Smart Nanocarriers Bearing Docetaxel for Improved Chemotherapy Cancer

By Gitu Pandey

Cancer is a term used for a collection of diseases in which some of the normal and somatic body 
cells start dividing and re-dividing out of control . It is a leading cause of  the  
morbidity  and  mortality  worldwide,  second  only  to  the  cardiovascular  diseases . The occurrence of cancer is not new to human kind and the world's oldest recorded case of breast cancer dates back to 1500 BC from ancient Egypt. Some evidences of human  osteosarcoma  were 
 also  found  in  mummies  in  ancient  Egypt  which  have  been recorded  in  the  manuscripts  
dated  about  1600  B.C  .  The  word  cancer, however, was first used by a physician 
Hippocrates  which finds its origin in a Greek word 'karkinos' to describe carcinoma 
tumors .

 

Our body is made up of the trillions of cells and any one of them could be the site of cancer 
initiation. During the normal physiological course, the human cells grow and divide to form new   
cells   as   and   when   the   need   arises   (McDonald,   Fielding,   &   Dedhar,   2008). 
Simultaneously,  the  old  and  damaged  cells  die  and  new  cells  replace  them.  This  process 
operates  in  a  checked  manner.  But  this  methodical  operation  breaks  down  when  cancer 
develops  and  cells  grow  in  an  unchecked  manner.  These  abnormal  cells  then  form  solid 
growths called tumors  . Some cancers like that of blood cells and known as leukemias 
do not form solid tumors and remain floating in the blood stream and may reach other  distal  
organs.  The  DNA  damage  repair,  which  is  an  inherent  mechanism  in  normal cells, is 
compromised in cancerous cells. This damaged DNA can often be inherited leading
to a genetic predisposition to cancer.

 

Cancer  can  be  classified  according  to  the  organ  of  origin  or  according  to  the  cells  
of  the origin of cancer . On the basis of the cells of origin,  major  types  of  clinically  identified  cancer  are  carcinomas,  sarcomas,  
leukaemias, lymphomas, myelomas, and brain and spinal cord cancer. Carcinomas are the most common 
type of cancer begins in the skin or in epithelial cells which lines the external body surface and 
covers the internal organs. These have been further classified into the subtypes such as, 
transitional     cell     carcinoma,     basal     cell     carcinoma,     squamous     cell     
carcinoma and adenocarcinoma etc. depending upon the epithelial layer of origin  .  

• Language: English
• Publisher: MOHAMMED ABDUL SATTAR
• Release date: Aug 25, 2023
• ISBN: 9798223106265

