a micro-nano particle system for sustained drug release in
TRANSCRIPT
University of South Florida University of South Florida
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Graduate Theses and Dissertations Graduate School
March 2020
A Micro-Nano Particle System for Sustained Drug Release in Lung A Micro-Nano Particle System for Sustained Drug Release in Lung
Cancer Therapy Cancer Therapy
Heta N. Jadhav University of South Florida
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A Micro-Nano Particle System for Sustained Drug
Release in Lung Cancer Therapy
by
Heta N. Jadhav
A thesis submitted in partial fulfillment
of the requirements for the degree of Master of Science in Pharmaceutical Nanotechnology
Department of Pharmacy Taneja College of Pharmacy University of South Florida
Major Professor: Shyam Mohapatra, Ph.D. Subhra Mohapatra, Ph.D. Eleni Markoutsa, Ph.D.
Date of Approval: March 19, 2020
Keywords: Microparticle, Nanoparticle, Lung Cancer, Telmisartan, Sustained release
Copyright © 2020, Heta N. Jadhav
ACKNOWLEDGMENT
I would like to thank my mentors and Committee members: Eleni Markoutsa, Ph.D.
Subhra Mohapatra, Ph.D. and Shyam Mohapatra, Ph.D., my (major professor), for
guiding me through the completion of my thesis project and facilitating an excellent
environment for success. I would also like to thank all the help I received from the
members of the Mohapatra laboratories at the University of South Florida Morsani College
of Medicine and Taneja College of Pharmacy.
i
TABLE OF CONTENTS
LIST OF FIGURES ........................................................................................................ iii
LIST OF TABLES ........................................................................................................... v
ABSTRACT ....................................................................................................................vi
CHAPTER 1: INTRODUCTION ..................................................................................... 1
Lung Cancer ......................................................................................................... 1
Risk Factors for Lung Cancer ............................................................................... 1
Lung Cancer : Molecular Biology .......................................................................... 2
Barriers for Drug Targeting ................................................................................... 3
Design of the Nano-in-Micro Particles .................................................................. 6
CHAPTER 2: MATERIAL AND METHOD ...................................................................... 7
Preparation Micro/ Nano Particles ........................................................................ 7
Synthesis of Telmisartan-Chitosan- TPP Loaded NP ................................ 8
Preparation FITC-Chitosan (TPP) NP. ....................................................... 8
Preparation of liposome. ............................................................................ 8
Encapsulation of NP into microparticle ...................................................... 8
Characterization of Telmisartan-Chitosan NPs ..................................................... 9
Size and Zeta Potential Measurement. ...................................................... 9
Morphology of NPs: ................................................................................... 9
Evaluation of telmisartan-chitosan NPs .............................................................. 10
Cell uptake of Chitosan-TPP NPs in monolayer cultures ................................... 11
Characterization of Micro-Nano Particle ............................................................. 12
Size and Morphology. .............................................................................. 12
In- vivo Biodistribution of Micro-Nano Particle ......................................... 12
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CHAPTER 3: RESULTS .............................................................................................. 14
Characterization of Telmisartan-Chitosan NPs ................................................... 14
Size distribution and zeta potential of chitosan- TPP NP ......................... 14
Size distribution and zeta potential of TEL. CS-TPP NP: ......................... 15
Morphology of TEL. CS-TPP NP ............................................................. 16
Drug Encapsulation Efficiency of TEL. CS-TPP NP. ................................ 16
Drug release from TEL. CS-TPP NP: ...................................................... 18
Cell Uptake Assay of cy3 conjugated CS-TPP NP ............................................. 19
Encapsulation of FITC CS-TPP NP into Liposome (micro-nano particle) ........... 20
Characterization of Micro-Nano Particle ............................................................. 22
In- vivo Biodistribution of Micro-Nano Particle. ........................................ 22
CHAPTER 4: CONCLUSIONS ...................................................................................... 25
REFERENCES .............................................................................................................. 26
APPENDIX A: ................................................................................................................ 29
IACUC Approval ................................................................................................. 29
