Multifunctional Nanoemulsion System for Combination Paclitaxel and Curcumin Delivery in

Human Glioblastoma Cells

Master’s Thesis

By

Ms. Sindhura Ganga

Advisor: Mansoor M. Amiji, PhD

to

The Bouve’ Graduate School of Health Sciences in Partial Fulfillment of the Requirement for the Degree of Master of Science

in Pharmaceutical Science with Specialization in Pharmaceutics and Drug Delivery

Department of Pharmaceutical Sciences Northeastern University

August, 2008

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PROJECT SUMMARY Glioblastoma multiforme (GBM) is an aggressive form of primary brain tumor that

afflicts as many as 12,000 patients per year in the United States. GBM is a very aggressive

disease and the average patient life-span is reduced to 6 months after diagnosis. Some of the

clinical challenges in GBM include difficulty in initial diagnosis, presence of tumor cell “islets”

in the critical areas of the brain, and the role of blood-brain barrier (BBB) in effective transport

and distribution of drugs in the brain. Due to the presence of tumor cell islets, complete surgical

resection is not always possible. Additionally, poor penetration of many chemotherapeutic agents

across the BBB limits availability and distribution for effective treatment of GBM. In addition to

the presence of a physical barrier in the form of tight endothelial cell junctions, BBB is also

comprised of chemical barriers in the form of expression of the efflux pump, P-glycoprotein (P-

gp), and the presence of drug metabolizing enzymes and the vascular endothelial cell surface.

GBM also has high incidence of resistance development due to the expression of multidrug

resistant protein (MRP).

In this study, our main objective was to evaluate co-administration of paclitaxel (PTX)

and curcumin (CUR), a modulator of MRP and an inhibitor of nuclear factor kappa B (NFκB)

signaling, in oil-in-water nanoemulsion formulations in brain tumor cells. CUR is known to

down-regulate both P-gp and MRP expression and also enhances apoptotic activity by inhibition

of NFκB mediated intracellular signaling. The nanoemulsion formulations in this study are

specifically composed of oils rich in polyunsaturated fatty acids (PUFA) and the surface was

modified to enhance BBB penetration.

As part of the study, PTX and CUR containing nanoemulsions have been formulated with

pine-nut oil, which is rich in linoleic and linolenic acids, an example of omega-6 and omega-3

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PUFA, respectively. The optimized formulations were characterized for oil droplet size, surface

charge, and observed with transmission electron microscopy (TEM). Human wild-type

glioblastoma cells U87 and the MRP-1 expressing resistant glioblastoma cells, T98G, were

grown in culture. Baseline expression of MRP-1 in T98G cells was confirmed by Western blot

and immunocytochemistry analysis. Intracellular delivery of fluorescently-labeled PTX and CUR

using nanoemulsion formulation was examined by fluorescence microscopy. The cell viability

upon treatment with PTX and CUR, either alone or in combination, was examined in both of

these cell lines using the MTT (conversion of tetrazolium salt to formazan) assay. MRP-1 down-

regulation and the inhibition of NFκB pathway was confirmed by Western blot analysis.

Qualitative apoptotic activity in U87 and T98G cells was evaluated using TUNEL staining.

Pine-nut oil nanoemulsions were successfully formulated with an approximate

hydrodynamic diameter of 120 nm and a -40 mV surface charge. Both PTX and CUR were

efficiently encapsulated in the oil phase of the nanoemulsion. TEM analysis showed that the oil

droplets of the nanoemulsion had a spherical shape and smooth surface morphology. Due to

rapid cellular uptake and internalization of PTX and CUR, as assessed by fluorescence

microscopy, the nanoemulsion formulations were able to significantly enhance cytotoxicity in

both cell lines. Combination of PTX and CUR were found especially to enhance cytotoxicity in

MRP-1 gene expressing T98G cells. CUR was found to down-regulate MRP-1 and was found to

inhibit NFκB pathway. TUNEL staining images confirmed the apoptosis in the treated U87 and

T98G cells. The overall results allowed us to understand the synergistic therapeutic effect upon

administration of PTX and CUR using nanoemulsions made with PUFA-rich oil.

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NORTHEASTERN UNIVERSITY

Graduate School of Bouvé College of Health Sciences

Thesis Title: Multifunctional Nanoemulsion System for Combination Paclitaxel and Curcumin Delivery in Human Glioblastoma Cells

Author: Sindhura Ganga

Department: Pharmaceutical Sciences

Approved for Thesis Requirements of the Master of Science Degree in Pharmaceutical Science

Dissertation Committee

____________________________________ __________________ Date

____________________________________ __________________

Date ____________________________________ __________________ (Chairman) Date ____________________________________ __________________

Director of Graduate School Date

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ACKNOWLEDGEMENTS

I have been waiting eagerly for this moment to express my heartfelt gratitude to those

people, who had a major role in the completion of my thesis project and to everyone who wished

good for me.

First and foremost, I would like to express my deepest gratitude, respect, and appreciation

to my advisor and my role model, Dr. Mansoor Amiji. If it weren’t for his ideas, constant

support, guidance, motivation, and patience, none of this would have been possible. Thank you

for making a difference in my career and for having faith in me that I could complete this

project.

I would like to thank my dissertation committee members, Dr. Rebecca Carrier and Dr.

Ralph Loring, for their time, support, and suggestions throughout the course of this work. I

would also like to thank Dr. Robert Campbell, Dr. Vladimir Torchillin and Bill Fowle for being

kind and for allowing me to use various analytical instruments in their laboratory.

I always felt like being with my family when I was working in the lab. I have received all

means of support, guidance, and encouragement from the lab members. I would like to first

thank Harikrishna Devalapally and Padmaja Magadala for helping me in starting with the

project. Lilian van Vlerken, Luis Brito, Lara Jabr-Milane, Mayank Bhavsar, Srinivas Ganta,

Thomas Barchet, and Sandra Chadwick for their valuable suggestions. I would like to specially

thank Sunaina Pai and Shardool Jain for their amazing company. Last, but not the least, I thank

my parents for all their love and sacrifices and for making my dreams come true. To them, I

dedicate this thesis.

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TABLE OF CONTENTS

PROJECT SUMMARY 2

ACKNOWLEDGEMENTS 5

TABLE OF CONTENTS 6

List of Tables

8

List of Figures

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1.

INTRODUCTION………………………………………………………………………

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1.1 Glioblastoma: Incidence and Mortality ……………………………………............ 11

1.2 Role of the Blood-Brain Barrier in GBM Therapy………………………………… 11

1.3 Paclitaxel and Curcumin in Glioblastoma Therapy………………………………… 13

1.4 Nanotechnology for Brain Delivery…………………………..……………………. 18

1.5 Multifunctional Nanoemulsions for PTX/CUR Combination Therapy …………… 20

2.

OBJECTIVES AND SPECIFIC AIMS…………………………………………………

23

2.1 Statement of the Problem……………………………………………………...........

23

2.2 Objectives and Hypotheses………………………………………………………….

23

2.3 Specific Aims………………………………………………………...……………..

24

3.

MATERIAL AND METHODS……………………………………………………….

26

3.1 Preparation of Oil-in-Water Nanoemulsions ………………………………………. 26

3.2 Characterization of the Nanoemulsions ………………………................................. 26

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3.3 Determination of Baseline MRP Expression in GBM Cells ……………………..

28

3.4 Fluorescence Microscopic Studies of Intracellular Delivery with Nanoemulsions... 30

3.5 Determination of MRP1 Down-Regulation by Western Blot Analysis ………..….. 31

3.6 Determination of NFκB pathway Inhibition by Western Blot Analysis ………..

31

3.7 Cytotoxicity of Single and Combination Treatments in GBM Cells………………. 33

3.8 Qualitative Apoptosis Study by TUNEL Staining ………………………………… 34

4.

RESULTS AND DISCUSSIONS……………………………………………………...

