active to a product, it should be added in a technical form that it develops sufficient dermal bioactivity in the skin. Therefore smart delivery systems to make problematic actives in the skin are an increasing de- mand of the industry. A meanwhile “old” delivery system are the liposomes which can incorporate lipid soluble actives in their phospholipid double layer, but they are not suitable for actives poorly soluble in water and simultaneously in lipids. A popular approach was for some time to use polymeric micro- or nanoparticles to incorporate these actives by using organic solvents in the production process. The polymer particle powders or aqueous suspensions can be admixed easily to a dermal product. However incorpora- tion of a poorly soluble active even worsens the situation. The poorly water soluble actives is firmly encaged in the polymer matrix, diffusion through the water phase in- to the skin is even retarded compared with the naked crystals of actives (e.g. mi- 5 Introduction cronized active) (Fig. 1 - 1 lower). A delivery technology is clearly required which enhances the solubility of actives. Figure 1 - 1 Microcrystals of a poorly soluble active have a low solubility Cs, thus a low concen- tration gradient to skin and low diffusion into skin (upper). Incorporation into a polymer matrix encloses the active like in a safe for money, thus even reducing skin penetration (lower) (sub- mitted to Household and Personal Care today - H&PC today, 2013) 6 Introduction Solubility enhancing technologies are solubilization in micelles or cyclodextrins (CDs). However many of the current actives do not have a sufficient solubilization ability, and do not fit into the space of the CDs. In addition the problem occurs, that actives are firmly incorporated in micelles (gold fish test by G. Levy 1965) or not re- leased from CDs (too high binding constant). A delivery technology would be ideal which creates an active diffusion pressure into the skin, and provides fast new dis- solved molecules from a depot, which replaces the molecules in the dermal formula- tion which are penetrated into the skin. Such a delivery technology is crystals below about 1 µm in size, the submicroncrystal and the nanocrystals. 1.2. Submicroncrystals & nanocrystals - definitions & prop- erties “Micronized powders” are technically employed to increase the dissolution velocity of actives. They possess a mean diameter roughly about 5 µm, with a size distribution ranging up to 10/20 µm. The increased dissolution velocity is due to the larger surface area compared to powders of “normal” size (rather 50-100 µm mean diameter). However these powders show no increased “saturation solubility” Cs! Physicochemical properties change when reducing the size of materials below 1 µm (= 1000 nm), which means generating crystals with a mean size > 100 nm to < 1000 nm (= “submicroncrystals”) or “nanocrystals” being 100 nm or below (≤ 100 nm). The saturation solubility Cs increases exponentially with decreasing size. This kinetic saturation solubility Cs can be 10-100 times higher than the thermodynamic sat- kinetic uration solubility Cs . A supersaturated system is generated. In addition the thermodynamic dissolution velocity dc/dt increases as well, due to the further increase in surface area A and in addition to the increase in Cs (dc/dt proportional A and Cs, Noyes-Whitney equation (Noyes and Whitney 1897). An additional benefit for dermal delivery is the increased adhesion of such fine-sized materials to surfaces in general; the skin is such 7