iii
LIST OF FIGURES
FIGURE 1 DESIGN AND COMPONENT OF THE MICRO/ NANO PARTICLE. ............ 6
FIGURE 2 SIZE DISTRIBUTION AND ZETA POTENTIAL OF TPP-CHITOSAN NANOPARTICLE. ....................................................................................... 14
FIGURE 3 SIZE DISTRIBUTION AND ZETA POTENTIAL OF TELMISARTAN CHITOSAN-TPP NANOPARTICLE ............................................................ 15
FIGURE 4 MORPHOLOGY OF TELMISARTAN CHITOSAN-TPP NANOPARTICLE BY TEM. ......................................................................... 16
FIGURE 5 DRUG ENCAPSULATION EFFICIENCY OF NANOPARTICLE. ................ 17
FIGURE 6 THE IN-VITRO DRUG RELEASE FROM CHITOSAN NANOPARTICLE. ....................................................................................... 18
FIGURE 7 THE CELL UPTAKE ASSAY OF CHITOSAN NANOPARTICLE. ............... 19
FIGURE 8 ENCAPSULATION OF FITC CS-TPP NP INTO LIPOSOME AND FORMATION OF MICRO-NANO PARTICLE ............................................. 20
FIGURE 9 SEPARATION OF MICROPARTICLE FROM SMALLER PARTICLE. ........ 21
FIGURE 10 TEM AND KEYENCE IMAGE OF MICRO/NANO PARTICLE SHOWING SIZE AND MORPHOLOGY OF PARTICLE. ............................ 22
FIGURE 11 STANDARD CURVE FOR LIPID LIPOSOME AND FITC CS. .................. 23
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FIGURE 12 MICRO-NANO-PARTICLE IN-VIVO BIODISTRIBUTION AND RELEASE USING IVIS MICROSCOPE. ................................................... 23
FIGURE 13 THE CONFOCAL IMAGE SHOWING LUNG TISSUE AND ACCUMULATION OF MICRO/NANO PARTICLE. ....................................... 23
FIGURE 14 LUNG TISSUE IMAGES AT DIFFERENT MAGNIFICATION. .................. 24
v
LIST OF TABLES
TABLE 1: TABLE (A) RATIO CONCENTRATION OF CHITOSAN, TPP AND
TELMISARTAN TO PREPARE NP (B)SIZE DISTRIBUTION, PDI AND
ZETA POTENTIAL. ...................................................................................... 17
vi
ABSTRACT
Lung cancer remains the leading cause of cancer-related mortality in men and
women worldwide (National Comprehensive Cancer Network). Hence, developing an
effective new therapy to treat lung cancer is under intense investigation. Specifically, the
sustained release of a drug in lung tumors is critically important. Previous studies at the
USF have shown that telmisartan exhibits synergistic properties when combined with
Actinomycin-D for lung cancer treatment. The objective of this study is to develop a novel
micro/nano system consisting of lipids and chitosan polymers that would be able to deliver
Tel directly to the lungs and release its payloads in a targeted and controlled way. To
achieve this, in this study we report the synthesis and in vitro characterization of a novel
nano-in-micro platform. First, telmisartan is entrapped in chitosan for sustained drug
release. For this purpose, Telmisartan-loaded tri-poly phosphate (TPP)- crosslinked
chitosan NPs are loaded onto liposomal microparticles for effective lung accumulation.
The size and morphology of this micro/nano-system were characterized via DLS and
TEM. The size distribution and zeta potential of telmisartan loaded chitosan-TPP
nanoparticle was found to be 160 ± 5 nm with PDI 0.1 ± 0.05 and 20mV, respectively.
The encapsulation of telmisartan into chitosan-TPP nanoparticle was 100 %. The
chitosan-TPP nanoparticle showed sustained release of telmisartan. In-vivo studies
demonstrated a high accumulation of this micro/nano-system in the lungs after
intravenous administration. Taken together, these results indicate that micro-in-
nanoparticles permit sustained release of telmisartan.
1
CHAPTER 1: INTRODUCTION
Lung Cancer
Lung cancer has become the second leading cause of mortality in humans. Every
day around 442 people die in America because of lung cancer (American Cancer Society,
2019). Some facts and figures state that the mortality rates in the poorest countries was
40% in 2012-2016 and have increased more responsible for the most cancer deaths at
around 1.76 million. It was seen that women but not men in industrialized countries have
more death rates compared to the developing countries (Siegel, Miller, & Jemal, 2019;
Torre et al., 2015). Also, the death rate due to lung cancer in men is declined by 48%
whereas in women it declined by just 23% (Meza, Meernik, Jeon, & Cote, 2015). Not only
America but also countries like Italy, the UK, Ireland, China, and Japan have a high
mortality rate (Siegel et al., 2019). The developing countries like India or Brazil, Russia,
and South Africa have lower mortality rates for lung cancer.
Risk Factors for Lung Cancer
Lifestyle, occupation, and the environment are the main causes of lung cancer. In
day-to-day life, humans are exposed to many pollutants coming from industries or
vehicles, radiation, micro-organism, tobacco smoke, noble gases, present in the
environment. These factors directly or indirectly affect human health. It was thought that
smoking is the main reason for lung cancer risk, but the trend has changed. While
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smokers and second-hand smokers are at high risk of getting lung cancer, even healthy
people can show the symptom of lung cancer. Early diagnosis is difficult and usually lung
tumors metastasize to other areas of the body before a doctor detects them in the lungs.
Cigarette smoking is the most critical hazard factor in lung cancer. Tobacco smoking,
specifically cigarettes, represents roughly 75%-90% of the lung cancer mortality.