36

4.1 Preparation and Characterization of Multifunctional Nanoemulsions……………...

36

4.2 Determination of Baseline MRP-1 Expression in GBM Cells…………………….. 37

4.3 Cellular Uptake and Distribution Studies………………………………………….. 39

4.4 Down Regulation of MRP-1 and the Inhibition of NFκB Pathway with CUR……. 41

4.5 Cytotoxicity of Single and Combination Treatments in GBM Cells………………. 43

4.6 Qualitative Apoptosis Study by TUNEL Staining…………………………………. 47

5.

CONCLUSIONS………………………………………………………………………..

51

6.

REFERENCES……………………………………………………………….................

53

7

LIST OF TABLES

Table 1. Physical characteristics of blank and drug-containing nanoemulsions

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LIST OF FIGURES

Figure 1. Schematic depiction of the blood-brain barrier in brain microvasculature

Figure 2. The chemical structures of paclitaxel and curcumin

Figure 3. Proposed multimodal mechanism of MDR modulation with curcumin

Figure 4. Transmission electron micrograph of pine-nut oil nanoemulsion for the co-administration of paclitaxel and curcumin

Figure 5. Immunocytometric analysis of MRP expression in U87 and T98G glioblastoma

cells Figure 6. Western blot analysis of MRP expression in U87 and T98G glioblastoma cells

Figure 7. Differential interference contrast (DIC) and epi-fluorescence microscopy images (20X) of control and PTX and CUR combination nanoemulsion (PTX+CUR-NE) treated U87 cells. The cells were incubated with the nanoemulsions for 8 hours at 37°C

Figure 8. Differential interference contrast (DIC) and epi-fluorescence microscopy images

(20X) of control and PTX and CUR combination nanoemulsion (PTX+CUR-NE) treated T98G cells. The cells were incubated with the nanoemulsions for 8 hours at 37°C

Figure 9: MRP-1 down-regulation and inhibition of NFκB pathway by Western blot

analysis. Figure 10: Cytotoxicity of PTX alone (A, B) and CUR alone (C, D) on U87 (A, C) and T98G

cells (B, D) when administered in the form of solutions and nanoemulsions Figure 11: Cytotoxicity of PTX and CUR (5, 10, 20 μM) combinations on U87 cells, when

administered in the form of solutions (A, B, C) and nanoemulsions (D, E, F) Figure 12: Cytotoxicity of PTX and CUR (5, 10, 20 μM) combinations on T98G cells, when

administered in the form of solutions (A, B, C) and nanoemulsions (D, E, F) Figure 13: Comparison of cytotoxicity of PTX and CUR combination, administered together

in the form of nanoemulsion with PTX alone on MRP-1 expressing T98G cells Figure 14: TUNEL staining images (20X) of untreated U87 and T98G cells

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Figure 15: TUNEL staining images (40 X) of apoptotic nuclei of U87 cells upon treatment with solutions (PTX-S, CUR-S, PTX+CUR-S) and nanoemulsions (PTX-NE, CUR-NE, PTX+CUR- NE)

Figure 16: TUNEL staining images (40 X) of apoptotic nuclei of T98G cells upon treatment

with solutions (PTX-S, CUR-S, PTX+CUR-S) and nanoemulsions (PTX-NE, CUR-NE, PTX+CUR- NE)

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1. INTRODUCTION

1.1 Glioblastoma: Incidence and Mortality

Primary brain tumors comprise only 2% of the malignant tumors in human beings (1, 2).

Glioblastoma multiforme (GBM) and anaplastic astrocytoma are the two malignant gliomas that

occur more frequently than any other types of primary brain tumors (3). They have a combined

incidence of 5-8 per 100,000 populations. Although the incidence of GBM is low, it comprises

the most aggressive form of brain tumor with a median survival rate of less than one year. The

standard treatment of malignant gliomas involve surgical excision followed by radiation therapy

with or without adjuvant chemotherapy (4). Even then, GBM and other brain tumors almost

always relapse and become refractory to traditional therapeutic strategies. Difficulty in early

detection, the low tolerance of the brain tissue to radiation, the presence of “islets” of tumor cells

disseminated in critical regions of the brain, and the presence of the blood-brain barrier (BBB)

makes it very challenging to treat malignant gliomas effectively (2).

1.2 Role of the Blood-Brain Barrier in GBM Therapy

The BBB was first observed by Ehrlich and Goldman (5), when they found that the

hydrophilic compound, tryptan blue, injected into the rat did not get distributed into and out of

the brain (Figure 1). The BBB is a dynamic interface separating blood from the extracellular

fluid in the brain parenchyma. It regulates the influx and efflux of biological molecules thus

eliminating the toxic substances from the endothelial compartment and supplying the brain with

nutrients and endogenous compounds (6). On the whole, the BBB serves as an important barrier

to protect the brain from insults and for regulation of its homeostasis. Lack of effective

permeability across the BBB is a major limitation for delivering drugs to treat brain tumors or

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other central nervous system disorders (7-9).

Figure 1. Schematic depiction of the blood-brain barrier in brain microvasculature. (ref.

http://www.stanford.edu/class/humbio103/ParaSites2005/Ivermectin/images/blood%20brain%20barrier.gif)

The physical barriers for the permeability is characterized mainly by the tight cellular

junctions, the absence of intercellular clefts, minor pinocytotic activity, a continuous non-

fenestrated endothelium, a high electrical resistance, and anionic sites at the luminal membrane

(6). The tightness of the endothelial cell junctions in brain capillaries is evident from the very

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high trans-endothelial electrical resistance of 1,500-2,000 Ωcm2 as compared to 3-33 Ωcm2 in

peripheral tissues (8, 9). Additionally, the astrocytes surround about 85% of the surface of the

capillaries, adding one more lipid barrier to the system. The endothelial cells that line the luminal

surface of the brain capillaries also express ATP-binding cassette (ABC) drug transporters, P-

glycoprotein (P-gp), and members of the multidrug resistance protein (MRP) family at a high

density (10). These efflux pumps remove a wide range of molecules from the intracellular

compartment of the endothelial cell before they can enter the brain parenchyma. Many cytotoxic

chemotherapeutic agents that have shown very high efficacy in brain tumor, such as paclitaxel

(PTX), vincristine, and vinblastine are pumped out by the P-gp efflux pump, leading to

inefficient anti-cancer therapy.

Drug transport across the BBB depends on its systemic pharmacokinetics, which include

absorption, distribution, metabolism and excretion. The physicochemical properties of the drug,

like lipophilicity, hydrophilicity, and hydrogen bonding potential plays an important role in the

passive transport of drugs across the BBB. Hydrophilic paracellular transport and the lipophilic

transcellular transport are the two kinds of passive diffusive transports, drugs undergo to cross

the BBB and they are mainly dependent on the size and charge of the molecule and the hydrogen

bonding potential. Drugs which are highly lipophilic and partially permeable through the tight

junctions of BBB are restricted because of the factors like P-gp efflux and cytochrome P-450

metabolizing enzymes, whereas hydrophilic drugs are completely impermeable by passive

diffusion through the tight junctions the BBB. Therefore, the BBB is an extremely important

physical and chemical hurdle that restricts delivery of drugs for CNS disorders (6).

1.3 Paclitaxel and Curcumin in Glioblastoma Therapy

Paclitaxel (PTX) is a very potent anticancer drug, originally derived from the bark of the

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pacific Yew tree (Taxus brevifolia). It has got tremendous potential against the breast, ovarian,

cervical and many other tumors. PTX interferes with the dynamic instability of the microtubules

by inducing tubulin polymerization and thereby interferes with the mitotic spindle formation and

arrests the cell cycle at G2/M phase, finally leading to apoptosis (11, 12). Although PTX is

highly effective in treatment of glioblastoma in vitro, the in vivo delivery to the brain is restricted

due to BBB presence and the expression of P-glycoprotein efflux mechanism (13). As such, PTX

efficiency in treating GBM and other brain tumors is hindered by lack of efficacy and potential

chemoresistance development. Most importantly, PTX activates NFκB pathway and hence

promotes cell survival, proliferation and metastatis (14-16). PTX is also found to down-regulate

the cellular proteins that promote cell survival and block apoptosis such as BcL-2 and Bcl-XL

(17) . Another important chemoresistance phenomenon acquired by cytotoxic drugs, like PTX, is

the resistance involving P-gp expression. PTX is a P-gp substrate and this allows less drug to get

accumulated inside the cell and, hence results in reduced cell-kill efficacy (18).

