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研究生:尼迪古普塔
研究生(外文):Nidhi Gupta
論文名稱:用於藥物輸送和生物成像應用的多隔室聚合物載體
論文名稱(外文):Multicompartmental Polymeric Carrier for Drug Delivery and Bioimaging Application
指導教授:詹揚翔Sampa Saha
指導教授(外文):Chan, Yang-HsiangSampa Saha
口試委員:李介仁廖尉斯張佳智林宏洲彭之皓Sampa Saha
口試委員(外文):Li, Jie-RenLiao, Wei-SsuChang, Chia-ChihLin, Hong-CheuPeng, Chi-HowSampa Saha
口試日期:2023-05-16
學位類別:博士
校院名稱:國立陽明交通大學
系所名稱:國際半導體產業學院
學門:工程學門
學類:電資工程學類
論文種類:學術論文
論文出版年:2023
畢業學年度:111
語文別:英文
論文頁數:141
中文關鍵詞:多隔室可生物降解藥物遞送生物成像電流體動力共噴射
外文關鍵詞:multicompartment particlesbiodegradable polymerdrug deliverybioimagingelectrohydrodynamic co-jetting
相關次數:
  • 被引用被引用:0
  • 點閱點閱:31
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CONTENTS
摘要 i
ABSTRACT ii
LIST OF FIGURES vii
LIST OF TABLES xi
LIST OF ABBERIVIATION xii
CHAPTER-1 1
INTRODUCTION AND LITERATURE SURVEY 1
1.1 Motivation and Background 1
1.2 Significance of carrier design 4
1.3 Fabrication Method 6
1.4 Application of Polymeric Carriers 10
1.4.4 Combinational therapy 15
1.5 Research Gap 16
1.6 Objective 16
1.7 Plan of Work 16
1.8 Thesis Format 18
CHAPTER-2 20
TRICOMPARTMENTAL MICROCARRIERS WITH CONTROLLED RELEASE FOR EFFICIENT MANAGEMENT OF PARKINSON'S DISEASE 20
2.1 Motivation and Background 20
2.2 Materials 22
2.3 Methodology 22
2.3.1 Fabrication of microparticles 22
2.3.2 Encapsulation of therapeutics 23
2.4 Characterization 24
2.4.1 Morphological Analysis 24
2.4.2 Atomic Force Microscope (AFM) 24
2.4.3 Confocal Laser Scanning Microscope (CLSM) 24
2.4.4 Micro-Raman Spectrometer 24
2.4.5 Differential Scanning Calorimetry 24
2.4.6 X-ray Diffraction 25
2.5 Invitro Study 25
2.6 Results and Discussion 25
2.6.1 Fabrication and Characterization of Tricompartmental particles 25
2.6.2 In-vitro drug release study 35
2.7 Summary 40
CHAPTER-3 42
INVIVO STUDIES OF DEVELOPED TRICOMPARTMENTAL CARRIER TO ANALYSE THE EFFICACY OF DEVELOPED SYSTEM. 42
3.1 Motivation and Background 42
3.2 Materials 44
3.3 Methodology 44
3.3.1 In vivo Study 44
3.3.2 Bodyweight evaluation: 45
3.4 Statistical Analysis 45
3.5 Pharmacokinetics 45
3.6 Histochemical Assessment 45
3.6.1 H&E staining: 45
3.6.2 Nissl staining: 46
3.6.3 Tyrosine Hydroxylase (TH)- 46
3.7 Results and Discussion 46
3.8 Summary 53
CHAPTER-4 54
OPTIMAL DESIGNING THE SHAPE AND CONTROLLED RELEASE OF THERAPEUTICS FROM THE MULTICOMPARTMENTAL CARRIER 54
4.1 Motivation and Background 54
4.2 Materials and Methods 56
4.2.1 Materials 56
4.2.2 Microparticle fabrication 57
4.2.3 Experimental design 59
4.3 Characterization 60
4.3.1 Morphological Analysis 60
4.3.2 Encapsulation Efficiency of therapeutics 61
4.3.3 Drug release Study 61
4.4 Result and Discussion 62
4.4.1 Morphological Analysis and in-vitro kinetics 62
4.4.2 Analysis of outcomes from Taguchi’s OA 67
4.4.3 Analysis of variance (ANOVA) for the Taguchi’s model 70
4.4.4 Validation of Taguchi’s model 70
4.4.5 Characterization of optimized microparticles 71
4.5. Regression analysis 73
4.5.1. Validation of regression model 73
4.5.2 Effect of ARDEV and Release factor on encapsulation efficiency 74
4.6 Summary 76
CHAPTER-5 77
TUNEABLE BICOMPARTMENTAL CARRIER SYSTEM FOR CO-DELIVERY OF MULTIPLE DRUGS WITH INDIVIDUAL RELEASE RATES. 77
5.1 Motivation and background 77
5.2 Materials 78
5.3 Methodology 78
5.3.1 Fabrication of microparticles 78
5.3.2 Encapsulation of therapeutics 79
5.4 Characterization 79
5.4.1 Morphological Analysis 79
5.4.2 Fourier Transfer Infrared Spectroscopy (FTIR) 79
5.4.3 Invitro Study 80
5.4.4 Cell-Viability study 80
5.5 Results & Discussion: 80
5.5.1 Fabrication and Characterization of Bicompartmental Particles 80
5.5.2 Co-delivery of Incompatible Drugs 85
5.5.3 Co-delivery of multiple drugs at tuneable release 86
5.5 Summary 89
CHAPTER-6 91
DESIGN OF PARTICLES FOR BIOIMAGING APPLICATION 91
6.1 Motivation and Background 91
6.2 Materials 92
6.3 Methodology 92
6.3.1 Fabrication of nanoparticles 92
6.3.2 Encapsulation of NIR-II dye 93
6.4 Characterization 93
6.4.1 Morphological Analysis 93
6.4.2 Particle Size and Zeta Potential 93
6.4.3 Stability Study 93
6.4.4 Brightness 94