Book preview

Chapter 1 : Introduction.....................................1

1.1  Introduction............................................2

1.2  Types of cancer.........................................3

1.3  Treatment modalities......................................4

1.3.1  Surgery............................................4

1.3.2  Radiation therapy......................................5

1.3.3  Chemotherapy.......................................5

1.3.4  Immunotherapy.......................................6

1.4  Conventional chemotherapy..................................6

1.5  Nanotechnology in chemotherapy...............................7

1.5.1  Advantages of nanoparticles...............................7

1.5.2  Types of nanoparticulate systems............................8

1.5.3  Tumor targeting with nano-systems...........................10

1.6  Nanocrystals..........................................12

1.6.1  Advantages of nanocrystals...............................12

1.6.2  Preparation methodologies................................13

1.7  Marketed nanocrystals.....................................15

1.7.1  Injectable nanocrystals..................................16

1.7.2  Targeted nanocrystals..................................16

1.8  Nanoemulsions.........................................17

1.8.1  Advantages of nanoemulsions..............................19

1.8.2  Preparation methodologies................................19

1.8.3  Fate of nanoemulsion after oral administration....................21

1.9  Nanocapsules.........................................21

1.9.1  Advantages of nanocapsules..............................22

1.9.2  Preparation methodologies................................23

1.9.3  Nanocapsules in cancer therapeutics..........................24

1.10  Drug Profile..........................................25

1.10.1  Docetaxel.........................................25

1.10.2  Mechanism of action...................................26

1.10.3  Pharmacokinetics....................................27

1.10.4  Indications and Clinical use...............................28

1.10.5  Adverse reactions....................................28

1.10.6  Drug interactions.....................................29

1.10.7  Drug warning.......................................29

1.10.8  Brand name:Taxotere®, Docefrez™.........................29

1.11  Specific objectives......................................29

Chapter 2 : Literature Review.................................32

Chapter 3 : Preformulation studies..............................43

3.1  Preformulation studies.....................................44

3.2  Docetaxel............................................44

3.2.1  Physical appearance...................................44

3.2.2  Melting point........................................44

3.2.3  Solubility determination..................................44

3.2.4  Partition co-efficient determination...........................45

3.2.5  Identification of docetaxel................................45

3.2.6  Analytical method development.............................48

3.2.7  Bio-analytical method development...........................49

3.2.8  Method validation.....................................50

3.2.9  Results...........................................51

Chapter 4 : Multifunctional Glycoconjugate Assisted Nanocrystalline Drug Delivery for Tumor Targeting and Permeabilization of Lysosomal-Mitochondrial Membrane . 57

4.1  Introduction...........................................58

4.2  Experimental section......................................61

4.2.1  Materials and methods..................................61

4.2.2  Synthesis of Chondroitin Sulphate A- Polyethylene Glycol conjugate.......61

4.2.3  Fabrication of docetaxel nanocrystals.........................61

4.2.4  Optimization of process parameters..........................62

4.2.5  Preparation of FITC tagged nanocrystals.......................62

4.2.6  Particle Size, Size distribution and Zeta potential...................63

4.2.7  Drug content........................................63

4.2.8  Surface morphology....................................63

4.2.9  In vitro dissolution..............................................63

4.2.10  Measurement of Fixed Aqueous Layer Thickness (FALT).............64

4.2.11  Storage stability.....................................64

4.2.12  Protein corona......................................64

4.3  In vitro cell line studies..............................................64

4.3.1  Cell culture conditions..................................64

4.3.2  Time dependent cell uptake...............................65

4.3.3  CD44 receptor blocking study..............................65

4.3.4  Cytotoxicity.........................................65

4.3.5  Cell cycle arrest......................................66

4.3.6  Apoptosis..........................................66

4.3.7  Mitochondrial membrane potential...........................66

4.3.8  Lysosomal membrane integrity.............................66

4.3.9  Scratch based cell migration assay...........................67

4.3.10  In vivo pharmacokinetics........................................67

4.3.11  In vivo efficacy studies..........................................67

4.3.12  Toxicological evaluation.................................68

4.4  Results and discussion....................................69

4.4.1  Synthesis of Chondroitin Sulphate A- Polyethylene Glycol conjugate.......69

4.4.2  Fabrication and characterization of DTX nanocrystals................69

4.4.3  Drug Content........................................73

4.4.4  Morphological characterization.............................73

4.4.5  Fixed aqueous layer thickness..............................74

4.4.6  In vitro dissolution..............................................75

4.4.7  Storage stability......................................76

4.4.8  Protein corona.......................................77

4.4.9  Cytotoxicity.........................................77

4.4.10  Time dependent cell uptake..............................78

4.4.11  CD44 receptor blocking study.............................81

4.4.12  Cell cycle arrest.....................................82

4.4.13  Apoptosis.........................................83

4.4.14  Mitochondrial Membrane Potential (MMP)......................84

4.4.15  Lysosomal Membrane Integrity (LMI).........................86

4.4.16  Cell migration assay...................................87

4.4.17  Pharmacokinetics....................................88

4.4.18  In vivo tumor regression.........................................90

4.4.19  Toxicological evaluation.................................91

4.5  Conclusion...........................................94

Chapter 5 : P-gp modulatory Acetyl-11-keto-β-

boswellic acid based nanoemulsified carrier system for augmented oral chemotherapy of docetaxel 95