Curcumin (CUR) is a yellow-colored polyphenolic compound extracted from the

turmeric rhizome (Curcumin longa) which is an Indian spice and also a coloring agent used in

variety of Asian foods. CUR widely exhibits antioxidant, anti-inflammatory, and anti-cancer

properties (19, 20). It has been found that free CUR induces apoptosis in many tumor cell lines

derived from breast, colorectal, lung and prostate carcinoma. CUR is also found to inhibit anti-

apoptotic, proliferative, and metastatic proteins in breast cancer cell lines (21). Additionally,

CUR has the potential to modulate multidrug resistance (MDR) in cancer (22, 23). MDR is a

phenomenon in cancer therapy, where the tumor cells develop resistance to a variety of

structurally and functionally dissimilar chemotherapeutic agents (24). MDR phenotype of cancer

cells is caused by various molecular mechanisms. They include over expression of the ATP-

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dependent drug efflux pump of ABC transporter family like P-glycoprotein (P-gp) and the

members of MRP family. MDR mechanisms also include modifications of the apoptotic

signaling, alterations in DNA repair mechanisms, and modifications in drug metabolism through

over-expression of glutathione-S-transferase (GST) and cytochrome P450 activity (24, 25).

Paclitaxel Curcumin

Figure 2. The chemical structures of paclitaxel and curcumin.

In this study, we hypothesize that co-administration of PTX with an MDR modulator,

like CUR, would be significantly beneficial by augmenting the therapeutic effects and improving

clinical outcomes, especially in GBM. We anticipate synergistic effect upon co-administration of

a chemotherapeutic agent that induces apoptosis, but is prone to P-gp and MRP resistance, along

with a modulator that inhibits NFκB pathway and can overcome the resistance.

Chemoresistance and enhanced survival in GBM, the primary brain tumor, is mainly due

to the over-expression of activator protein-1 (AP-1) and the transcription factor nuclear factor κB

(NFκB) (26). NFκB regulates a number of genes, which play an important role in inflammation,

apoptosis, angiogenesis, and tumor progression. NFκB is present in its inactive state bound to the

IκB family proteins in the cytoplasm (27, 28). Once when NFκB activated by specific stimuli,

which include growth factors, cytokines, lymphokines, radiation, and stress, IκBα kinase (IKK)

phosphorylates and then degrades the IκBα bound to the NFκB, thus releasing the NFκB into the

15

nucleus, which in turn binds to the DNA and activates transcription of various genes, which

helps in cell proliferation and survival. In other way, AP-1 a transcription factor is activated by

JNK pathway (29). C-Jun N-terminal kinases (JNKs) phosphorylate c-Jun, which combines with

c-Fos to form the AP-1. Together, AP-1 and NFκB were found to be the potential targets for

GBM therapy and CUR has been found to decrease the activity of AP-1 and inhibit the NFκB

pathway (26). In this case, the apoptosis induced by CUR was found to be p53 and caspase

independent. CUR inhibits the NFκB by inhibiting the activation of IKK and, thus inhibiting the

subsequent phosphorylation and degradation of IκBα. Additionally, CUR is known to down-

regulates the NFκB-regulated gene products such as Bcl-2, Bcl-XL, cyclin D1, matrix

metalloproteinase-9, cyclooxygenase-2, and interleukin-6, resulting in cell cycle arrest,

suppression of proliferation, and induction of apoptosis (26). Curcumin also inhibited the AP-1

signalling pathway by decreasing the constitutive phosphorylation of the AP-1 transcription

factor, c-Jun.

The other factors making the glioma therapy ineffective is the intrinsic or the acquired

drug resistance phenomenon with the over expression of P-gp and other members of the MRP

family, especially MRP-1, MRP-3 and MRP-5 and glutathione-S-transferase pi (GST-pi) (30). P-

gp is a 170-kDa membrane glycoprotein with 12 membrane spanning domain and is encoded by

the mdr1 gene (31, 32). P-gp serves as a xenobiotic pump in lung, intestine, kidney, and placenta

and is overly expressed on physiological barriers including the BBB. Many cationic and neutral

compounds, in addition to many anti-cancer drugs like taxanes and vinca alkaloids, are substrates

for the drug transporters. Over-expression of P-gp, especially in tumor cells, will not allow drugs

to accumulate in the cells and results in reduced cytotoxicity. Combination of P-gp inhibitors and

conventional chemotherapeutic drugs was found to be efficacious in treating tumors.

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Curcuminoids were found to inhibit both P-gp function and expression in KB-V1 cells and can

reverse the multidrug resistance phenomenon (33, 34). Commercially available curcumin is a

mixture of curcuminoids, curcumin I, II & III. All three of these curcuminoids inhibit P-gp, but

curcumin I is more potent due to the presence of two methoxy groups and two hydroxyl groups,

which helped in binding more to P-gp. It was also found that P-gp does not confer resistance to

CUR and also modulation of P-gp by CUR was not cell type dependent. On the other hand,

members of the MRP family show the MDR phenomenon, being capable of carrying wide range

of anticancer drugs. MRP-1 is a 190-kDa protein with 17 transmembrane spanning domains

encoded by mrp1 gene (32, 35). Its expression was also found high in BBB. It mainly effluxes

drugs, that are either conjugated or co-transported with glutathione. It prefers transporting

organic anions. Its function is found entirely different from the P-gp of the same ABC transporter

family.

Figure 3. Proposed multimodal mechanisms of MDR modulation with curcumin.

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CUR and its glutathione conjugates are found to inhibit MRP-1 and MRP-2 (36). The

mechanism of inhibition is still unknown. Even the MRP modulators can be given along with

anti-cancer agents for effective cancer chemotherapy.

In the effective management of GBM, therefore, PTX can be given in combination with

CUR, which can show the synergistic effect, by inhibiting the NFκB pathway and

downregulating the P-gp and other multidrug resistance proteins, which are over-expressed in the

tumor cells and the blood brain barrier. It was found that in breast cancer, PTX activated NFκB

pathway involving IKK activation, IκBα phosphorylation and degradation and NFκB-regulated

gene expression, whereas, CUR completely suppressed IKK activation and thus suppressing

NFκB activation. CUR also suppressed the various PTX-induced anti-apoptotic gene products

(14). So it is expected that CUR is having the ability to sensitize the GBM cells to PTX- induced

apoptosis.

1.4 Nanotechnology for Brain Delivery

Nanotechnology involves the design of materials, devices and the delivery systems that

have the functional organization at least in one dimension on the nano scale, typically around

100 nm (12, 37, 38). In medicine and biology, nanosystems are engineered in such a way to

interface the cells and tissues at the molecular level. They can carry out many functions at a time

or in a preferred sequence and in a controlled way, an important property for the successful

delivery of drugs across anatomical and physiological barriers, including BBB (39, 40).

Significant research in finding nanotechnology approaches to cross the BBB is done in effective

delivery of anti-neoplastic drugs and other treatments for CNS therapy. This system should cause

less toxic side effects to the healthy tissues, and then cross the BBB, reach the tumor site, and

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then be effective in killing the tumor cells. Many of the anti-neoplastic drugs have poor

physicochemical properties. PTX, for instance, is poorly soluble in water and the drug has very

low bioavailability. As such, there is a critical need to develop nanotechnology-based strategies

for effective delivery of PTX across the BBB.

Nanoparticles like liposomes, micelles and polymeric systems are extensively developed

in the recent years, which provided organ- and tissue-specific as well as intracellular delivery of

drugs and genes (41). Because of their small size and surface modification, they can cross the

barriers and penetrate through the capillaries into the cells and allow efficient accumulation of

the drug at the target site. In general, nanotechnology-based delivery systems passively or

actively target the tumor site. Passive targeting relies on the size, charge, and surface properties

of the nanocarrier, while active targeting involves attachment of a specific ligand that is

recognized by the cells at the disease site. Using passive targeting, nanocarriers can

preferentially accumulate at the tumor site due to leaky and fenestrated vasculature and poor

lymphatic drainage, which is termed as enhanced permeability and retention (EPR). The leaky

vasculature allows for accumulation of nanoparticles, whereas the poor lymphatic drainage helps

in retention at the required site (42). A number of examples of polymeric-, lipid-, and dendrimer-

based nanocarriers have utilized the EPR effect to concentrate the drug at the tumor mass and

enhance the residence time of the drug (43).

