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研究生：	鄭家亘 Cheng, Chia-Hsuan
論文名稱：	建構吸入劑型的PLGA奈米載體攜帶雙重藥物並評估其在動物模型上減緩肺纖維化的效果 Construct an Inhalation Formulation of the PLGA Nanocarrier to Carry Dual Drugs and Evaluate its Effectiveness in Reducing Pulmonary Fibrosis in an Animal Model
指導教授：	吳炳慶 Wu, Ping-Ching
學位類別：	碩士 Master
系所名稱：	工學院 - 生物醫學工程學系 Department of BioMedical Engineering
論文出版年：	2020
畢業學年度：	108
語文別：	英文
論文頁數：	67
中文關鍵詞：	肺纖維化、奈米粒子、奈米藥物載體、聚乳酸-甘醇酸共聚合物、二甲雙胍、培尼皮質醇
外文關鍵詞：	Pulmonary fibrosis, nanoparticles, nano-carrier, poly (lactic-co-glycolic acid) (PLGA), metformin, prednisolone
相關次數：	點閱：84 下載：1
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肺部纖維化的死亡率近年來有顯著上升的趨勢，目前在臨床上並無任何有效藥物或方法可反轉肺纖維化，對於肺纖維化的病人僅能給予支持性治療以減少肺功能喪失為目標，台灣胸腔暨重症加護醫學會指出，在歐美地區，其年發生率約每10萬人有6-7人罹患，台灣則每10萬人約有1-2人會得到此疾病，且其存活期平均只有0.9年，5年存活期甚至低於乳癌、結腸癌等癌症。臨床上造成肺纖維化之原因很多，如各種肺部感染和因職業吸入石綿、矽、煤礦，又或者是抽煙或空汙，以及藥物、放射線治療所引起，更有一部分的病患是由自體免疫疾病或不明原因的原發性肺部纖維化等，因此一項能反轉或減緩肺纖維化的治療的方法在臨床上是迫切需求的。
抗糖尿病二甲雙胍類藥物Metformin近年來有許多研究發現其能有效提升AMPK去抑制TGF-β並去抑制肺纖維化的產生，而類固醇藥物Prednisolone能有效的減少細胞內活化氧化物並減少肺內的發炎反應。本研究將利用具有生物可分解性高分子聚合物PLGA作為一項能搭載親水性及親油性藥物雙層藥物的奈米載體，並將Metformin與Prednisolone兩種藥物包覆於奈米藥物載體內，以達到同時減緩肺部纖維化並減少肺部發炎的功效，並且配合奈米藥物載體的使用能讓藥物具有緩釋的功能。接著以肺纖維母細胞(IMR-90)以及肺上皮細胞(L2)和C57BL/6小鼠使用Bleomycin引發纖維化後，再給予雙重藥物奈米載體治療去驗證藥物效果及臨床治療的可行性。
研究結果顯示，使用雙重藥物奈米載體包覆藥物後可以獲得121 ± 29.1奈米大小的奈米粒子，並可分別包覆39.3%的水溶性藥物Metformin和64.83%的脂溶性藥物prednisolone，且使用此包覆雙重藥物的奈米載體能在細胞實驗上有效的抑制ROS的生成來減少更多的肺部發炎，以及減少肺上皮細胞的細胞凋亡。而在動物之肺臟病理切片分析中，發現給予bleomycin確實可以誘導肺泡間質增厚以及膠原蛋白的增加，並在給予雙重奈米藥物載體後能有效的減少肺泡間質厚度以及小支氣管周圍膠原蛋白，以及減少TGF-beta、NF-kB等發炎因子的表現。透過實驗結果顯示此雙重奈米藥物載體確實具有減少發炎之能力並能減緩肺纖維化的產生。
本研究成功發展出一項能同時攜帶親水性及親油性藥物的雙重藥物奈米載體，並藉由包覆Metformin和Prednisolone兩種藥物來成功對抗和減緩肺纖維化，並作為臨床醫師之參考。目的希望可以大幅提升肺纖維化患者生活品質並減少本國對於肺纖維化在健保上的花費及醫療社會成本。

Pulmonary fibrosis (PF) defined as an unknown progressive and irreversible disease that may be induced by cigarette, radiation, chemical substance, autoimmune diseases, and even idiopathic. Moreover, the mortality rate of PF has increased significantly in recent years. However, there is no clinically effective drug or therapy can reverse PF until now. For patients with pulmonary fibrosis, only supportive treatment can be given to reduce lung function loss. The Taiwan Society of Pulmonary and Critical Care Medicine Association pointed out that the annual incidence rate is about 6-7 people per 100,000 people in Europe and the United States, and about 1-2 people per 100,000 people in Taiwan. The average survival period is only 0.9 years, and the five-year survival rate is lower than breast cancer, colon cancer and other cancers. Therefore, a treatment that can alleviate or slow down pulmonary fibrosis is urgently needed clinically relevant patients.