5.1  Introduction...........................................96

5.2  Materials and Methods....................................99

5.2.1  Materials..........................................99

5.2.2  Solubility determination of DTX..............................99

5.2.3  Preparation of nanoemulsion..............................100

5.2.4  Optimization parameters................................100

5.2.5  Particle size, Size distribution and Zeta potential..................101

5.2.6  pH measurements....................................101

5.2.7  Percentage transmittance................................101

5.2.8  Stress test........................................102

5.2.9  Morphological characterization by TEM........................102

5.2.10  Encapsulation Efficiency................................102

5.2.11  In vitro release study..........................................103

5.2.12  Stability studies.....................................103

5.3  Cell culture...........................................104

5.3.1  Cell uptake study by flow cytometry..........................104

5.3.2  P-gp inhibition studies..................................104

5.3.3  Cytotoxicity on MDA-MB-231 cells...........................105

5.3.4  Cell cycle distribution..................................106

5.3.5  Apoptosis.........................................106

5.4  In vivo animal studies..............................................106

5.4.1  In vivo pharmacokinetics........................................107

5.4.2  Liquid-liquid extraction and LC-MS/MS methodology................107

5.4.3  In vivo antiproliferative activity....................................108

5.5  Statistical analysis......................................108

5.6  Results and discussion....................................108

5.6.1  Solubility of docetaxel in different oils.........................108

5.6.2  Optimization of process parameters and surfactant concentration.........109

5.6.3  pH and Transmittance..................................112

5.6.4  Stress test.........................................113

5.6.5  Morphological characterization.............................113

5.6.6  Encapsulation efficiency.................................114

5.6.7  In vitro drug release............................................114

5.6.8  Stability studies......................................115

5.6.9  Cell uptake study.....................................119

5.6.10  P-gp inhibition study by flow cytometry and confocal microscopy........119

5.6.11  P-gp inhibition study via HPLC............................120

5.6.12  Cytotoxicity study....................................122

5.6.13  Cell cycle arrest.....................................122

5.6.14  Apoptosis........................................123

5.6.15  Pharmacokinetics....................................125

5.6.16  In vivo anti-proliferative activity...................................126

5.7  Conclusion...........................................128

Chapter 6 : Fucose ligated docetaxel bearing frankincense oil nanocapsules for neo- vasculature inhibition and improved tumor suppression  129

6.1  Introduction..........................................130

6.2  Experimental section.....................................131

6.2.1  Materials and methods.................................131

6.2.2  Preparation of nanocapsules..............................132

6.2.3  Optimization of process parameters..........................132

6.2.4  Fucosylation of nanocapsules.............................133

6.2.5  Particle size, Size distribution and Zeta potential..................133

6.2.6  Surface morphology...................................133

6.2.7  Drug loading and Encapsulation efficiency......................134

6.2.8  In vitro drug release............................................134

6.2.9  Storage stability.....................................134

6.3  Cell based studies......................................135

6.3.1  Cell uptake........................................135

6.3.2  Competitive binding assay...............................136

6.3.3  Cytotoxicity studies in MDA-MB-231.........................136

6.3.4  Apoptosis.........................................136

6.3.5  Mitochondrial membrane potential...........................137

6.3.6  Lysosomal membrane integrity.............................137

6.3.7  Anti angiogenic activity.................................137

6.3.8  Cell migration assay...................................138

6.3.9  Pharmacokinetics....................................138

6.3.10  In vivo efficacy study..........................................139

6.3.11  Toxicity analysis....................................139

6.4  Results and Discussion...................................140

6.4.1  Preparation and characterization of nanocapsules.................140

6.4.2  Fucosylation of nanocapsules.............................142

6.4.3  Surface morphology...................................143

6.4.4  Drug loading.......................................144

6.4.5  In vitro drug release............................................144

6.4.6  Storage stability.....................................145

6.4.7  Cell uptake........................................146

6.4.8  Cytotoxicity........................................148

6.4.9  Apoptosis.........................................150

6.4.10  Mitochondrial membrane potential..........................151

6.4.11  Lysosomal membrane integrity............................152

6.4.12  Angiogenesis inhibition................................154

6.4.13  Cell Migration Assay..................................155

6.4.14  Pharmacokinetic study.................................156

6.4.15  In vivo tumor regression study...................................157

6.4.16  Toxicity studies.....................................158

6.5  Conclusion...........................................160

Chapter 7 : Summary and Conclusions...........................161

References......................................................165

List of Figures

Figure 1-1: Various clinically used treatment options for different types of cancer. 4