6.4.5 Cell Viability 94
6.5 Result & Discussion 94
6.5.1 Fabrication of bicompartmental nanoparticles 94
6.5.3 Brightness study 101
6.5.4 Cell-Viability 102
6.6 Summary 103
CHAPTER-7 104
SUMMARY AND FUTURE SCOPE 104
7.1 Summary 104
7.2 Future Outlook 106
REFERENCES 108
List of Publications 140

LIST OF FIGURES
Figure 1.1
Timeline showing FDA-approved DDS in the market.[15]
4
Figure 1.2 (A) Schematic diagram of the interactions between the photons and tissue when executing fluorescence imaging. (B) absorption spectrum of water in the range of 400–1800 nm measured through a 1-mm-long path. (C) reduced scattering of different biological tissues and intralipid solution in the 400–1700 nm range. (D) autofluorescence spectra of ex vivo mouse liver (black), spleen (red), and heart tissue (blue) under 808 nm excitation. (E) timeline of significant milestones in the development of NIR-II fluorophore 13

Figure 1.3
Flow chart showing the Work Plan followed to fabricate the multicompartmental carrier 17

Figure 2.1 A. Schematic of Electrohydrodynamic co-jetting (EHDC) set-up to fabricate tricompartmental particles. B. Photograph of the Taylor cone, exhibiting the black color of the liquid-1 (loaded with iron nanoparticles) and white color of Liquid-2 and 3 (loaded with TiO2 ) to display three distinct phases maintained during EHDC 26

Figure 2.2
A) Optical Micrograph B) Atomic Force Micrograph of microparticles, the phase contrast shows the presence of three distinct compartments. C) SEM Micrograph (zoom-in-image inset) D) Particle size distribution (average particle diameter 4 ± 0.99 μm; standard deviation 0.99) and (E-G) CLSM micrographs of tricompartmental particles (system, T-4). E Blue channel represents PLA+PCL phase loaded with L.D. and F red channel represents LPLGA phase of particle loaded with CD on one side and PEG+ENT on the other. G Overlays of both blue and red channels. Scale bars represent A, C and E-G 10 μm and 5 μm for AFM micrograph 28