Carcinogen present in tobacco such as polycyclic aromatic hydrocarbon incite mutation
in the p53 gene, which is essential for cell-cycle dysregulation and carcinogenesis. Noble
gas like radon which naturally present underground or dirt underneath structures can
come through the ground into a house causing lung cancer to people exposed to it. Other
environmental risk factors are exposure to asbestos, chromium, radon, cadmium, silica,
arsenic, nickel and beryllium. Biological factors can also result in developing lung cancer.
Aging, genetic history, disease condition can lead to lung cancer development. Disease
conditions like HIV, COPD can cause lung cancer. Also, constant exposure to radiation
while working or during any radiation treatment for other diseases or cancer treatment
leads to lung cancer (McErlean & Ginsberg, 2011; Meza et al., 2015; Siegel et al., 2019).
Lung Cancer : Molecular Biology
Lung cancer is an uncontrolled growth of abnormal lung cells. They can be
epithelial cells or squamous cells. DNA regulates the cell growth and cell regulation but if
DNA damaged or mutated because of the factor discussed previously then healthy cells
turned into cancerous cells. The lungs are vital to normal body function and thus the
abrogation of their normal function is particularly detrimental to health. The lungs are
hypervascular and possess a very rich oxygenated blood supply, increasing the likelihood
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of the cancer spreading (metastasis) to other body regions. There are two types of lung
cancer, small cell lung cancer also called oat cell lung cancer and non-small cell lung
cancer. Small cell lung cancer occurs almost exclusively in heavy smokers and is less
common than non-small cell lung cancer. Non-small cell lung cancer is an umbrella term
for several types of lung cancers that behave similarly. Non-small cell lung cancers
include squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. Currently,
adenocarcinoma is the most common lung cancer in humans (Lortet-Tieulent et al., 2014;
Siegel et al., 2019; Travis, 2011; Travis et al., 2011). Biological markers are biological
components released by the cancer cells. Most of the ongoing studies are concentrating
on molecular-based therapies for non–small cell lung malignant growth. The KRAS
mutation and epidermal growth factor receptor (EGFR) are the two somatic mutations
demonstrated from the profiling of tumor DNA mutations that can be act as biomarkers
against anticancer drugs (McErlean & Ginsberg, 2011).
Barriers for Drug Targeting
During respiration, pathogens and airborne particles are drawn inside the lungs
(Patil & Deshpande, 2018)(Iyer et al., 2015). The defense mechanism or/and clearance
mechanism are the huge obstacles in the treatment of lung-related diseases or disorders.
First, the walls of the trachea are lined with the thick ciliated mucous, this lining continues
till the tertiary bronchi. These mucosal layers act as a defensive barrier and obstruct the
entry of foreign substances. This is known as mucociliary clearance. Furthermore,
alveolar linings are made up of proteins and lipids. The tight junction between epithelial
cells also block the entrance of other pathogenic agents. The particles that can conquer
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these obstructions are either destroyed by the cells or phagocytosed by the alveolar
macrophages (Paranjpe and Muller-Goymann, 2014). Sometimes, drug particles are also
expelled out from the lung if given by the nasal route therefore achieving sustained
release and deep lung penetration of drugs is difficult (Kumar et al., 2011). Hence drug
delivery by the nasal route is problematic.
The premise of the present Research
Previously, (Das et al., 2017) and (Kumar et al., 2011) showed that Sertoli cells
can effectively accumulate to the lungs and deliver drugs to the deep lung. This cell-
mediated drug delivery approach, which is based on the use of live Sertoli cells has some
disadvantages. Live cells are able to degrade nanoparticles (NPs) that have been taken
up and, in that case, the drug will be released inside the lysosomes. FDA clearance is
also difficult because cells are derived from animals. There will be batch to batch variation
in preparation of sertoli cells and it is very inconvenient and time consuming. To overcome
the drawbacks of Sertoli cells, we reasoned that a synthetic microparticle with a size of
Sertoli cell could be developed to deliver NPs to the deep lung.
Further, using a nanofiber scaffold that amplifies cancer system cells, it was
previously discovered that a combination of actinomycin–D and telmisartan effectively
treat total lung cancer, i.e together they eliminate cancer cells and cancer stem cells
(Green et al., 2019). But there is a need to develop a targeted drug delivery system that
will carry these drugs specifically to the lung and release the payload in a controlled way.
This study mainly focused on the preparation of microparticles loaded with Telmisartan.
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Hypothesis & Specific Aims
We hypothesized that a micro/nano-particle drug delivery system can be
synthesized comprising chitosan NPs in a lipid microparticle (Figure 1) that will allow
sustained release of drugs. We plan to test this using telmisartan, a drug that is used in
combination with actinomycin-D, which synergistically effect on reducing lung cancer
tumor volume significantly ( Ryan et al, 2019).