Figure 1-2: Schematic represent of tumor targeting via passive and active mechanisms (SalmanOgli, 2011).  11

Figure 1-3: Pictorial representation of nanonization of drug crystals leading to increased surface exposure.  12

Figure 1-4: Bench-top model of Microfluidizer M-100P. 14

Figure 1-5: Schematic representation of a nanoemulsion droplet and nanocapsule particle. 22

Figure 1-6: Chemical structure of Docetaxel. 26

Figure 1-7: Mechanism of anticancer action of docetaxel at high and low dose. At low dose it inhibits the microtubule dynamics whereas at high dose it directly inhibits the microtubule polymerization. MT: Microtubule (Seruga, Ocana, & Tannock, 2011).  27

Figure 3-1: UV spectra of DTX indicating the absorption maximum. 46

Figure 3-2: Fourier Transformed Infrared (FT-IR) spectrum of DTX. 47

Figure 3-3: Mass spectrum of DTX. 47

Figure 3-4: Proton NMR spectrum of DTX. 48

Figure 3-5: Representative HPLC chromatogram for blank sample. 52

Figure 3-6: Representative HPLC chromatogram for DTX in analytical sample. 53

Figure 3-7: Representative LC-MS/MS chromatogram for blank plasma sample. 53

Figure 3-8: Representative LC-MS/MS chromatogram for DTX in plasma sample. 53

Figure 3-9: Calibration curve of DTX plotted between peak area and concentration at 228 nm. 54 Figure 4-1: Diagrammatic illustration of the proposed mode of action of the mPEG-CSA coated nanocrystals.  60

Figure 4-2: Synthetic scheme of conjugation between chondroitin sulfate A and polyethylene glycol via carbodiimide chemistry and the NMR spectra of chondroitin sulfate A and prepared pegylated chondroitin (mPEG-CSA).  69

Figure 4-3: Effect of surfactant concentration (PEG and mPEG-CSA) on particle size and zeta potential. The particle size decreases and the absolute value of zeta potential increases with increase in the surfactant concentration. All results are expressed as mean± SD (n=3).  71

Figure 4-4: (A) & (B) The size distribution and zeta potential curve of the optimized DTX@CSA-NCs.

73

Figure 4-5: The scanning electron microscopic and (B) atomic force microscopic image of the optimized DTX@CSA-NCs clearly displaying the rod shape of the nanocrystals.  74

Figure 4-6: A) Zeta potential versus Debye-Huckel plot for DTX@CSA-NCs after dispersing them in various concentration of sodium chloride. B) In vitro DTX release from various formulations in pH 7.4 phosphate buffer saline.  75

Figure 4-7: (A) Change in particle size and PDI of DTX@PEG-NCs over a period of 8 weeks (B) The change in zeta potential of DTX@PEG-NCs with respect to temperature over a period of 8 weeks. 76 Figure 4-8: Change in particle size and PDI of DTX@CSA-NCs over a period of 8 weeks (B) The change in zeta potential of DTX@CSA-NCs with respect to temperature over a period of 8 weeks.  77

Figure 4-9: Dot plot via FACS showing percent FITC positive MDA-MB-231 cells after treating with FITC tagged formulations at the end of 1 hour, 2 hour and 4 hour.  79

Figure 4-10: Fluorescence microscopic images showing FITC positive cells after treating MDA-MB- 231 cells with FITC tagged formulations at the end of 1 hour, 2 hour and 4 hour.  80

Figure 4-11: Dot plot via FACS showing percent FITC positive 4T1 cells after treating with FITC tagged formulations at the end of 1 hour and 4 hour.  81

Figure 4-12: Flow cytometry histogram displaying uptake of DTX@CSA-NCs in (A) MDA-MB-231 and

(B) MCF-7 cells; black: control cells, green: uptake in presence of HA, red: uptake in absence of HA.