Figure 2.3
A)Represented Raman Spectra of tricompartmental microparticles loaded with (A) LD in the middle PLA/PCL phase-1, (B) CD in LPLGA phase-2 (C)) and ENT in LPLGA/PEG phase-3. Corresponding spectra of the neat PLA, PCL, LPLGA, PEG, and drugs LD, CD, and ENT were given to compare drugs' presence in the localized compartment. (D) Raman Optical Micrograph of tricomaprtmental microcarrier irradiated at different positions of a particle 30

Figure 2.4
A. DSC thermogram of pure PLA, tricompartmental microparticles (neat and drug-loaded), and simple blend (PLA / PLGA / PCL / PEG / drugs). B XRD 32

Figure 2.5 (A)NMR spectra of neat LD, neat CD corresponding to the LD and CD after extraction from monophasic microparticles in D2O . (B) NMR spectra of neat ENT and ENT encapsulated in monophasic microparticles.
34

Figure 2.6 In vitro release study of therapeutics (LD, CD and ENT) from different formulations A) T-1, B) T-2, C) T-3, D) T4, E) T5, F) T-6.
37


Figure 2.7 : (A) SEM micrograph of drug loaded particles after24h of in vitro release (B) Water uptake study of drug loaded microparticles
38

Figure 2.8 In vitro release study of therapeutics from A) system T-7, B) Commercially available tablet (syncapone)
39

Figure 2.9 A: Zero Order, B: First Order, C: Higuchi, D: Hixon-Crowell, E: Rigter-Peppas
40

Figure 3.1 A) Body Weight evaluation, B) Rotarod C) Catalepsy D) Swim Test E) Passive avoidance. Values are mean ±SEM values (n =5 in each group). ααα(P <0.001) compared to saline-treated normal group; *(P < 0.05), **(P <0.01), ***(P <0.001) compared to treatment group.
48

Figure 3.2 Plasma concentration-time profile of Levodopa (LD) in the market and microparticle formulation. Results are expressed as the mean ± SD (n=4).
50

Figure 3.3 Hematoxylin and Eosin staining of the nigral neuron of the brain depicting the pathological manifestation in various rats. Blue Circle- represents the prominent nucleoli, black arrow-Pyknotic nuclei. (Original magnification 40X)
52

Figure 3.4 (A)Nissl-Staining of nigral neuron (B) Statistical graph representing the no. of viable neurons, quantify through ImageJ software (C )TH-immunoreactivity in the SNc. Photomicrographs of immuno-staining sections of rat SNc from Group 1 (microparticles), Group 2 (Market), Group 3 (Rotenone),and Group 4 (Control) were shown. Dark brown staining gives a qualitative indication of TH presence. (Original magnification: 40×)
52

Figure 4.1 Schematic representation of electro-jetting
58

Figure 4.2 RBC AR 1.25 ± 0.1 (calculated using ImageJ)
64

Figure 4.3 SEM images for experimental trials according to Taguchi L9 (P-1 to P-9), the zoom-in image inset at left corner, scale bar: 1μm.
64

Figure 4.4 In vitro release study of LD and CD from a)P1, b)P2, c)P3, d)P4, e)P5, f)P6, g)P7, h)P8, i)P9 in SGF for 5 h followed by release into SIF for 24 h at 37 °C for L9 Taguchi experiments
66

Figure 4.5 Main effect of SN ratios on ARDEV
68

Figure 4.6 Main effect of SN ratios for Release factors
69

Figure 4.7 Control variables' relative contribution on the a) ARDEV and b) Release factor
69

Figure 4.8 A) SEM image of optimized microcarrier, the zoom-in image inset left corner, scale bar-0.5um b) particle size distribution c) confocal image tricompartmental microcarrier loaded with dye in PLGA phase, 1 exhibited the blue fluorescence irradiated by 405nm UV laser, 2- demonstrated the DIC micrograph, 3-overlay of DIC and blue fluorescence d) In-vitro release of LD, CD, and ENT of the optimized electro-jetted particle
72

Figure 4.9 Confocal laser scanning microscopy (CLSM) image of low ARDEV and RF and High ARDEV and RF
75

Figure 5.1 Experimental set-up of bicompartmental particles
81

Figure 5.2 Design of bicompartmental polymeric carrier
81

Figure 5.3 SEM micrograph of biphasic microparticles at A) 0%, B) 10%, C)25%, and D) 50% PEI concentration in one phase.
82