There are three aims to test this hypothesis. The first aim of this study is to
synthesize and characterize Telmisartan loaded NP. The second aim involves loading
of telmisartan NP into microparticle. The third aim involves in vivo evaluation of micro-
nano particle.
Design of the Nano-in-Micro Particles
The design represents the microparticle encapsulating NPs in it. The microparticle
is made up of lipid bilayer and carries the telmisartan loaded NP to deliver at the lung.
The size of the microparticle will be 30 µm which will lead this particle to reach and get
accumulate the lung when administered via IV. The NP is made of Low Molecular Weight
Chitosan (LMW CS) cross-linked with tri-poly phosphate (TPP). Chitosan is
biocompatible, biodegradable, water-soluble, positively charged, easy to prepare NPs,
low cost. TPP is an anionic cross-linker. Hence when TPP reacts with chitosan, the
anionic group of TPP binds with amines groups present on chitosan. And since CS binds
ionically, it’s a very strong bond that will result in a stable NP with sustained release of
drugs.
6
When TPP cross-links with chitosan it creates a small gap where telmisartan will get
entrapped. The microparticle is made up of DPPC.
Figure 1 Design and Component of the Micro/ nano Particle. Telmisartan loaded ionically crossed -linked chitosan-Tri-Poly Phosphate nanoparticles encapsulated onto liposomal
microparticle.
7
CHAPTER 2: MATERIAL AND METHOD
Preparation Micro/ Nano Particles
Synthesis of Chitosan- TPP NPs (CS-TPP NP): A solution of 2mg/mL water-
soluble LMW chitosan (CS) (10kD) was dispersed in sterile H2O. A stock of the
Tripolyphosphate Penta-sodium (TPP) (Sigma–Aldrich Co., 238503-25G) solution was
dissolved in sterile H2O at a concentration of 10mg/mL. 1mL of chitosan solution was
taken in the V-bottom vial and 20µL of TPP from stock was added to the CS solution, the
solution was ultrasonic for 15minutes at 6-7 speed. The NP solution was then centrifuged
using Thermo scientific legend X1R centrifuge at 5000 RPM for 5minutes. The loaded
NPs were stored at 4°C until further use.
Synthesis of Telmisartan-Chitosan- TPP Loaded NP (TEL. CS-TPP NP): A
solution of 2mg/mL water-soluble LMW chitosan (CS) (10kD) was dispersed in sterile
H2O. A stock of the Tripolyphosphate Penta-sodium (TPP) (Sigma–Aldrich Co., 238503-
25G) solution was dissolved in sterile H2O at a concentration of 10mg/mL. Additionally,
Telmisartan (Selleckchem.co, Cat. No. S1738) was prepared at a concentration of
5mg/mL in DMSO. 1mL of chitosan solution was taken in the V-bottom vial and 20µL of
TPP from stock was added to the CS solution along with 40µl of Telmisartan, the solution
was ultrasonic for 15minutes at 6-7 speed. The NP solution was then centrifuged using
8
Thermo scientific legend X1R centrifuge at 5000 RPM for 5minutes. The loaded NPs were
stored at 4°C until further use.
Preparation FITC-Chitosan (TPP) NP (FITC CS-TPP NP): FITC-labeled NPs
were prepared by using NHS-ester hydrolysis conjugation reaction of FITC to chitosan. A
basic solution of chitosan (1mg/mL) pH 7.5 and FITC was kept under stirring conditions
overnight at 4°C. Nonconjugated FITC was removed by centrifugation at 12000 RPM for
15mins. Following the procedure described above, FITC conjugated chitosan-TPP NPs
were prepared. The NPs were characterized for size and zeta.
Preparation of liposome: The liposomes were prepared using thin film hydrating
method (Varona et al., 2011). Here film was hydrated with the NP solution. A solution of
1mL of cholesterol (10mg/mL) and 251µL of DPPC (10m/mL) was prepared in
chloroform:methanol (2:1). All lipids are added in a round-bottom flask, then 25µL of
rhodamine (1mg/mL) was added to it. The flask was connected to the rotary evaporator
initially heated at 50°C. The flask was inclined in such a way that the solution of the lipid
cover most of the surface of flask and form the uniform film. The flask was rotated at 100
RPM until the solution forms a uniform film in the flask.
Encapsulation of NP into microparticle: The lipid film was hydrated with 2mL solution
of FITC CS-TPP NP (10mg/mL) using a sonication bath and vortex. It is continued
until the pink lipid film gets dispersed in the FITC CS-TPP NP solution and gives the
orangish color solution. The solution was vortex for extra 2mins. The solution was taken
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in Eppendorf tubes and using liquid nitrogen solution was freeze. Using
lyophilizer, the product was lyophilized and stored at -80°C. Some quantity was taken
from the lyophilized product and dissolved in the sterile water. Using the Keyence
microscope and TEM, the size and morphology of the micro-nano particle were checked.