Nanotechnology based formulations also include, encapsulating the drug in the form of a

nanoemulsions (39). Edible plant-seed and fish oils containing high percentage of

polyunsaturated fatty acids (PUFA) have the potential to cross the BBB, thus mimicking the

uptake of natural fatty acids in the brain (44). Nanoemulsions made with oils rich in

polyunsaturated fatty acids (PUFA) were found to solubilize PTX efficiently in the oil droplet

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and enhanced its oral bioavailability (12). There are two important PUFAs found abundantly in

the brain and are essential for maintaining the neurological health. They are 1) omega -3 PUFA

and the 2) omega-6 PUFA (45).

One of the proposed models for the mechanism of uptake of fatty acids (FA) explains that

the FA diffuses across the BBB and reaches brain cells, without the involvement of transporters.

They cross the luminal and the transluminal leaflets of the brain endothelium and the plasma

membrane by reversible flip-flop. Once the FA reaches the neural cells, acetyl-CoA synthetase

traps the FA by forming acetyl-CoA. This model works when the plasma membrane is

permeable to the FA, but there is also significant of evidence explaining that the transport of FA

across the BBB is derived from FA/albumin complexes (44).

Our preliminary studies have shown that nanoemulsions made with pine-nut oil and

Lipoid® E80 were found to enhance the transport across the BBB (12). Pine-nut oil is rich in

omega-3 and omega-6 PUFAs and specifically pinolenic acid and linoleic acid, respectively. Of

the total fat content in commercially-available extra virgin pine-nut oil, at least 45% of its total

unsaturated fatty acid content as pinolenic acid and 87% as linoleic acid (46). In a study reported

by Edmond (47), it was shown that linoleic acid with 18-carbon backbone and two cis-double

bonds was imported to the brain, while oleic acid containing 18 carbons and one cis-double bond

was not. This suggests exclusive selectivity of essential PUFA across the BBB.

1.5 Multifunctional Nanoemulsions for PTX/CUR Combination Therapy

Nanoemulsions are defined as thermodynamically stable dispersions of oil-in-water or

water-in-oil stabilized by an interfacial film of surfactant and co-surfactant molecules, with the

droplet size in the nanoscale ranging from 100 to 200 nm (37). This drug delivery system mainly

helps in overcoming the difficulty in delivering poorly soluble species and those that have poor

20

bioavailability, by encapsulating them in the inner phase (37, 38). The properties of

nanoemulsions are governed by the factors like the particle size and shape, surface charge,

surfactant resident time at the nanoemulsion surface and particle to particle interactions. These

factors in turn can be varied with the selection of oil phase, water phase and the surfactants. The

versatility of the nanoemulsions is also well described from the fact that the surface of the

droplet can also be modified with target-specific ligands for disease-specific localization.

Nanoemulsions are spontaneously formed when the surface tension is reduced approximately to

zero by applying mechanical energy input. Microfluidizers, ultra-sound and ultra-high shear high

pressure mixing devices are needed to provide significant energy for producing nano-scale

emulsions. Higher the energy applied results in smaller particle size of the oil droplet (37).

In the drug formulation and product optimization, another important reason for the

rejection of many new chemical entities at formulation stage itself is related to poor aqueous

solubility of the species and, thus the difficulty in delivering to systemic targets. PTX, for

instance, is formulated in an ethanolic solution containing Cremophor® EL, which is found to

cause adverse reactions upon systemic administration (48). Apart from the poor solubility, being

a P-gp substrate, PTX also has poor bioavailability and cannot cross BBB. In addition, CUR is

also a poorly soluble drug and has very low oral bioavailability. Its therapeutic effects are hence

limited to the gastrointestinal tract. In a Phase I clinical study, it was reported that patients were

administered with 8.0 grams of CUR per day orally in order to achieve detectable systemic levels

(21). Administration of CUR intravenously will enhance its potential as an anticancer agent.

Nanoemulsions can enhance the solubility of PTX and CUR by specifically dissolving in the oil

phase.

In this study, our objective was to evaluate co-administration of PTX and CUR in

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nanoemulsion formulations. We anticipate that PTX and CUR will be effectively solubilized in

the oil phase of the emulsions and allow us to achieve intracellular availability. Furthermore, co-

administration of CUR will significantly improve PTX therapeutic effective, especially in MRP-

1 expressing T98G glioblastoma cells. Lastly, the use of PUFA-rich oil for preparation of

nanoemulsion also has an advantage in enhancing BBB transport.

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2. OBJECTIVES AND SPECIFIC AIMS

2.1 Statement of the Problem

Glioblastoma multiforme (GBM), the most lethal form of brain tumor, affects as many as

12,000 patients in the United States and more than 200,000 world-wide every year (1). The high

mortality rate of GBM is partly due to lack of efficacious therapeutic outcomes upon systemic

chemotherapy administration. The blood-brain barrier (BBB) restricts optimum permeability and

availability of chemotherapeutic agents in the effective treatment of GBM. Nanotechnology-

based delivery systems, such as oil-in-water nanoemulsions, have been shown to enhance drug

availability in the brain upon systemic administration. As such, nanoemulsion formulations can

be very effective in therapeutic management of GBM, especially when combination therapies are

used that can augment the efficacy and improve clinical outcomes.

2.2 Objectives and Hypotheses

The main objective of this Master’s thesis project was to evaluate the potential of oil-in-

water nanoemulsion delivery system for combination therapy in GBM. The nanoemulsions in

this study are specifically prepared with oils rich in omega-3 and omega-6 polyunsaturated fatty

acids (PUFA) to enhance drug transport across the BBB. An additional objective is to evaluate

the potential of combination chemotherapeutic agent (i.e., paclitaxel) and a multidrug resistant

(MDR) modulator (i.e., curcumin). Curcumin is known to have several beneficial effects

including down-regulation of MRP-1 gene and enhancement of apoptosis in tumor cells through

inhibition of the NFκB signaling. Our experimental hypothesis is that the combination of

paclitaxel (PTX) and curcumin (CUR), when administered in nanoemulsions, will significantly

enhance therapeutic efficacy in GBM by down-regulation of drug resistance and augmentation of

cellular apoptotic response.

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Our experimental hypotheses for this project were: (1) PUFA-rich oil-in-water

nanoemulsions can efficiently solubility PTX and CUR and allow for enhanced intracellular

delivery in GBM cells and (2) co-administration of CUR with PTX will enhance therapeutic

efficacy in GBM cells by simultaneous down-regulation of MRP-1 expression and inhibition of

NFkB signaling.

2.3 Specific Aims

The specific aims of this MS thesis project were:

Aim 1: Preparation, Characterization, and Optimization of Pine Nut Oil-Containing

Oil-in-Water Nanoemulsions:

(a) Blank, PTX- and CUR-containing nanoemulsions, as single agent and in combination,

will be prepared using pine-nut oil as the internal oil phase.

(b) The nanoemulsion formulations will be characterized for particle size, surface charge,

and oil droplet morphology.

(c) Optimization of the nanoemulsion formulations based on droplet size and stability.

Aim 2: Intracellular Delivery in U87 (wild-type) and T98G (MRP positive) GBM

Cells

(a) Establishment of U87 and T98G human glioblastoma cells in culture.

(b) Qualitative evaluations of intracellular PTX and CUR delivery with nanoemulsion

formulations by fluorescence microscopy.

Aim 3: Down-Regulation of MRP-1, Inhibition of NFκB pathway and Enhancement

in Cytotoxicity and Apoptosis in U87 (wild-type) and T98G (MRP-1 positive) GBM Cells

24

(a) Determination of baseline MRP-1 expression in U87 and T98G cells by

immunocytometry and Western blot analysis.

(b) Analysis of MRP-1 expression down-regulation and Inhibition of NFκB pathway with

CUR treatments using Western blot analyses.