82

Figure 4-13: Histograms showing percent of cells at various stages of cell cycle after treating with different DTX formulations for 24 hour and 48 hour.  83

Figure 4-14: The dot plot and bar graph representing the percent of MDA-MB-231 cells undergoing early (lower right quadrant) and late apoptosis (upper right quadrant) after treatment with different DTX formulations.  84

Figure 4-15: (A) & (B) Dot plot and bar graph showing mitochondrial depolarization at the end of 24 and 48 hour after treatment with Taxotere®, DTX@PEG-NCs and DTX@CSA-NCs. The red dots and green dots correspond to normal and depolarized mitochondria respectively.  85

Figure 4-16: (A) & (B) The confocal microscopic images and associated corrected total cell fluorescence showing the disruption of lysosomal membrane in MDA-MB-231 cells after treatment with different DTX formulations and AO staining.  87

Figure 4-17: (A) The scratch based wound healing assay representing metastasis inhibiting potential of the mPEG-CSA stabilized nanocrystals after 24 hour and 48 hour.  88

Figure 4-18: Plasma DTX concentration versus time profile curve of Taxotere®, DTX-NCs, DTX@PEG- NCs and DTX@CSA-NCs. Formulations were injected through intravenous route in rats at a dose equivalent to 10 mg/kg of DTX.  89

Figure 4-19:The mean tumor growth curves during the study, B) The body weight of the animals for the duration of the treatment (C) & (D) The tumor burden and mean tumor volume at the end of the study showing least burden in DTX@CSA-NCs treated group. E) Representative images of the morphology of excised tumors at the end of the study. The data represents mean±SD, n=5.  91

Figure 4-20: The percent hemolysis resulting with different formulations after incubating them for 4 hour with 2% red blood cell suspension.  92

Figure 4-21.(A) The evaluation of motor co-ordination studied using rotarod apparatus on day 12 and day 26 (B) Pain response in hot plate test after 12 and 26 days.  93

Figure 4-22: The histological sections of different organs after treatment with different DTX formulations for 15 days.  94

Figure 5-1: Schematic representation of the P-gp blockade by the boswellic acid constituents of the FO present in the nanoemulsion leading to the higher intracellular DTX concentration and consequently increased efficacy.  98

Figure 5-2: (A) The effect of homogenization pressure on particle size and polydispersity index displaying gradual decrease in both parameters, (B) Effect of surfactant PVA concentration on particle size just after preparation of formulation on same day and after 2 days(All values represent mean ± SD, n=3).  110

Figure5-3: (A)The average particle size and average zeta potential of optimized batch. 111

Figure 5-4: TEM image of optimized DTX-FO-NE displaying spherical droplet of the nanoemulsion.

114

Figure 5-5: The release profile of DTX-NE, DTX-FO-NE and marketed formulation Taxotere® in pH 7.4 buffer up to 48 hour.  115

Figure 5-6: The change in particle size and PDI of the DTX-FO-NE upon storage at 4 ±1 °C and 22 ±1

°C for a total period of 90 days. 116

Figure 5-7:The change in particle size and PDI of the DTX-NE upon storage at 4 ±1 °C and 22 ±1 °C for a total period of 90 days.  117

Figure 5-8: Cell uptake graph showing the higher uptake of the prepared nanoemulsion (red) than the FITC solution (green). The black graph is representing cinherent fluorescence of cells.  119

Figure 5-9: The CLSM images of Caco-2 cells displaying the enhanced uptake of Rho-123 in FO treated group as compared to non treated control group. The highest fluorescence is displayed by verapamil (100μM) treated group which is potent inhibitor of P-gp pump.  120

Figure 5-10: (A) The cellular uptake of DTX quantified via HPLC after treating with different formulations with or without FO and verapamil. (B) The flow cytometric data depicting internalization of Rho-123( green- Rho-123, pink- Rho-FO-NE, Violet- Rho + verapamil).  121

Figure 5-11: The cell cycle distribution in MDA-MB-231 cells after incubation with Taxotere®, DTX-NE and DTX-FO-NE for 24 and 48 hour at a dose equivalent to 0.5μM DTX.  123

