Title Page

Summary of this study

Contents

Chapter Ⅰ. Self-assembled nanodrops of hyaluronic acid-oleic acid conjugates for improving the ocular delivery of cyclosporine A 24

1. Introduction 24

2. Materials and Methods 27

2.1. Materials 27

2.2. Synthesis of oleic acid-hyaluronic acid conjugates (HOCs) 28

2.3. Preparation of CsA-loaded self-assembled HONs (CsA-HONs) 29

2.4. Physicochemical characterizations of HOCs and CsA-HONs 30

2.5. In vitro drug release study 33

2.6. In vitro drug permeation study 34

2.7. In vitro cytocompatibility of CsA-HONs for ocular application 35

2.8. Assessment of anti-inflammatory potential of CsA-HON 36

2.9. Assessment of anti-angiogenic potential of CsA-HON 38

2.10. Statistical analysis 39

3. Results and Discussion 40

3.1. Synthesis and characterization of HA-CYS 40

3.2. Synthesis and characterization of HOCs 49

3.3. Characterization of HONs and CsA-HONs 54

3.4. In vitro drug release 61

3.5. In vitro drug permeation 63

3.6. Cytocompatibility evaluation of CsA-HONs for ocular use 65

3.7. Evaluation of the anti-inflammatory activity of CsA-HON1 71

3.8. Assessment of the antiangiogenic potential of CsA-HON1 75

3.9. Commentary for the significance of study 79

4. Conclusions 80

Chapter Ⅱ. Redox-sensitive oleic acid-hyaluronic acid nanoparticles induce overexpression of lipid accumulation as a synergistic effect to boost the anticancer activity of doxorubicin 81

1. Introduction 81

2. Materials and Methods 83

2.1. Materials 84

2.2. Preparation of DOX-loaded HON 84

2.3. DLS measurement 85

2.4. FE-TEM analysis 85

2.5. In vitro drug release study 86

2.6. Cell culture and maintenance 86

2.7. Oil red O staining and neutral lipid quantification 86

2.8. Neutral lipid droplets staining using BODIPY 493/503 87

2.9. In vitro cytotoxicity 87

2.10. Statistical analysis 88

3. Results and Discussion 89

3.1. Characterizations of GSH-triggered disassembly of HON 89

3.2. Characterizations of DOX-HONs 91

3.3. In vitro drug release study 93

3.4. OA-induced lipid accumulation caused cell death 96

3.5. HOC enhanced the cancer targeting of OA 99

3.6. In vitro cytotoxicity of DOX-loaded formulations 103

3.7. Commentary for the significance of study 106

4. Conclusions 107

References 108

List of Tables

Chapter Ⅰ. Self-assembled nanodrops of hyaluronic acid-oleic acid conjugates for improving the ocular delivery of cyclosporine A 17

Table 1. Characterization of HA-CYS with different DSs of CYS 46

Table 2. Physicochemical characterizations of blank HON and CsA-HON solutions 57

Chapter Ⅱ. Redox-sensitive oleic acid-hyaluronic acid nanoparticles induce overexpression of lipid accumulation as a synergistic effect to boost the anticancer activity of doxorubicin 18

Table 1. Characterization of DOX-HONs (mean ± SD, n＝3) 92

Table 2. The IC₅₀ and SI values of DOX-loaded formulations in MCF-7 and HFF-1 cell lines 105

List of Figures

Chapter Ⅰ. Self-assembled nanodrops of hyaluronic acid-oleic acid conjugates for improving the ocular delivery of cyclosporine A 19

Figure 1. Schematic illustration of a two-step reaction forming the HA-CYS-OA conjugates (HOCs) 41

Figure 2. ¹H-NMR spectra of HA, CYS and 4 conjugates of HA-CYS 43

Figure 3. FTIR spectra of HA, CYS and 4 conjugates of HA-CYS 44

Figure 4. UV-VIS spectra of HA and 4 conjugates of HA-CYS 44

Figure 5. Cytocompatibility of HA-CYS with different DSs of CYS. (A) HPCEC viability after treatment with CYS at different concentrations for 24... 48

Figure 6. ¹H-NMR spectra of three HOCs 51

Figure 7. FTIR spectra of HA, HA-CYS 4 and three HOCs 52

Figure 8. CMC determination of three HOCs 53

Figure 9. FTIR spectra of CsA, three HOCs and three CsA-HONs 58

Figure 10. PXRD patterns of CsA, three HOCs and three CsA-HONs 58

Figure 11. FE-SEM and FE-TEM morphologies of three CsA-HONs 59

Figure 12. Digital camera images of blank HONs, CsA-HONs and Restasis® solutions 60

Figure 13. Absorbance measurement of blank HONs, CsA-HONs and Restasis® solutions 60

Figure 14. Profiles of drug release from different CsA-HONs compared to Restasis®. 62

Figure 15. Profiles of drug permeation from different CsA-HONs compared to Restasis®. 64

Figure 16. Viability of HPCECs after 24 h of treatment with (A) free CsA, (B) free OA, and (C) three HOCs (n＝3). 68