(c) Cytotoxicity of single and combination PTX and CUR therapy in solution and in

nanoemulsion formulations using the MTT (formazan) assay.

(d) Qualitative apoptosis measurements by TUNEL staining.

25

3. MATERIALS AND METHODS

3.1 Preparation of Oil-in-Water Nanoemulsions

Oil-in-water nanoemulsion formulations were prepared using an ultrasonication method

that has been optimized in our laboratory (12). Briefly, the aqueous phase was prepared using 4

mL of deionized distilled water and 120 mg of egg phosphotidylcholine (Lipoid® E80, Lipoid

GmbH, Ludwigshafen, Germany), which were mixed and stirred for at least 30 minutes to insure

complete dissolution. Separately, the oil phase, consisting of 1 mL of extra virgin pine-nut oil

(Siberian Tigers Inc., Springfield, VT) was heated to 75-80oC for 2-3 minutes. Gradually, the

aqueous phase was added to the oil phase under stirring conditions. The oil droplet particle size

in the course emulsion formed was further reduced by ultrasonication at 21% amplitude and 50%

duty cycle using Vibra-Cell VC 505® (Sonics Instruments, Newtown, CT) ultrasound instrument

for 10 minutes. The stable nanoemulsions thus formed were stored in a refrigerator at

approximately 4°C for further use.

For the preparation of drug-containing nanoemulsions, stock solutions of both the drugs,

PTX and CUR, were made in dehydrated ethanol. Desired amount of PTX and CUR powders

were weighed and dissolved in ethanol. The stock solution was added to the oil phase (1 mL).

Ethanol in the oil phase was evaporated under high vacuum prior to the mixing of aqueous

phase. The mixture was then ultrasonicated to obtain the 1.0 mg/mL PTX- and CUR-containing

nanoemulsions.

3.2 Characterization of the Nanoemulsions

3.2.1 Particle Size Analysis: Blank and drug-containing nanoemulsions were

characterized for the hydrodynamic particle size and size distribution using dynamic light

scattering method with the Brookhaven Instrument’s ZetaPALS® 90Plus (Holtsville, NY). The

26

nanoemulsion sample was diluted a 1,000 times with deionized distilled water and the oil droplet

particle size was determined at 90° scattering angle and 25°C temperature. Average count rate

was maintained constant between 50-500 kcps to achieve reproducibility in the particle size

measurements. The hydrodynamic diameter of the oil droplet is determined and compared by

considering the mean effective diameter on log-normal size distribution mode.

3.2.2 Surface Charge Measurements: Blank and drug-containing nanoemulsions were

diluted with deionized distilled water and the zeta potential values were measured using

Brookhaven Instrument’s ZetaPALS® 90Plus (Holtsville, NY). The refractive index was kept at

1.33 and the viscosity at 1 cps to mimic the conditions of water. Out of the diluted nanoemulsion

(2.5 mL), small volume of approximately 1.5 mL was connected to an electrode at 4.0 volts and

2.0 Hz field frequency for surface charge determination. Electrophoretic mobility of the nano-

droplets was measured and converted to zeta potential values with a built-in software that uses

the Smoluchowski equation to calculate zeta potential values.

3.2.3. Transmission Electron Microscopy (TEM): To observe the morphology of the oil

droplets in the nanoemulsions, each batch was also characterized by TEM using a negative

staining technique. Approximately, 50 µL of the control and drug-loaded nanoemulsion

formulations were added to the 200 mesh Formwar-coated copper grids (Electron Microscopy

Science, Hatfield, PA). The samples were allowed to dry by draining off the excess liquid with

Whatman filter paper, which was placed on the edge of the copper grid. Then they were

negatively stained with 50 µL of 1.5% uranyl acetate and allowing the staining to proceed for 10

minutes at room temperature. Then the excess uranyl acetate was drained off and the copper grid

containing the nanoemulsion sample as a dry film was placed on a TEM sample holder and

27

observed with a JEOL 100-X (Peabody, MA) transmission electron microscope equipped with a

20 µm aperture and at 60 kV accelerating voltage.

3.3 Determination of Baseline MRP-1 Expression in GBM Cells

3.3.1. Cell Culture Conditions: Wild type human GBM cells (U87-MG) and MRP-1

expressing resistant human GBM cells (T98-G), purchased from American Type Culture

Collections (ATCC, Rockville, MD), were cultured in Minimum Essential Medium (MEM) at

37°C in 5.0% CO2 atmosphere. When the cells in the flask reached confluency, they were

detached with trypsin-EDTA and were then splitted into two flasks. For the immunocyto-

chemistry experiment, when the cells reached confluency, they were detached and plated into 6-

well microplates with a sterile glass coverslip at a density of 5,000 cells per well.

3.3.2 Immunocytometric Evaluation of MRP Expression: Cultured U87 and T98G cells

were grown on sterile glass cover slips overnight at 37oC. On the following day, the cover slips

were gently washed with sterile PBS. The adherent cells were fixed with 10% formalin in PBS

for 10 minutes while keeping the cells wet and the excess formalin was rinsed with subsequent

washes with sterile PBS. To avoid any non-specific binding of the immunoglobulin, the cover

slips were incubated for 30 minutes in normal goat serum blocking solution. After which, the

cover slips were incubated with primary rabbit MRP-1 antibody (Cell Signaling, Danvers, MA)

diluted in 1:100 primary antibody dilution buffer for an hour at room temperature. The cover

slips were then rinsed twice for 2 minutes each in washing buffer. To block the endogenous

peroxidase activity, the cover slips were further incubated in peroxidase blocking solution for 10

minutes and rinsed three times with the washing solution. The cells were then incubated for 30

minute at room temperature with the secondary antibody 1:200 (i.e., goat anti-rabbit antibody)

28

conjugated with horse radish peroxidase (Abcam, Inc., Cambridge, MA) and washed three times

with the washing solution. Subsequently, diaminobenzidine (DAB) solution was added and

allowed to incubate for 5-10 minutes for the peroxidase catalyzed reaction to occur resulting in

the formation of brown precipitates. The samples were observed with a bright field microscope

under 20X magnification.

3.3.3. Western Blot Analysis: In order to determine the MRP levels in U87 and T98G

glioblastoma cells, we have carried out Western blot analysis as follows.

The first step involves extraction of cytosolic proteins from U87 and T98G cells using

freshly prepared lysis buffer (i.e., 0.5 ml of 1.0 M Tris pH 7.4, 0.2 ml of 0.5 M EDTA pH 8.0,

1.5 ml of 5.0 M NaCl, 4.38 ml of 10%, Brij®-97, 0.625 ml of 10% Tween®-20 dissolved in 50 ml

of deionized distilled water) for a period of 5 minutes followed by centrifugation at 3,000 rpm

for an additional 5 minutes. The supernatant was then collected and the protein concentration

was estimated using NanoOrange® (Invitrogen, Carlsbad, CA) protein quantitation assay. A

standard curve for NanoOrange® quantitation was constructed using bovine serum albumin. The

total protein extract was then diluted 1:100 and stored at -80oC. The second step involves loading

and running the gel where 100 micrograms and 25 micrograms of the protein extract was

denatured at 90oC for 10 minutes in a water-bath, followed by 1:1 dilution with Laemmeli’s

buffer and then loaded onto pre-cast 4-15% SDS-polyacrylamide gel electrophoresis (PAGE)

gradient gel (Bio-Rad, Hercules, CA).

The gel was then run using Tris/Glycine/SDS running buffer at 125 volts for 90 minutes.

Subsequently, the protein bands on the gel were transferred onto a nitrocellulose membrane

(BioRad, Hercules, CA) in Tris/Glycine/SDS transfer buffer supplemented with 10% methanol at

25 volts for two on ice-bath. The transferred protein bands on the membrane were then blocked

29

with 3% milk in Tween®-containing Tris buffer saline (T-BST) for 45 minutes. The

nitrocellulose membrane was incubated overnight with agitation at 4oC with 1:1,000 dilution of

the primary mouse MRP-1 specific monoclonal antibody in milk. Following morning, the milk

was decanted and the membrane was washed twice with water and then incubated with 1:2000

dilution of the secondary anti-mouse HRP-conjugated IgG in T-BST for 1 hour at room

temperature. After rinsing the excess secondary antibody with T-BST and water, the reagent was

added that is cleaved by perodixase to give a chemiluminescent product. The chemiluminescent

bands were then visualized with a Kodak imager (Carestream Health, Rochester, NY).