Figure 5.4 SEM micrograph of bicompartmental microparticles with A) 10w/w %, B) 25w/w%, and C) 50w/w% PEI along with 5 w/w% BTDA of PEI in one phase.
83

Figure 5.5 FTIR spectra of PLGA, PEI, PEI+crosslinker(BTDA), and PLGA+PEI+Crosslinker (BTDA).
84

Figure 5.6 SEM Micrograph of bicompartmental particles after incubating in pH-5 for 24h
85

Figure 5.7 CLSM Micrograph of bicompartmental microparticles with A) PLGA / PLGA, B)PLGA+CdSe / PLGA+BLUE dye, C) PLGA+CdSe / PLGA + PEI + BTAD+Blue dye, D) PLGA+Blue dye / PLGA+PEI+BTAD+CdSe in different phase.
86

Figure 5.8 Cumulative release profile of model drug Rhodamine B(RB) and Ibuprofen (IB) from PLGA+RB in one phase and Ibuprofen in with A) PLGA B)PLGA+5w/w%PEI, C) PLGA + 5 w/w% PEI + BTDA, D) PLGA+10 w/w% PEI+ BTDA, E) PLGA + 25w/w% PEI + BTDA, and F) PLGA+50w/w%PEI+BTDA in another phase.
87

Figure 5.9 MTT-assay of microparticles with PLGA in one phase and various concentrations of PLGA+PEI in the other phase. A) PEI10 : PLGA+10w/w% PEI, PEI10X: PLGA+10w/w% PEI+BTDA, PEI25: PLGA+25w/w%, B) PEI, PEI25X: PLGA+25w/w% PEI+BTDA, PEI50: PLGA+50w/w% PEI, PEI50X: PLGA+50w/w% PEI+BTDA, Control: PLGA (no PEI).
89

Figure 6.1 Biphasic nanoparticles through EHDC technique. The polymer concentration of 3, 2,1 w/v% in CHCl3: DMF (1:1) at a flow rate of 50 μl/h and 100 μl/h.
95

Figure 6.2 Biphasic nanoparticles through EHDC technique. The polymer concentration of 1, 0.8, 0.5 w/v% in CHCl3: DMF (1:1) at a flow rate of 30μl/h.
95

Figure 6.3 Biphasic nanoparticles through EHDC technique. The polymer concentration of 1 w/v% in CHCl3: DMF (6:4) at flow rates of 50μl/h, 30μl/h and 20μl/h.
96

Figure 6.4 The SEM micrograph of nanoparticles at a polymer concentration of 1 w/v % in solvent concentration (DMF : CHCl3) of 7:3, 8:2 and 95:5 at a flow rate of 20μl/h.
97

Figure 6.5 SEM micrograph of particles dispersed in A) acetone, B) water (lyophilized), C) TEM micrograph D) Particle size-distribution of nanoparticles fabricated from polymer concentration of 1 w/v % at a solvent concentration (DMF :CHCl3) of 6:4 at a flow rate of 20μl/h, by passing through syringe filter of 0.22μm.
97

Figure 6.6 A) SEM micrograph B) TEM micrograph C) Particle size distribution of biphasic particles with crosslinked PEI.
98

Figure 6.7 SEM micrograph of particles fabricated at 1 w/v % polymer concentration with A) 0.5w/w%, B) 1w/w%, C) 1.5w/w% of CTAB. D) SEM micrograph of optimized biphasic nanoparticles with 10% NIR-II dye and the particle size distribution given in (E).
100

Figure 6.8 SEM micrograph of biphasic nanoparticles with 25w/w% of PEI in one phase after dispersing in ethanol.
100

Figure 6.9 A) SEM micrograph of biphasic nanoparticles with 40% NIR-II dye, B) particle size distribution of individual particles measured through DLS, C) Scheme showing conjugation of DSPE-PEG-CM to nanoparticles by EDC/NHS coupling D) Particle size distribution of modified nanoparticles, E) Zeta-potential of developed nanoparticles system.
101

Figure 6.10 Brightness Intensity of nanoparticles loaded with 10% NIR-II dye in PLGA phase, irradiated by 793nm laser, using 1100 long pass filter at an exposure of 100s at a concentration of A) 30mg/ml, B) 15mg/ml, C) 10mg/ml, and D) 7.5mg/ml.
102