Recently, metformin, an anti-diabetic drug, was found to help reverse fibrosis by activating AMPK to inhibit TGF-β and Smad signaling pathway. Furthermore, prednisolone, a steroid, can be used as an anti-inflammation drug by inhibiting reactive oxidants and pro-inflammatory molecules. In this study, we used Poly(lactic-co-glycolic acid) (PLGA), a biocompatible and FDA approved polymer, as a dual-therapeutic nano-carrier which is capable of carrying with the hydrophilic and hydrophobic drug, metformin and prednisolone, respectively. Then, nanoparticles (NPs) were given to bleomycin-induced lung fibroblasts (IMR-90), lung epithelial cells (L2) and C57BL / 6 mice to verify the drug effect and the feasibility of clinical treatment.
According to the result, the NPs attain an average size of 121 ± 29.1 nm. The encapsulation rate of NPs is 39.3% and 64.83%, respectively, with metformin and Prednisolone. Moreover, it can control drug-releasing, which takes three days to reach the maximum drug releasing. In in vitro experiments, administration with NPs can effectively inhibit the generation of ROS from reducing more lung inflammation and the apoptosis of lung epithelial cells. In in vivo experiments, bleomycin-induced fibrosis can significantly thicken alveolar interstitial and increase collagen around bronchi through mice lung sections. Furthermore, the alveolar interstitial became thicker and bronchi collagen decrease after administration of drug-encapsulated NPs. Also, the administration of drug-encapsulated NPs reduces the expression of inflammatory factors, such as TGF-beta and NF-κB. From the above result, the dual-therapeutic PLGA NPs can reduce inflammation and alleviate or slow down pulmonary fibrosis deterioration.
In this study, we successfully developed a dual-therapeutic PLGA NPs that simultaneously carry both hydrophilic and hydrophobic drugs. Through encapsulation with metformin and prednisolone alleviated and slowed down the deterioration of pulmonary fibrosis, and served as a reference for clinicians. The purpose of this study is to greatly improve the patient’s quality of life with pulmonary fibrosis and reduce the expense of National health insurance and societal costs.

Contents
摘要 I
Abstract III
致謝 V
Contents VII
List of Table X
List of figures XI
Abbreviation List XIV
Chapter 1 Introduction 1
1.1. The severity and duration of pulmonary fibrosis 2
1.1.1. The prevalence and risk factors of pulmonary fibrosis(PF) 2
1.1.2. Current management of pulmonary fibrosis	3
1.2. Benefits and applications of drug delivery via inhalation 3
1.3. Techniques and benefit of inhalational dual-therapeutic nanoparticles (NPs) 4
1.3.1. Materials for inhalational dual-therapeutic nanoparticles 4
1.3.2. Manufacture methods of inhalational dual-therapeutic nanoparticles 5
1.3.3 Benefits of inhalational dual-therapeutic nanoparticles 6
1.4. The specific aims of this study 6
Chapter 2 Material and Methods 8
2.1. Materials 9
2.2. Experimental instruments 10
2.3. Synthesis of inhalational dual-therapeutic NPs 10
2.4. Physicochemical properties of the nanoparticle 11
2.4.1. The morphology, size and zeta potential of NPs 11
2.4.2. Determination the functional groups on NPs 11
2.5. Determination drug encapsulating rate in NPs 12
2.5.1. Determination encapsulating rate of NPs 12
2.5.2. in vitro Drug release study 12
2.6. Performance of NPs on Cell Lines 13
2.6.1. Cell culture 13
2.6.2. Cell counts 14
2.6.3. Induce inflammation on cell line 14
2.6.4. Cytotoxicity verification 14
2.6.5. Total ROS measurement 15
2.6.6. Apoptosis measurement 15
2.7. Performance of NPs on Animals 16
2.7.1. Animals 16
2.7.2. The process of intra-tracheal instillation with drugs 16
2.7.3. Evaluation of inhalation distribution 17
2.8. Ultrasonic aerosol inhaler devise for animal model and verification with IVIS 21
2.9. Data Analysis and Statistical Evaluation 21
Chapter 3 Results 23
3.1. Characteristics of inhalational drug-encapsulated NPs 24
3.2. Determination of drug concentration in NPs 24
3.3. Verify drug-encapsulated NPs toxicity and safety on different cell line and animal model 24
3.4. The anti-inflammation effect of drug-encapsulated NPs on cell line and animal model 25
3.5. The apoptosis rate of bleomycin-induced fibrosis cell line after treatment with drug-encapsulated NPs 26
