A Micro-Nano Particle System for Sustained Drug Release in

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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

ii

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

iv

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

2

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

3

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

4

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.

5

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

9

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.

17

Sample Chitosan conc.

TPP conc. Telmisartan conc.

1 2 mg/mL 0.2 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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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

Documents

A Micro-Nano Particle System for Sustained Drug Release in