Figure 6.11 Brightness Intensity of nanoparticles loaded with 40% NIR-II dye in PLGA phase with both DSPE-PEG modified and unmodified, irradiated by 793nm laser, using 1100 long pass filter at an exposure of 100s at a concentration of A) 30mg/ml, B) 20mg/ml, C) 12.5mg/ml, D) 10mg/ml, E) 5mg/ml and F) 2mg/ml.
102

Figure 6.12 MTT-Assay of nanoparticles loaded with 40% NIR-II polymer in PLGA phase with A) PEG conjugation and B) unmodified surface, respectively.
103




LIST OF TABLES
Table 2.1 Wavenumber Assingment of Raman Spectroscopy of Copartment-1 (LD)
31

Table:2.2 Wavenumber Assignment of Raman Spectroscopy of Compartment-2(CD)
31

Table:2.3 Wavenumber Assignment of Raman Spectroscopy of Compartment-3(ENT) 32

Table 2.4 Transition temperatures and crystallinity obtained from DSC thermograms (second heating transition) 33

Table 2.5 Composition of compartment, particle size and drug encapsulation efficiency in various systems
35

Table 2.6 Coefficient of determination (R2 correlations) for several mathematical models and n (release exponent) values from Ritger– Peppas model for all the three drugs
39

Table 3.1 Different animal groups studied in vivo 47

Table 3.2 Pharmacokinetics parameter of LD after drug solution from Microparticles and Market formulation after oral administration in rats (12hrs). 50

Table 4.1 Control factors with three different level
58

Table 4.2 Taguchi L9 OA experimental design
59

Table 4.3 Encapsulation efficiency and cumulative release (5 h) for particles 67

Table 4.4 Design of Experiment (DOE Taguchi’s L9 OA) of aspect ratio deviation, release factor, and S/N ratio 68

Table 4.5 ANOVA analysis for the Taguchi’s designed
70

Table 4.6 Validation test results for minimization of ARDEV and release factor
71

Table 4.7 Regression validation analysis
74

Table 4.8 ARDEV and Release factor on the encapsulation efficiency of LD, CD, and ENT
75



LIST OF ABBERIVIATION
ACN Acetronitrile
ANOVA Analysis of variance
ARdev Aspect ratio
AFM Atomic Force Microscope
BTDA 1,2,3,4 Butanetertacarboxylic dianhydride
BSA Bovine serum albumin
CD Carbidopa
CV Coefficient of variance
COMT Catechol-O-methyltransferase
CT Computed Tomography
CLSM Confocal Laser Scanning Microscope
DOE Design of Experiment
DCM Dichloromethane
DMSO Dimethyl Sulfoxide
DDC Dopa decarboxylase
EHDC Electrohydrodynamic co-jetting
EE Encapsulation Efficiency
ENT Entacapone
FI Fluorescence Imaging
H&E Hematoxylin and Eosin
HCL Hydrochloric acid
IB Ibuprofen
LD Levodopa
MM Malignant Mesothelioma
NMP N, methyl-2-Pyrrilidinone
DMF N, N-dimethylformamide
ONC Onconase
OA Orthogonal Array
PD Parkinson's disease
PRINT Particle Replication in Nonwetting Template
PAI Photoacoustic Imaging
PVA Poly (vinyl alcohol)
PEG Poly(ethylene glycol)
PVP Poly(vinylpyrrolidone)
PCL Polycaprolactone
PEI Polyethyleneimine
PLA Polylactic acid
PLGA Polylactic-co-glycolic acid
PET Positron Emission Tomography
QD Quantum Dots
QY Quantum Yield
RB Rhodamine B
RENP Rare-Earth-Doped Nanoprobes
RBC Red Blood cell
RF Release Factor
SEM Scanning Electron Microscopy
S/N Signal-to-Noise ratio
SGF Simulated Gastric Fluid
SIF Simulated Intestinal Fluid
SPECT Single-Photon Emission Computed Tomography
SWCNT Single-Walled Nanotubes
SDS Sodium Dodecyl Sulfate
SN Substantia Nigra
TH Tyrosine Hydroxylase
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