3.6. M1 macrophage and M2 macrophage expression after treatment with drug-encapsulated NPs in fibrosis-induced C57BL/6 mice lung tissue 26
3.7. Type I collagen and MMP-9 manifestation on C57BL/6 mice 27
Chapter 4 Discussion 28
4.1. To establish a dual therapeutic drug-encapsulated PLGA NPs carrier 29
4.2. Establishment of pulmonary fibrosis model 30
4.3. The technique of intra-tracheal instillation (I.T.) and the distribution of drug-encapsulated NPs in lung 31
4.4. The effect of dual-therapeutics PLGA NPs on pulmonary fibrosis 32
4.4.1 Anti-inflammation effect of NPs on pulmonary fibrosis 32
4.4.2 The role of NPs on apoptosis rate of pulmonary fibrosis 32
4.4.3 The role of M1 and M2 macrophage on pulmonary fibrosis 33
4.4.4 Type I collagen deposition in pulmonary fibrosis	33
4.5. The benefit of inhalational dual-therapeutics PLGA NPs 34
4.6. Toxicity and degradation of PLGA NPs 35
4.7. Ultrasonic aerosol inhaler device for animal and its limitation of the design 35
4.8. Limitation and prospect 36
Chapter 5 Conclusion 38
Tables 40
Figures 44
Reference 63

List of Table
Table 1. Characterization of the modified scale 40
Table 2. Overview the manufacture and particle size of inhalational nanoparticle 40
Table 3. Drug encapsulation rates were determined by HPLC. The concentrations of metformin and prednisolone were determined at wavelengths of 233 and 254 nm. 41
Table 4. Reviews of dual-drug-encapsulated NPs formula 42
Table 5. Overview of the dosage of therapeutic agents on bleomycin-induced PF animal 43

List of figures
Figure 1. Risk factors for pulmonary fibrosis(PF) and other conditions can further induce PF 44
Figure 2. Strategy for pulmonary fibrosis treatment by using metformin and prednisolone 44
Figure 3. Effect of inhalation nanoparticles as combo-therapeutic carriers 45
Figure 4. Emulsion-evaporation method for the synthesis of inhalation double-drug encapsulated NPs. 45
Figure 5. TEM photomicrograph of drug-encapsulated NPs under solution at 60k (left, middle) and 15k (right). 46
Figure 6. Particle size distribution of NPs measured by DLS (Mean particle size (SD): 121 ± 29.1 nm). 46
Figure 7. The charge of NPs was determined by zeta potential (-8.06 ± 0.2 mV) 47
Figure 8. FTIR spectrum of (A) prednisolone, (B) metformin, (C) PLGA empty NPs and (D) drug-encapsulated PLGA NPs with wave numbers from 400-4000 cm-1	48
Figure 9. Encapsulating the rate of prednisolone (10x diluted NPs with DMSO) was determined by HPLC with 25 cm reversed phase C-18 column under the condition of the mobile phase consisting of acetonitrile: deionized water, 28:72 (v/v). Flow rate of the mobile phase was maintained at 1 mL/min. Tube temperature was set at 25 degrees Celsius and the UV absorption was measured at 254 nm. 49
Figure 10. Encapsulating rate of metformin (10x diluted NPs supernatant) was determined by HPLC with 25 cm reversed phase C-18 column under the condition of mobile phase consisting of acetonitrile: deionized water, 34:66 (v/v). Flow rate of the mobile phase was maintained at 1 mL/min. Tube temperature was set at 25 degrees Celsius and the UV absorption was measured at 233 nm . 49
Figure 11. Drug-encapsulated NPs underwent drug release at 1x PBS and 37℃. The concentration of metformin and prednisolone was determined at wavelengths of 232 and 247 nm. 50
Figure 12. Cell viability of different concentrations of NPs or pure drugs (2x: metformin 4.54 mg/mL, prednisolone 1.95 mg/mL; 10x: metformin 0.908 mg/mL, prednisolone 0.389 mg/mL; 100x: metformin 90.8 μg/mL, prednisolone 38.9 μg/mL) were measured by CCK-8 kit and detected at wavelength of 450 nm and the cell viability was calculated by the ratio to the absorbance value of control. * p value < 0.05 50
Figure 13. Cell viability of different cell lines were measured by the CCK-8 kit was detected at wavelength of 450 nm and the cell viability was calculated by the ratio to the absorbance value of the control. 51
Figure 14. Using the ROS-GloTM H2O2 Assay to measure the amount of total ROS in IMR-90 and L2 cell lines treated with different concentrations of NPs or pure drugs (2x: metformin 4.54 mg/mL, prednisolone 1.95 mg/mL; 10x: metformin 0.908 mg/mL, prednisolone 0.389 mg/mL; 100x: metformin 90.8 μg/mL, prednisolone 38.9 μg/mL) after 24 hours bleomycin induced.  ** p< 0.005, *** p< 0.001;  # p< 0.05,  ## p< 0.005,  ### p< 0.001 compared with bleomycin group by one-way ANOVA. 52