The dispersion of the micro-nano particle was heterogeneous. To obtain a
monodispersed sample the particle dispersion was centrifuged at different speed for
15mins. The solution was centrifuged at 800, 1000 and 1400 RPM. The pellet and
supernatant were separated and observed under the microscope to ensure the separation
of large particles from smaller particles.
Characterization of Telmisartan-Chitosan NPs
Size and Zeta Potential Measurement: The particle size distribution of
telmisartan-chitosan NPs was determined using dynamic light scattering (DLS)
technique. The particle size distribution and Zeta potential were measured using a
Malvern Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). The telmisartan-
chitosan NP solution was diluted to 1:10 with deionized water and placed in disposable
folded capillary cell cuvette and the scattering intensity was measured in triplicate for each
sample at 25◦C (Upasana, Angshuman Ray, Sumanta Kumar, Nuzhat, & Qamar, 2017).
Morphology of NPs: Telmisartan chitosan NPs were observed under a
transmission electron microscope (TEM). All samples for TEM were prepared in 1:30
dilution and a single drop of NP suspension is placed on the Formvar® coated 150
10
Cat.No. 215-412-8400) to dry overnight at room temperature and analyzed the next day.
(Antonio Rampino, 2013).
Evaluation of telmisartan-chitosan NPs
Determination of % drug encapsulation: The drug encapsulation was obtained
by determining the concentration of the free drug in the supernatant. Loaded and
unloaded NPs prepared as explained before and then ultra-centrifuged at 120000 RPM
using Beckman optima max tl tabletop ultracentrifuge for 30bminutes. The supernatant
was separated from the pellet and the absorbance was estimated using a Synergy H4
Plate-reader. The amount of drug-loaded in the NPs was calculated as the difference
between the initial concentration of drug and the concentration of drug in the supernatant.
The supernatant of unloaded NPs was used as a blank. % drug encapsulation was
calculated using the following equation.
% 𝑑𝑟𝑢𝑔 𝑒𝑛𝑐𝑎𝑝𝑠𝑢𝑙𝑎𝑡𝑖𝑜𝑛 = 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑐𝑜𝑛𝑐. − 𝐹𝑖𝑛𝑎𝑙 𝑐𝑜𝑛𝑐.
𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑐𝑜𝑛𝑐. 𝑋100
Drug Release Assay: In-vitro drug release was studied by equilibrium dialysis
membrane method in triplicate. Telmisartan chitosan NPs were prepared and 5ml of this
solution was taken in the seal dialysis membrane. The bag was then suspended in the
1liter of deionized sterile water at 37°C being stirred at 180 RPM (Upasana et al., 2017).
The non encapsulated or drug released from the NP will be removed by the dialysis. At
predetermined time points, 0.5ml is taken out from the dialysis bag and kept for freeze
11
drying in a preweighed glass vial. The lyophilized product is dissolved in the 100µL
NaOAc-AcOH buffer, 80µL chitosanase, and 20µL lysozyme to find the amount of drug
left to release from NP. The vial kept in 37°C being stirred at 200 rpm for 4hours (Mao et
al., 2001). After 4hrs, 800µL DMSO is added and the solution is centrifuged at maximum
speed for 30mins. Absorbance is recorded using Synergy H4 Plate-reader. The % drug
release is calculated using the following equation.
% 𝐷𝑟𝑢𝑔 𝑟𝑒𝑙𝑒𝑎𝑠𝑒 = (𝐹𝑖𝑛𝑎𝑙 𝑐𝑜𝑛𝑐. − 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑐𝑜𝑛𝑐. ) 𝑋100 /100
Cell uptake of Chitosan-TPP NPs in monolayer cultures
Cy-3 Chitosan-TPP NP Preparation: First, the NPs were prepared using the cy3 sulfo-
NHS chitosan. The solution of chitosan and cy-3 was kept under stirring for overnight at
4°C. The next day, excess cy-3 was removed by first centrifuging the solution at the
highest speed for 15mins and then washing the pellet until colorless supernatant is not
obtained. Following the procedure described above, cy-3 conjugated chitosan-TPP NPs
were prepared. The NPs were characterized for size and zeta before dosing into the cell.
Lewis lung carcinoma (LLC) cell line was used for the cell uptake assay. The LLC
cells were cultured in T75 Filtered Flasks using Dulbecco’s modified Eagle’s medium
(DMEM) growth media with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin
(P/S). The flasks were incubated at 37⁰C in a humidified incubator with 5% CO2/95%. The
cells were passaged after the 80- 100% confluency achieved. The cells were washed with
sterile PBS and then detached from the flask by treating with 3mL of trypsin. The cells
were centrifuged at 1,250 RPM for 3 mins. The supernatant was discarded, and the cell
12
pellet was resuspended in 10mL of fresh growth media. 10μl of the re-suspended cell
suspension was added to the Newbauer chamber to find the count of the cells per mL. In
96 well plate, 5000 cells per well were seeded to get initial 50- 60% confluency for the
experiment on the following day. Five such plates were prepared for each time point.