Figure 5-12: (A) Representative dot plots of the apoptosis analysis via flow cytometer at the end of 48 hours, (B) Bar graph representing percentage apoptotic cells after 24 hour and 48 hour.  124

Figure 5-13: The bright field images showing morphology of cells after treatment with various formulations for 24 and 48 hour.  124

Figure 5-14.Comparative plasma concentration – time profile of DTX after oral administration of Taxotere® and DTX-FO-NE at a dose equivalent to 20mg/kg of DTX (inset showing the PK profile from 0-4 h). The values represent mean ± SEM ( n=3 per group).  125

Figure 5-15: (A) The tumor shape and morphology at the end of the study (B) The mean tumor growth curves during the study period from the starting of dosing (10mg/kg). Each value in graph is mean±SD, n=5.  127

Figure 5-16: (A) & (B) The mean tumor volume and tumor burden at the end of the study showing least burden in DTX-FO-NE treated group. The values represent mean ±SD (n=5).  128

Figure 6-1: A & B) The representative size and zeta potential curves of the optimized batch of nanocapsules.  143

Figure 6-2: The scanning electron microscopic images of the optimized and fucose coated nanocapsules.  144

Figure 6-3: The atomic force microscopic images of prepared nanocapsules in two dimensional and three dimensional view.  144

Figure 6-4: The comparative profile of DTX release from Taxotere®, DTX@FO-NCaps and Fu- DTX@FO-NCaps at conditions mimicking systemic pH (pH 7.4) and tumor microenvironment (pH 5.4).  145

Figure 6-5: (A)The stability profile in terms of change in particle size, polydispersity index and (B) zeta potential during storage at two different temperatures over a duration of seven weeks.  146 Figure 6-6: The fluorescence microscopic images of the MDA-MB-231 cells treated with free dye and dye loaded in non-targeted (FITC@FO-NCaps) and fucose ligated nanocapsules (Fu-FITC@FO- NCaps).  147

Figure 6-7: (A) The histograms obtained via flow cytometry showing distinct shift in fluorescence intensity after treatment with different FITC loaded formulations; black: FITC solution, Green: FITC@FO-NCaps, Red: Fu-FITC@FO-NCaps (B) The corrected total cell fluorescence of the cells after endocytosis of the dye FITC.  147

Figure 6-8: The FACS histogram obtained after incubating FITC loaded naocapsules with fucose saturated and non saturated cells ( Black: FITC@FO-NCaps; Red: Fu-FITC@FO-NCaps with fucose pretreated cells; Green: Fu-FITC@FO-NCaps without fucose pretreatment).  148

Figure 6-9: The cell viability after treating MDA-MB-231 cells with different formulation as a function of concentration and time.  149

Figure 6-10: The microscopic photographs showing the morphology of the cells after various treatments.  150

Figure 6-11: A) The dot plot representing the degree of early apoptosis, late apoptosis and necrosis after treating the cells with different formulations for the time duration of 48h B) The percent apoptotic cells after treatment with different formulations for 48 hours at the dose of 0.5μM equivalent to DTX.  151

Figure 6-12: A) and B) Dot plot and bar graph representing the change in mitochondrial membrane potential after 48h incubation with different DTX containing formulations at the dose of 0.5μM equivalent to DTX.  152

Figure 6-13: The confocal microscopic images of MDA-MB-231 cells treated with different formulation and stained with dye acridine orange to assess the integrity of lysosomal membrane at the dose of 0.5μM equivalent to DTX.  153

Figure 6-14: The formation of tubes with HUVEC cells treated with different formulations. Here, the positive control group and Taxotere treated group showed conspicuous tubular network. The groups treated with formulations containing FO significantly inhibited the tube formation.  154

Figure 6-15: The recovered matrigel plug from mice after six days of subcutaneous implantation. New blood vessels are present in positive control and Taxotere treated group while absent in nanocapsules treated group.  155

Figure 6-16: The cell migration inhibition in the FO containing nanocapsules at the