3.4 Fluorescence Microscopic Studies of Intracellular Delivery with Nanoemulsions

3.4.1 Preparation of Fluorescently-Labeled Nanoemulsions: PTX was replaced by

rhodamine-labeled paclitaxel conjugate (rhodamine-PTX) (at 0.1% (w/w)) and was added to the

oil phase. The aqueous phase was added to the oil phase and ultrasonicated, as mentioned above,

to get the fluorescently labeled formulation. CUR is intrinsically fluorescent in the visible green

region (21) and therefore, was directly added to the oil phase and the nanoemulsions were

formulated as previously described with the final CUR concentration to 1 mg/mL.

3.4.2 Fluorescence Microscopy Studies: Nanoemulsion containing rhodamine-PTX and

CUR were diluted with MEM and aliquots were added to the U87 and T98G cells and were

compared with the fluorescently labeled solutions of PTX and CUR respectively. At regular time

intervals, the medium was removed and the cells were washed with fresh medium and then with

sterile PBS. After the final wash, the coverslips were mounted on glass slides with Fluromount-

G® mounting medium. Differential interference contrast (DIC) and epi-fluorescent images were

30

acquired at 2 0X magnifications using fluorescence microscope and the digital images were

processed using the Adobe Photoshop® software.

3.5 Determination of MRP1 Down-Regulation with CUR by Western Blot Analysis

The U87and T98G cells were treated with 20 µM CUR in the form of solutions and

Nanoemulsion formulation and incubated for 24 hours at 37ºC. Following treatments, the total

protein was extracted and the protein content was measured with NanoOrange® assay. Thirty-

micrograms of extracted protein was mixed in a 1:1 ratio with Laemmeli’s buffer and then

loaded onto a pre-cast 4-15% SDS-polyacrylamide gel electrophoresis (PAGE) gradient gel. The

gel was then run for 90 minutes at 125 V and the protein bands on the gel were transferred onto a

nitrocellulose membrane using transfer buffer supplemented with 10% methanol at 25 volts for

two-and-half hours on ice-bath. The transferred protein bands on the membrane were then

blocked with 3% milk in Tween®-containing Tris buffer saline (T-BST) for 45 minutes and then

incubated overnight with agitation at 4oC with 1:1,000 dilution of the primary mouse monoclonal

MRP-1 antibody in milk. The next day, milk was decanted and the membrane was washed twice

with water and then incubated with 1:2,000 dilution of the secondary anti-mouse horse radish

perodixase-conjugated IgG in T-BST for 1 hour at room temperature. After rinsing the excess

secondary antibody with T-BST and water, the ECL reagent was added, which was cleaved by

the horseradish perodixase enzyme to give a chemiluminescent product. The chemiluminescent

bands were visualized using a Kodak imager.

3.6 Determination of NFκB Pathway Inhibition with CUR by Western Blot Analysis

U87and T98G cells were treated with 20µM CUR in the form of solutions and

Nanoemulsion formulation and incubated for 24 hours at 37ºC. Following treatments, the total

31

protein was extracted and the protein content was measured with NanoOrange® assay. Thirty

micrograms of extracted protein was mixed in a 1:1 ratio with Laemmeli’s buffer and then

loaded onto a pre-cast 4-15% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-

PAGE) gradient gel. The gel was then run for 90 minutes at 125 V and the protein bands on the

gel were transferred onto a nitrocellulose membrane using transfer buffer supplemented with

10% methanol at 25 volts for two-and-half hours on ice-bath. The transferred protein bands on

the membrane were then blocked with 3% milk in Tween®-containing Tris buffer saline (T-BST)

for 45 minutes and then incubated overnight with agitation at 4oC with 1:1,000 dilution of the

primary rabbit NFκB p65 (Cell Signaling, Danvers, MA) antibody in milk. The next day, milk

was decanted and the membrane was washed twice with water and then incubated with 1:2,000

dilution of the secondary anti-rabbit horse radish perodixase-conjugated IgG in T-BST for 1 hour

at room temperature. After rinsing the excess secondary antibody with T-BST and water, the

ECL reagent was added, which was cleaved by the horse radish perodixase enzyme to give a

chemiluminescent product. The chemiluminescent bands were visualized using a Kodak imager.

The entire procedure was repeated for Primary antibody incubation with rabbit polyclonal

antibody against IκBα followed by secondary antibody incubation.

In addition, to visualize the control beta-actin bands, the nitrocellulose membrane with

proteins was cut into two halves and the upper half was incubated with primary antibody against

NFκB and the lower second half was then incubated overnight with primary antibody against

beta-actin. Following these treatments, the secondary antibody was added and the bands were

visualized as described above.

32

3.7 Cytotoxicity of Single and Combination Treatments in GBM Cells

The cytotoxicity studies were performed with both solutions and the nanoemulsion

formulations containing different concentrations of PTX, CUR and combination of PTX and

CUR. Solutions were prepared by dissolving the PTX, CUR in DMSO and then adding them to

culture medium to obtain the desired concentrations of PTX (1 nM, 5 nM, 10 nM, 50 nM, 100

nM, and 500 nM) and CUR (5 µM, 10 µM, and 20 µM) respectively and maintaining the DMSO

at 0.1% concentration in the well. Similarly nanoemulsions were also prepared at the above

mentioned concentrations of both PTX and CUR in MEM. The U87 cells and resistant T98G

cells were allowed to adhere on the surface of 96-well microplates at a density of 5,000 cells per

well. After 48 hours, when the cells in the wells were approximately confluent, the culture media

was replaced with the solutions and nanoemulsions of PTX and CUR separately and also in

combinations. After dosing, the U87 cells and T98G cells were incubated at 37°C for 3 days.

Since PTX is a cell cycle specific drug, the 3-day time period was used to insure that all cells in

the microplates can succumb to the drug effect. Cells treated with MEM containing 0.1% DMSO

and also cells treated with MEM containing Blank nanoemulsion formulation alone was used as

negative controls in determining the cytotoxicity of solutions and nanoemulsion formulations,

respectively, and those treated with 250 µg/mL of poly(ethyleneimine) (Mol. Wt. 10 kDa), a

cytotoxic cationic polymer, were used as positive control in both the cases.

For the cytotoxicity study, the MTT reagent was prepared at a concentration of 1 mg/mL

by dissolving the yellow dye (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a

tetrazole dye) in MEM. Following a 3-day incubation period, the media from each well was

replaced with 50µL of freshly prepared MTT reagent into each well and again incubated for 2

hours in the CO2 chamber at 37°C. Then 150 µL DMSO was added into each well to dissolve

33

the insoluble purple-colored formazan crystals formed in the mitochondria of living cells. The

absorbance in each well was measured using a BioTek-HT (Winooski, VT)

UV/Visible/fluorescence microplate reader at 570 nm. The percentage cell viability was

calculated at each concentration of PTX, CUR and at each combination of PTX and CUR by

dividing the absorbance of the treated cells over the control cells and multiplying it by one

hundred. Then IC50 was found for the solutions and the nanoemulsions on both U87 cells and

T98G cells with the help of bar charts plotted using Microsoft Excel® and Graph-Pad Prism®

softwares. The IC50 was also calculated for the PTX and CUR combinations on both wild-type

and MRP-1 expressing glioblastoma cell lines.

3.8 Qualitative Apoptosis Studies by TUNEL Staining

The TUNEL assay (terminal dUTP nick-end labeling) is an assay used to detect apoptosis

in-situ by measuring nuclear DNA fragmentation, a biochemical indicator of apoptosis at the

single-cell level. This system, end-labels fragmented DNA with biotinylated nucleotide at the

3’OH ends using the terminal deoxynucleotidyl transferase, recombinant (rTdT) enzyme.