To determine cellular uptake, the cell plate was observed under the microscope
for cell morphology and confluency. The cells were washed with the PBS and fresh PBS
was added in each well. The cells were dose with different concentration chitosan and
incubated at 37⁰C in a humidified incubator with 5% CO2/95%. After each time point, the
cells were washed trice to remove excess NP and stop the uptake action. The 100μl PBS
with DAPI was added to each well and incubated for more than 10 mins. Cells were
washed with PBS and observed using the Keyence microscope.
Characterization of Micro-Nano Particle
Size and Morphology: The microparticles were observed by the Keyence
microscope and transmission electron microscope (TEM) for size and morphology. All
samples were prepared in 1:30 dilution and checked under the Keyence microscope for
size and encapsulation. For TEM - a single drop of NP suspension is placed on the
Formvar® coated 150-mesh copper grid to dry overnight at room temperature and
analyzed the next day.
In- vivo Biodistribution of Micro-Nano Particle: The solution of the Micro-nano-
particle was prepared and characterized using the Keyence microscope to ensure size
and encapsulation. C57/B6 mice were ordered and 4 mice in each cage were placed.
13
The 3 mice were used for sample and 1 was control. The 100 µL sample was injected
into sample mice. After 23 hours, mice were sacrificed, and organs were collected. Using
IVIS microscope, images of the collected organs were taken.
Immunohistochemistry: The organs collected from mice were placed in the 10%
sucrose solution for 24 hours. The lung tissues were taken out from sucrose solution and
place in the mold containing the OCT embedding medium. The sections were taken on
poly-lysin coated slides using the cryostat. The slides were air-dried for more than 15-20
mins and then washed with the 1x PBS thrice. Few drops of hard-set mounting media
containing DAPI were added on the sections and coverslip was placed on it. The slide
was allowed to dry overnight. The images of lung sections were taken using the Keyence
and confocal microscope.
Statistical Analysis: All the data were analyzed using standard statistical
methods through mean ±.S.E.M. A p value < 0.05 was considered statistically significant
(Green et al., 2019; Quarni et al., 2019).
14
CHAPTER 3: RESULTS
Characterization of Telmisartan-Chitosan NPs
Size distribution and zeta potential of chitosan- TPP NP (CS-TPP NP): TPP
crosslinked chitosan NPs were prepared using the ultrasonication method. The NP
solution was then centrifuged, and the supernatant is checked for size distribution and
zeta potential of telmisartan loaded chitosan NP. The size distribution was found to be
200.10 ± 5 nm with PDI 0.1 (FIG. 1A). And zeta potential was found to be +38 ±8 (FIG.
1B) which confirms that when chitosan and TPP were mixed firms a positively charged
particles.
Figure 2 (A) Size distribution of TPP-chitosan nanoparticle was found to be 200nm±5, PDI was 0.1±0.05; (B) Zeta potential of TPP-chitosan nanoparticle was found to be +38.8mV.
15
Size distribution and zeta potential of telmisartan loaded chitosan NP (TEL. CS-
TPP NP):
Telmisartan loaded chitosan NPs were prepared by crosslinking chitosan with TPP
crosslinker using the ultrasonication method. The NP dispersion was then centrifuged to
remove big particles. The size distribution was found to be 160 ± 5 nm with PDI 0.1 (FIG.
2A). And zeta potential was found to be 25 ±1 (FIG. 2B) which confirms that when
chitosan and TPP were mixed, it forms a positively charged particles.
When CS- TPP NPs were loaded with Telmisartan, the NP size was smaller compared to
empty NP. Also, the zeta potential was decreased indicating that more amines were
occupied by the TPP forming more compact NPs.
160 nm + 20 mV
Figure 3 (A) Size distribution of telmisartan chitosan-TPP nanoparticle was found to be 160nm±5 and PDI was 0.1± 0.05 and (B) Zeta potential of telmisartan chitosan-TPP
nanoparticle was +20mV
16
Morphology of TEL. CS-TPP NP: TEM images confirmed the size and
morphology of the NP. The surface of the NP looks smooth without any aggregation.
Drug Encapsulation Efficiency of TEL. CS-TPP NP: The Drug Encapsulation
Efficiency of TEL. CS-TPP NP was estimated by determining the concentration of the free
drug in the supernatant. TEL. CS-TPP NPs were prepared using different concentrations
of TPP solution keeping the chitosan concentration constant. dispersions were Ultra-
centrifuged at 120000 RPM using Beckman optima max tl tabletop ultracentrifuge for
30minutes. The amount of telmisartan-loaded in NPs and the encapsulation efficiency
was calculated using a standard curve of telmisartan. Different TPP/chitosan ratios were
tested, and the size and zeta potential were estimated.