Figure 17. (A) HPCEC viability after a short exposure time (3, 5, 10 min) to different formulations at 500 μg/mL CsA (n＝3). (B) Live/dead staining... 69

Figure 18. Viability of HPCECs after prolonged incubation with lower concentrations of CsA by dilution, including (A) 50 μg/mL CsA and (B) 5... 70

Figure 19. (A) Viability of M0 macrophages after 24 h of treatment with different concentrations of CsA (n＝3). (B) Viability and (C) morphological... 73

Figure 20. (A) Fluorescent images of immunocytochemistry staining of the polarized macrophages with CD163 (M2 marker, green) and DAPI (nuclear,... 74

Figure 21. (A) and (B) Viability of HUVECs after 24 h of treatment with varying concentrations of free CsA and different formulations equivalent to 5... 77

Figure 22. Characterizations of tube formation assay on GRF Matrigel of HUVECs. (A) Morphological behaviours, (B) the tube length (px), (C) the branching points and (D) the covered area. 78

Figure 23. The schematic diagram illustrates the significance of study 79

Chapter Ⅱ. Redox-sensitive oleic acid-hyaluronic acid nanoparticles induce overexpression of lipid accumulation as a synergistic effect to boost the anticancer activity of doxorubicin 22

Figure 1. GSH-triggered disassembly of HON. (A) Particle size distribution and digital camera image of HON before and after 24 h incubation with or... 90

Figure 2. FE-TEM morphology of blank HON and DOX-HONs 92

Figure 3. Drug release profiles of DOX-loaded formulations in PBS (10 mM, pH 7.4) solutions. (A) without GSH and (B) with GSH (1-10 mM) 95

Figure 4. OA-induced lipid accumulation caused cell death. (A) Images of ORO staining of MCF-7 (cancer cells) and HFF-1 (normal cells) after 48 h... 98

Figure 5. HOC enhanced lipid accumulation and caused cell death. (A) Cell viability of two cell lines after 48 h treatment of different concentrations of... 101

Figure 6. The roles of OA, CYS, and HA in conjugate potentiate cancer targeting. (A) Cell viability of the glutathione monoethyl ester (GSH-OEt,... 102

Figure 7. Cytotoxicity of DOX-loaded formulations. (A) Human breast cancer cells (MCF-7) and (B) Human normal fibroblasts (HFF-1) 104

Figure 8. Schematic illustration of the redox-sensitive oleic acid-hyaluronic acid nanoparticles for boosting the anticancer activity of DOX. 106

 A fattigation platform pioneered by our group has shown to be a promising technology for the modification and improvement of druggability of various therapeutic drugs. In this platform, a number types of fatty acids (FAs) were conjugated into biomacromolecules such as gelatin, albumin, apotransferrin as biocompatible nanocarriers for drug delivery, or they were directly conjugated into peptide drugs like leuprolide to develop prodrugs (Amin et al., 2017; Park et al., 2020a; Park et al., 2015; Park et al., 2022; Tran et al., 2013a). There are two distinct features of FAs used in the fattigation platform technology, including the carbon length of the FAs and their degree of saturation. By modulating the types of conjugated fatty acids, a variety of structures of conjugated fatty acids were obtained, which could then be readily assembled into nanoparticles with enhanced physicochemical properties such as deformability, improved stability, and permeability of drugs, as well as biopharmaceutical properties, such as enhanced degradation in human plasma and selective cytotoxicity for cancer targeting with different lengths of FAs (Park et al., 2020a; Park et al., 2022). However, there are still remaining potential mechanisms that could underline the use of fatty acids in this platform, since FAs have been shown to be bioactive molecules with many functions affecting the human body, particularly cell signaling and regulation. For example, the polyunsaturated fatty acids (PUFAs) such as the mixture of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and alpha-linolenic acid (ALA) found in omega 3 products are responsible for modulating neurotransmitters, reducing inflammation, reducing oxidative stress, and providing a wide range of biological functions (Zhou et al., 2022). In addition, a number of other unsaturated fatty acids (UFAs), including oleic acid (OA) and linoleic acid (LA), have been proven to have anti-inflammatory, anti-oxidant, and antitumor properties. (Farag and Gad, 2022; Hernández et al., 2021). Furthermore, saturated fatty acids (SFAs) play a crucial role in energy production as well as being vital components of cellular membranes (Ferreri et al., 2016). There is no doubt that FAs are able to exert a variety of effects, however their application has been limited due to their low aqueous solubility and toxicity issues. Herein, I suggest that the fattigation platform could be considered to be an effective method of solubilizing FAs and preventing burst leaching of FAs which may cause cellular damage. Therefore, this study was aimed to explore and evaluate the roles of conjugated FAs not only in improving the physicochemical modifications of drugs but also in exerting their potential bioactive effects on boosting the activity of therapeutic drugs. Among different types of FAs, OA was selected in this study owing to its versatility. Since OA is a monounsaturated long chain FA, it has a higher thermal stability and a lower oxidative burst than other long chain PUFAs, such as linoleic acid and omega 3 FAs with higher degree of unsaturation (Hatanaka et al., 2013). In addition, the use of long chain FAs has been shown to be more advantageous than the use of short chain FAs for enhancing the stability of conjugates and thereby prolonging the degradation of those conjugates against human plasma (Park et al., 2020b; Park et al., 2019). Furthermore, among three major FAs (palmitic acid, oleic acid, and stearic acid) that are present in human plasma, OA has a greater water miscibility at elevated temperatures than the other long chain SFAs due to the introduction of a cis double bond on the fatty chain, which is advantageous for achieving a high conjugation efficiency with hydrophilic biomaterials using fattigation platform technology. Lastly, and perhaps most importantly, at certain concentrations of application, OA also exhibits a variety of biological effects such as anti-inflammation, anti-angiogenesis, and anti-tumor properties (Farag and Gad, 2022; Gao et al., 2014; Giulitti et al., 2021; Lamy et al., 2014). Hyaluronic acid (HA) was selected as a biomacromolecule for fatty acid conjugation in this study. HA is a natural and biocompatible macromolecule composed of linear and repetitive units of glucuronic acid and acetyl glucosamine (Mattheolabakis et al., 2015). Owing to its high biocompatibility and biodegradability, high capacity for retaining water, lack of toxicity and low immunogenicity, HA has a wide range of applications in pharmaceutical fields. Further, HA is also capable of exerting its bioactive effects as a result of its binding to receptors that are located on the surface of the cell membrane, such as CD44, and the receptor for HA-mediated motility (Misra et al., 2015). Also, HA contains a variety of chemical groups, including carboxylic, hydroxyl, as well as N-acetyl, which are available for conjugation with FAs (Burdick and Prestwich, 2011).