Horseradish peroxidase-conjugated streptavidin (streptavidin-HRP) bound to the biotinylated

nucleotide was detected using the peroxidase substrate, hydrogen peroxide and the chromogen

diaminobenzidine, which stains apoptotic nuclei dark brown. A DeadEndTM Colorimetric

TUNEL assay system kit (Promega Corporation, Madison, WI) was used for this assay.

Both U87 and T98G glioma cells grown on Lab-Tek® chamber slides were incubated

with solutions and Nanoemulsions of both 20 µM CUR, 100 nM PTX and combination of 20

µM CUR and 100 nM PTX for 24 hours at 37oC and 5% CO2 to induce apoptosis. The cells were

then washed twice with PBS and were fixed by immersing them in 10% buffered formalin for 25

minutes at room temperature followed by immersing twice in PBS for 5 minutes each at room

34

temperature. The cells were then permeabilized by immersing in 0.2% Triton® X-100 solution in

PBS for 5 minutes at RT. The cells were again immersed twice in PBS for 5 minutes each at RT.

The cells were then equilibrated for 5-10 minutes at RT with 100 µL of equilibrium buffer. One

hundred-μL of rTdT reaction mix per slide was prepared using 98 μl of equilibration buffer + 1

μL of biotinylated nucleotide mix + 1 μL of rTdT enzyme. One hundred-μL of the rTdT reaction

mix was then added to the previously equilibrated sections on the slide without allowing the

sections to dry. Plastic coverslips were used to cover the sections to ensure even distribution. For

the end-labeling reaction to occur the coverslips were then incubated at 37oC, 5% CO2 for 60

minutes. The reaction was then terminated by removing the coverslips and immersing the slides

is 2X sodium chloride-sodium citrate buffer (SSC) buffer for 15 minutes at RT. The slides were

then washed twice by immersing in PBS for 5 minutes at room temperature. The slides were

again immersed in 0.3% hydrogen peroxide solution in PBS at room temperature to block

endogenous peroxidases and were then washed twice by immersing in PBS for 5 minutes at

room temperature. The streptavidin-HRP provided is diluted 1:500 in PBS and 100 μL of it was

added to each slide followed by 30 minute incubation at RT. The slides were then washed twice

by immersing in PBS for 5 minutes at room temperature. DAB solution was prepared just before

use and 100 μL of it was added to the sections until a light brown color develops. The sections

were then rinsed several times in deionized water. The slides were then mounted in permanent

mounting medium and observed under a light microscope.

35

4. RESULTS AND DISCUSSIONS

4.1 Preparation and Characterization of Multifunctional Nanoemulsions

Oil-in-water pine-nut oil-containing nanoemulsions were formulated using egg

phosphotidylcholine as the surfactant, which surrounded the oil droplet as an interfacial film and

helped in reducing the surface tension and the high shear conditions of the ultrasound instrument

made it possible to make a stable dispersion of nanoscale oil droplets in aqueous phase. The

mean hydrodynamic diameter of the oil droplets of the nanoemulsion was in the range of 100-

120 nm for all of the samples as shown in Table 1. The zeta potential values of all the

nanoemulsion formulations were found to be around -40 mV as shown in Table 1, which is

obtained due to the presence of the ampiphathic egg phosphotidylcholine molecules adsorbed at

the oil-water interface.

Table 1: Physical characterization of the blank and drug-containing nanoemulsions

Abbreviations Formulations Hydrodynamic Particle Size (nm)

Zeta Potential (mV)

NE-BLK Blank Nanoemulsion

111.9 ± 0.2*

-36.8 ± 0.7

NE-PTX Paclitaxel- Containing

Nanoemulsion

115.9 ± 2.0

-36.6 ± 4.0

NE-CUR Curcumin- Containing

Nanoemulsion

114.8 ± 2.3

-42.8 ± 2.8

* Mean ± S.D. (n = 5)

PTX and CUR were efficiently encapsulated in the form of an oil droplet and the TEM

analysis confirms the spherical shape and the smooth surface morphology of the oil droplet as

shown in Figure 4. TEM image also confirms the diameter in the range 100-120 nm.

36

Figure 4: Transmission electron micrograph of pine-nut oil nanoemulsion for the co-administration of paclitaxel and curcumin

4.2 Determination of Baseline MRP-1 Expression in GBM Cells

4.2.1 Immunocytometric Evaluation of MRP-1 expression: Qualitative baseline

expression of MRP-1 in U87 and T98G human glioblastoma cells was examined by

Immunocytometric analsyis. U87 and T98G cells which are not treated with anti-body against

MRP-1, but treated with DAB stain are used as control. U87 cells and T98G cells were treated

with anti-MRP-1 primary antibody and HRP-linked secondary antibody and were then treated

with DAB stain. The peroxide catalyzed reaction that occurs following the addition of DAB

results in the formation of a brown precipitate on the cell surface and within the cell, indicating

the localization of MRP-1 in U87 and T98G cells as shown in Figure 5. Dark brown precipitate

observed on T98G cells as shown in Figure 5 confirms the over-expression of MRP-1 in

37

accordance with the Western blot results as shown in Figure 6. Controls cells did not show any

brown precipitates indicating the absence of non-specific binding of primary antibody.

A B

C D

A B

C D

Figure 5. Immunocytometric analysis of MRP-1 expression in U87 and T98G glioblastoma cells. The figures represent T98G cells (A&B) and U87 cells (C&D). A&C are images of unstained

control cells, while B&D represent cells that were treated with MRP-1 antibody and stained with diaminobenzidine

4.2.2 Western Blot Analysis: The main purpose of this study is to further confirm the

over-expression of MRP-1 in resistant T98G cells when compared to that of wild-type U87

human glioblastoma cells. As shown in Figure 6, MRP-1 expression was observed in both U87

(lane 1) and T98G (lane 3), but there was far higher level of expression in T98G cells as

compared to U87 cells. Hence the results of Western blot analysis, confirms that T98G cell line

would serve as a best model to study the down regulation of MRP-1 by CUR.

38

39

Figure 6: Western blot analysis of MRP-1 expression in U87 and T98G glioblastoma cells. (The bands are: (1) 100 µg of protein in the well extracted from U87 cells,

(2) 25 µg of protein in the well extracted from U87 cells, (3) 100 µg of protein in the well extracted from T98G cells, and

(4) 25 µg of protein in the well extracted from T98G cells).

4.3 Cellular Uptake and Distribution Studies

Cellular uptake and intracellular trafficking studies were carried out to confirm that the

NE can transfect U87 and T98G human glioblastoma cells. For this study, rhodamine-labelled

paclitaxel conjugate and CUR itself were encapsulated in the oil phase. CUR is naturally

fluorescent in the visible green region. Both U87 cells and T98G cells were incubated with PTX

and CUR combination nanoemulsion formulation for 3 hours, 6 hours and 8 hours time-points.

Since CUR is naturally fluorescent, it has got a very weak signal, which quenches rapidly.

Therefore, DXRED and FITC images were not overlayed. Cells treated with blank nanoemulsion

were used as control. Higher intracellular fluoroscence was observed for both PTX-NE and

CUR-NE after 8 hours of incubation in U87 and T98G cells as shown in Figure 7 and Figure 8

respectively. There was no fluoroscence observed in control cells, confirming that U87 and

T98G cells don’t exhibit auto fluoroscence. These results suggest that the oil droplets were found

stable and accumulated within the cell and were not present on the cell surface. This also

suggests that NE can deliver both PTX and CUR in wild-type U87 and resistant T98G human

glioblastoma cells.