Figure 4 Morphology of Telmisartan chitosan-TPP nanoparticle by TEM.
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Sample Chitosan conc.
TPP conc. Telmisartan conc.
1 2 mg/mL 0.2 mg/mL 0.2 mg/mL
2 2 mg/mL 0.4 mg/mL 0.2 mg/mL
3 2 mg/mL 0.8 mg/mL 0.2 mg/mL
4 2 mg/mL 1 mg/mL 0.2 mg/mL
Table 1: Table (A) Representation ratio concentration chitosan, TPP and Telmisartan used to prepare nanoparticles. (B)Size distribution, PDI and Zeta Potential of nanoparticle prepared using different
concentration of TPP Crosslinker.
Figure 5 (A) Drug Encapsulation Efficiency of nanoparticle prepared using different concentration of TPP Crosslinker, (B) Drug Encapsulation Efficiency of nanoparticle prepared using different concentration of
TPP Crosslinker, (C) Standard curve for Telmisartan.
A
V
B
18
Sample 1 and 2 showed 243.4 size distribution and 0.1 PDI. Also, the solution of
NP was clear when compared to another sample. Sample two showed the highest
encapsulation. When the experiment was repeated with the same concentration
encapsulation efficiency was found to be 100%.
Drug release from TEL. CS-TPP NP: The concentration of free drug in the
supernatant was obtained using the standard curve of the drug Telmisartan. It was
expected that TEL. CS-TPP NP show sustained release of the Telmisartan. Figure 6
shows the percentage of drug releases at different time points. These chitosan derived
NPs show sustained release of the drug. In the first 4 hours, 18 percent of the drug is
released from the NP. After 24 hours, approximately 45 percent of the drug was released.
On 5th day i.e after 96 hours, 100 percent drug was released, proving the sustained
release of Telmisartan from CS- TPP NP.
Figure 6 The in-vitro drug release from chitosan nanoparticle. In distilled water (37°C) Telmisartan release from the chitosan-TPP nanoparticles.
19
Cell Uptake Assay of cy3 conjugated CS-TPP NP
The CS-TPP NP was prepared using the cy-3 labeled chitosan. A low
concentration (50mg/mL chitosan) and a high concentration (100 mg/mL chitosan) of
labeled NPs were added to the cells for different time points. The cell uptake was
analyzed using the Keyence microscope. The NP uptake was analyzed using
fluorescence emitted from cy-3 chitosan NP. At 30 min time point, there was no cell
uptake seen in the low dose group whereas few particles were taken by the cells in the
high dose group. After 1-hour of incubation, the low dose group started showing some
NP uptake whereas a high dose showed an increase in the NP uptake. After 4-hour, cells
showed significantly increased cell uptake compared to earlier time points.
.5 1 2 4
0.0
0.5
1.0
1.5
Cell Uptake
Hours
Inte
nsit
y
Low Dose
High Dose
****
Figure 7 The Cell uptake of Chitosan-TPP NPs in LLC monolayer cultures. The significant cell uptake is seen after 4hours (D)
20
Encapsulation of FITC CS-TPP NP into Liposome (micro-nano particle)
The lipid film was hydrated with the solution of FITC CS-TPP NP using a sonication
bath and vortex which results in the encapsulation of NP into the lipid microparticle. The
microparticle size and the NP encapsulation to microparticles were confirmed using
fluorescence microscopy. The dispersion of the micro-nano particle was also observed
under a microscope before and after lyophilization. Both samples were polydispersed. It
was observed that Lyophilization not only increases the encapsulation of NP but also
results in a bigger and more spherical microparticle. When the size was measured, most
of the particles were in the range of 30-50 µm. The separation larger particles from
smaller was necessary and hence a separation step was performed.
Separation step: To obtain a more homogeneous dispersion of microparticles, the
microparticle dispersion was centrifuged for 15mins at different speeds. The supernatant
was collected, and the pellet was redispersed in 1 mL of distilled water. The first sample
was centrifuged at 1400 RPM and supernatant of the sample was observed under a
Figure 8 Encapsulation of FITC CS-TPP NP into Liposome and formation of micro-nano particle.
21
microscope. The solution was a mixture of smaller particles. The particles were smaller
in the expected size range. The second sample was centrifuged. The solution was a
mixture of larger particles and few smaller particles. Very small particles were separated.
Then it was observed that self-precipitated particles were in the expected size range. The
size of the microparticle was found to be 30 µm. The microparticles appeared individual
and spherical in shape.
Figure 9 Separation of Microparticle from smaller particle by centrifugation.