In chapter I, cyclosporine A (CsA), which is well-known as the lipidbased emulsion of the Restasis® ophthalmic product, is FDA-approved for treating dry eye disease (DED); however, the treatment causes side effects, such as ocular pain, eye redness and blurred vision, after administration. In this study, we designed and evaluated new conjugates of oleic acid (OA) and hyaluronic acid (HA), via a fattigation platform. Cystamine (CYS) as a diamine linker was used to bridge carboxylic groups of HA and OA via a two-step reaction using EDC/NHS coupling agents. CYS was first modified on HA (HA-CYS) in a sufficient amount to maintain the same cytocompatibility as HA before OA conjugation. Following that, OA was conjugated to HA-CYS to generate HA-CYS-OA conjugates (HOCs) with controlled degrees of substitution (DSs) of OA, including 4.6%, 8.3% and 12.2%. These conjugates could easily self-assemble into nanoparticles (HONs) to encapsulate CsA for ocular drug delivery. Our results showed that compared to Restasis, CsA-loaded HONs significantly improved the transparency and in vitro drug permeation. Among the three HOCs, the higher DS of OA led to smaller HONs with a higher drug loading efficiency, while the lower DS of OA resulted in more transparency and ocular cytocompatibility. Interestingly, OA conjugation conferred the optimal nanoformulation abilities for stimulating macrophage polarization into the M2 phenotype and inhibiting VEGF-promoted human endothelial cells proliferation and capillary-like tube formation, which potentiates exerting the anti-inflammation and anti-angiogenesis, respectively, in a synergistic manner with CsA. The new finding in our study indicates that a promising design was developed with multiple potential for ophthalmic applications, especially for the treatment of multifactorial DED.

As a result, chapter II examines the mechanisms leading to the toxicity caused by the application of high concentrations of OA and provides a new approach to control and enhance the anti-tumor effect of OA with regards to cancer treatment. OA has been shown to facilitate the formation of lipid droplets that store and regulate neutral lipids in the cells to maintain energy homeostasis and support cellular functions. The overexpression of exogenous OA, however, results in the massive neutral lipid accumulation associated with reduced autophagy, which in turn reduces cell viability. Taking advantage of unique properties of OA and HA for cancer targeting, we developed a biocompatible OA-conjugated hyaluronic acid via a disulfide bond-contained linker that could self-assemble into nanoparticles for drug encapsulation and impart a redox-sensitive behavior. Doxorubicin (DOX) was selected as a model chemotherapeutic drug in this study. Conceptually, the GSH concentration in cancer cells is significantly higher than in normal cells, the disulfide bond is susceptible to cleavage, resulting in OA and DOX being released together, thus killing the cancer cells in a synergistic manner. Human normal fibroblasts (HFF-1) and human breast cancer cells (MCF-7) were used to evaluate the selective anticancer activity of free OA, HOC and DOX-loaded HON. As a result, it is significant to note that although OA displayed higher cytotoxicity towards normal cells, HOC showed a reverse trend with higher cytotoxicity towards cancer cells. Additionally, it was observed that there was a positive correlation between the modulation of intracellular GSH concentration using a GSH enhancer/reducer and the viability of cancer cells. Taken together, we found that the redox-sensitive nanoformulation can exhibit a dual release of OA from conjugates, inducing a higher lipid accumulation as a synergistic effect to boost the anticancer activity of DOX with greater selectivity. Based on the results of this study, HOC appears to be a potential nanocarrier for boosting the performance and efficiency of therapeutic drugs.