Figure 15. Using Annexin V/ PI to measure apoptosis rate in L2 cell lines treated with different concentrations of NPs or pure drugs (2x: metformin 4.54 mg/mL, prednisolone 1.95 mg/mL; 10x: metformin 0.908 mg/mL, prednisolone 0.389 mg/mL; 100x: metformin 90.8 μg/mL, prednisolone 38.9 μg/mL) after 24 hours bleomycin induced. * p< 0.01;  # p< 0.001 compared with the bleomycin group by one-way ANOVA. 53
Figure 16. The process of intra-tracheal instillation with the drug. (A) Lay the anesthetized mice on a slope and fix the front teeth onto the wire to open the mouth. (B) Use tweezers to pull out the tongue and keep it with the tongue depressor. (C) Observe the vocal cord with intense light from the body surface around the trachea site and then give drugs with a 20 μL pipet. 54
Figure 17. (A) Mice lungs were dissected to determine the particle distribution after 80 μL 0.4% trypan blue NPs given by intratracheal instillation(I.T.). (B) Mice was given 80 μL cypate NPs (390 μg/mL) and determined the lung distribution of nanoparticle by IVIS. 54
Figure 18. Outline of the design of the treatment group for evaluation of reversing pulmonary fibrosis with established fibrosis following bleomycin-induced lung injury. Bleomycin only gave at day 0 to induce fibrosis and the following treatment were gave every other day from day 7. 55
Figure 19. (A) Survival rate and (B) body weight percentage of mice in response to bleomycin (1.25 U kg–1, i.t.) and nanoparticles (Metformin 50 mg/kg, Prednisolone 5 mg/kg) or pure drugs (Metformin 50 mg/kg, Prednisolone 5 mg/kg) (N=9). 55
Figure 20. Evaluation of bleomycin-induced pulmonary fibrosis effect on mice after 7, 14 , and 21 days bleomycin (1.25U/kg) administration by H&E and Masson’s trichrome staining (40x and 200x). 56
Figure 21. The effect of nanoparticle treatment (Metformin 25mg/kg, prednisolone 10mg/kg) on bleomycin-induced mice after 21 days by H&E and Masson’s trichrome staining (A) Alveoli (B) Bronchi (40x and 200x). 57
Figure 22. Masson’s trichrome stain was quantified by using a modified Ashcroft scale. (A) The distribution of grades from 0 to 8 (B) Evaluated of the severity of pulmonary fibrosis between four groups. *** p< 0.001 57
Figure 23. (A) The anti-inflammation effect of nanoparticles on bleomycin-induced  fibrosis mice by DAB staining with TGF-beta antibody (200x) and NF-κB antibody (400x). (B) The magnified figure of DAB staining of NF-κB to show the nuclear signaling of NF-κB (red arrow). 58
Figure 24. IHC stain was quantified by using ImageJ with the IHC profiler plugin (Varghese et al., 2014) to quantify the TGF-beta data with percentage contribution of the score. * p< 0.05 ** p< 0.01 58
Figure 25. (A) Lung sections were subjected to IHC staining with anti- CD80 and anti- CD163 for detecting M1 and M2 macrophages in the lung, respectively (400x). (B) The average intensity of anti- CD80 and anti- CD163 were analyzed by ImageJ software. *** p<0.001 59
Figure 26. (A) Lung sections were subjected to IHC staining with anti-type I collagen and anti- MMP-9 for evaluating the effect of alleviation of the lung (400x). (B) The average intensity of anti- type I collagen and anti-MMP-9 were analyzed by ImageJ software. *** p<0.001 60
Figure 27. Ultrasonic aerosol inhaler device design for animals. (A) Baffle type nebulizer by 3D printing (B) 1.2MHz Ultrasonic driven aerosol generator with micro-nozzle plate 16mm plate (C) 1.2MHz Ultrasonic driven aerosol generator with the micro-nozzle plate 61
Figure 28. (A) Drug distribution by using intra-tracheal(I.T.) instillation (80 μL) or ultrasound inhaler (15 minutes) to administrate cypate NPs (390 μg/mL) and determined the fluorescence by in vivo imaging system (IVIS) (Ex: 745/Em:840). (B) IVIS image after lung harvesting. (C) The devise design of ultrasound inhaler. 61
Figure 29. The schematic of metformin and prednisolone on treating pulmonary fibrosis. 62
Figure 30. The schematic of dual-therapeutic nanoparticles to treating pulmonary fibrosis. 62



                                    

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