22
Characterization of Micro-Nano Particle
Size and Morphology of Micro-Nano Particle: The microparticles were
characterized using the Keyence microscope and transmission electron microscope
(TEM) for size and morphology. The TEM images confirmed the size and encapsulation
NP into microparticle. The size of the micro- nano particle was found to be 30 µm and the
TEM image clearly showed individual NP distributed in the microparticle.
In- vivo Biodistribution of Micro-Nano Particle: The sample of the micro-nano
particle was evaluated before injecting it into the mice. The size and morphology were
confirmed using a Keyence microscope. Using the Stewart assay and FITC standard
curve, the concentration of lipid and chitosan was calculated. The lipid concentration
was 70M in 100 µg/mL and FITC CS-TPP NP was 5 µg/mL. The mice look healthy after
post-injection and there were no unusual signs were seen. The IVIS images showed the
accumulation of microparticle in the lung tissue.
Figure 10 TEM and fluorescence image of micro/nano particle showing size and morphology of particle.
23
Figure 11 (A)Standard curve for lipid liposome using stewart assay, (B) Standard curve for FITC CS.
Figure 12 Micro-nano-particle in-vivo biodistribution and Release using IVIS microscope.
Figure 13 The confocal image showing lung tissue and accumulation of micro/nano particle.
24
Figure 13 shows the release of NP from microparticle and accumulation in the lung.
The red signal represent rhodamine from microparticle whereas green represents FITC
NP. The confocal images (figure 14) at different magnification showed the morphology of
the lung and accumulation of the micro/nano particle in the lung tissue after 24 hours.
Figure 14 Lung Tissue Images at Different Magnification.
25
CHAPTER 4: CONCLUSIONS
The obtained results supported the hypothesis and satisfied the three aims of this
study. According to Aim 1 and 2, a nano-in-micro particle platform was successfully
developed utilizing chitosan-derived NP encapsulated in lipid-based microparticles. The
core of the microparticle carried Chitosan-TPP NPs for sustained release of the Tel. The
TEM images confirm the morphology of NP and micro/nano particles as well as
encapsulation of NP into microparticle. The drug encapsulation efficiency of CS-TPP NPs
was found to be 100%. In vitro studies proved that Chitosan-TPP NPs show a controlled
release of a Tel drug. Chitosan-TPP NP labeled with cy3 shows cell uptake within 4 hours.
Regarding Aim 3, in-vivo studies demonstrated that the micro/nano particle does not show
any toxicity in mice even after 24 hours. The lipid microparticles do accumulate and
release the NP within the lung for increased targeting effect. Based on obtained results,
in the future, the development of this novel formulation may provide an excellent approach
to combine another drug NPs to a synergistic effect. The final goal will be to achieve a
pH-induced drug release of a second drug, which may be useful for Act-D and Tel
combination therapy.
26
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25. Lortet-Tieulent, J., Soerjomataram, I., Ferlay, J., Rutherford, M., Weiderpass, E., & Bray, F. (2014). International trends in lung cancer incidence by histological
28
subtype: adenocarcinoma stabilizing in men but still increasing in women. Lung Cancer, 84(1), 13-22. doi:10.1016/j.lungcan.2014.01.009
26. McErlean, A., & Ginsberg, M. S. (2011). Epidemiology of lung cancer. Semin Roentgenol, 46(3), 173-177. doi:10.1053/j.ro.2011.02.002
27. Meza, R., Meernik, C., Jeon, J., & Cote, M. L. (2015). Lung cancer incidence trends by gender, race and histology in the United States, 1973-2010. PLoS One, 10(3), e0121323. doi:10.1371/journal.pone.0121323
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31. Torre, L. A., Bray, F., Siegel, R. L., Ferlay, J., Lortet-Tieulent, J., & Jemal, A. (2015). Global cancer statistics, 2012. CA Cancer J Clin, 65(2), 87-108. doi:10.3322/caac.21262
32. Travis, W. D. (2011). Pathology of lung cancer. Clin Chest Med, 32(4), 669-692. doi:10.1016/j.ccm.2011.08.005
33. Travis, W. D., Brambilla, E., Noguchi, M., Nicholson, A. G., Geisinger, K. R., Yatabe, Y., . . . Yankelewitz, D. (2011). International association for the study of lung cancer/american thoracic society/european respiratory society international multidisciplinary classification of lung adenocarcinoma. J Thorac Oncol, 6(2), 244-285. doi:10.1097/JTO.0b013e318206a221
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FORMULATION OF NANOPARTICLES OF TELMISARTAN INCORPORATED
IN CARBOXYMETHYLCHITOSAN FOR THE BETTER DRUG DELIVERY AND
ENHANCED BIOAVAILABILITY. Asian Journal of Pharmaceutical and Clinical
Research, 10(9), 236-241. https://doi.org/10.22159/ajpcr.2017.v10i9.19162
29
APPENDIX A:
IACUC Approval