1 2 3 4

Control-DIC Control-DXRED Control-FITC

PTX+CUR- NEDIC

PTX+CUR- NEDXRED

PTX+CUR- NEFITC

Figure 7: Differential interference contrast (DIC) and epi-fluorescence microscopy images (20X) of control and PTX and CUR combination nanoemulsion (PTX+CUR-NE) treated U87

cells. The cells were incubated with the nanoemulsions for 8 hours at 37°C

Control-DIC Control-DXRED Control-FITC

PTX+CUR- NEDIC

PTX+CUR- NEDXRED

PTX+CUR- NEFITC

Figure 8: Differential interference contrast (DIC) and epi-fluorescence microscopy images (20X) of control and PTX and CUR combination nanoemulsion (PTX+CUR-NE) treated T98G

cells. The cells were incubated with the nanoemulsions for 8 hours at 37°C

40

4.4 Down Regulation of MRP-1 and Inhibition of NFκB Pathway with CUR

There was literature evidence suggesting that Curcumin and its glutathione conjugates

have the potential to inhibit MRP-1 (36). From the above results, it was also shown that U87 and

T98G cell line can serve as a best model to study MRP-1 down-regulation. Both U87 and T98G

cells were treated with CUR solution and CUR nanoemulsions and the cytosolic proteins were

extracted from both treated and untreated cells (control). The chemiluminiscent bands observed

for the MRP-1 were shown in Figure 9. Lanes 1, 2 and 3 indicates protein extracted from U87

cells. Lanes 4, 5 and 6 indicates protein extracted from T98G cells. Lane 1 and 4 indicate protein

extracted from untreated cells. Lanes 2 and 5 indicate protein extracted from CUR solution

(CUR-S) treated cells. Lanes 3 and 6 indicate protein extracted from CUR nanoemulsion (CUR-

NE) treated cells. MRP-1 bands were observed at 190 kDa. Bands in lane 2 and 3 were less

intense when compared to that of lane 1 and band in lane 6 was less intense when compared to

that of lane 4 and 5 suggesting the MRP-1 down-regulation in CUR treated U87 and T98G cells.

Bands in lane 3 & 6 were less intense when compared to that of 2 and 5 indicating that,

nanoemulsion formulation was much more efficient in MRP-1 down-regulation.

For studying the inhibition of NFκB pathway, two different antibodies were choosen.

One antibody was against NFκB p65 domain. Most abundantly occurring NFκB dimers in

cytoplasm contain p65 domain. For certain stimuli, IKK gets activated in a tumor cell, which

further phosphorylates and degrades IκBα and thus NFκB translocates into the nucleus and

initiates the transcription of several genes responsible for tumor cell survival. CUR inhibits

NFκB pathway by inhibiting IKK. Therefore high intense NFκB bands at 64 kDa were observed

in CUR treated U87 cells when compared to that of untreated U87 cells as shown in Figure 9.

There was not such inhibtion observed in T98G cells treated with CUR. This result confirms the

41

presence of more NFκB in cytosol in an inactive state in CUR treated U87 cells. In untreated

cells, NFκB gets translocated into the nucleus and hence less intense bands were observed. The

other antibody choosen for this study was against IκBα. Figure 9 also illustrates the IκBα bands

at 36 kDa. Same as in the case of NFκB, high intense bands of IκBα from CUR treated U87 cells

which confirm the presence of more IκBα in cytosol and the inhibition of the NFκB pathway.

Again, there was no such inhibition obseved in T98G cells, as there was no differenc in the

intensity of bands. CUR inhibits IKK and thus inhibits the degradation of IκBα . NFκB remains

in the inactive states as long as IκBα is not degraded. Beta-actin bands with same intenstiy in all

the 6 lanes served as a loading control in this study. This Western blot analysis confirms the

MRP-1 down-regulation and inhibition of NFκB pathway in U87 cells by CUR. Further

investigation is needed to confirm the MRP-1 down-regulation and inhibition of NFκB pathway

in T98G cells by CUR

NFκB(64 kDa)

IκBα(36 kDa)

β-actin(42 kDa)

6 5 4 3 2 1

MRP-1(190 kDa)

Figure 9: MRP-1 down-regulation and inhibition of NFκB pathway by Western blot analysis. Lane 1, 2 and 3 represent the protein extracted from U87 cells. Lane 4, 5 and 6 represent the protein extracted from T98G cells. Lane 2 and 5 is treatment with CUR-S. Lane 3 and 6 is

treatment with CUR-NE. Thirty-μg of protein was added into each well.

42

4.5 Cytotoxicity of Single and Combination Treatments in GBM Cells

The cytotoxicity experiments were carried out with both solutions and nanoemulsion

formulations. IC50 values were determined and the efficiency of nanoemulsions over solutions

and the single therapy over co-therapy was evaluated. From semi-logarthmic plots as shown in

Figure 10, it was clearly observed that the IC50 of PTX solution alone when administered on

U87 cells was found approximately between 100 nM and 500 nM, whereas the PTX-NE (PTX

nanoemulsion) was found approximately between 10 nM and 50 nM showing a 10-fold increase

in the cytotoxicity with NE when compared to that of solutions. Figure 10 also showed that there

was an increase in cell cytotoxicity with NE (IC50 between 50 nM and 100 nM) on T98G cells

when compared to that of the solution (IC50 above 100 nM). No significant change in cell

viability at three different concentrations 10 nM, 50 nM and 100 nM (around 60% cell viability)

was probably due to the resistance shown by MRP-1 to PTX. At low concentrations of CUR,

there was no significant cell death as shown in Figure 10. CUR was found to have some

cytotoxicity in the sensitive U87 cell line, when administered in the form of NE.

43

Cytotoxicity of PTX alone on U87 cells

0

20

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0 0.5 1 1.5 2 2.5 3

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Cytotoxicity of PTX alone on T98G cells

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Cytotoxicity of CUR alone on U87 Cells

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Cytotoxicity of CUR alone on T98G cells

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60

80

100

120

0 0.2 0.4 0.6 0.8 1 1.2 1.4

log (conc of CUR)

% c

ell v

iabi

lity

CUR solutionCUR-NE

A B

C D

Figure 10: Cytotoxicity study of PTX alone (A, B) and CUR alone (C, D) on U87 (A, C)) and T98G cells (B, D), when administered in the form of solutions and nanoemulsions.

Mean +/- S.D. (n=6) ( p<0.05 when compared with the control)

For combination therapy, different concentrations of PTX were administered with a fixed

concentration of CUR at 20 μM and the cell viability was calculated as shown in Figure 11 and

Figure 12. There was some synergism shown with the co-administration of PTX and CUR in the

form of solutions on the sensitive U87 cells when compared to that of nanoemulsions. However,

in the resistant T98G cell line, there was significant synergism shown in both the cases of

solutions and NE. These results suggest the potential of MDR modulation with addition of CUR.

44

45

Figure 11: Cytotoxicity of PTX and CUR (5, 10, 20 uM) combinations on U87 cells, when administered in the form of solutions (A, B, C) and nanoemulsions (D, E, F).

Mean +/- S.D. (n=6) (* p<0.05 when compared with the PTX alone)

Effect of PTX + 5 uM CUR solution on U87 cells

0

20

40

60

80

100

120

140

Blnk PEI 5nM 10nM 50nM 100nM

conc of PTX in nM

% c

ell v

iabi

lity

PTX alonePTX + 5 uM CUR

A

Effect of PTX + 10 uM solution on U87 cells

0

20

40

60

80

100

120

140

Blnk PEI 5nM 10nM 50nM 100nM

conc of PTX in nM

% c

ell v

iabi

lity

PTX alonePTX + 10 uM CUR

B

Effect of PTX + 20 uM solution on U87 cells

0

20

40

60

80

100

120

140

Blnk PEI 5nM 10nM 50nM 100nM

conc of PTX in nM

% c

ell v

iabi

lity

PTX alonePTX + 20 uM CUR

C

Effect of PTX + 5 uM CUR emulsion on U87

0

20

40

60

80

100

120

140

Blnk PEI 5nM 10nM 50nM 100nM

conc of PTX in nM

% c

ell v

iabi

lity

PTX alonePTX + 5 uM CUR

D

*

** *

* * * *

*

Effect of PTX + 10 uM CUR emulsion on U87

0

20

40

60

80

100

120

140

Blnk PEI 5nM 10nM 50nM 100nM

conc of PTX in nM

% c

ell v

iabi

lity

PTX alonePTX + 10 uM CUR

E

* *

Effect of PTX + 20 uM emulsion on U87 cells

0

20

40

60

80

100

120

140

Blnk PEI 5nM 10nM 50nM 100nM

conc of PTX in nM

% c

ell v

iabi

lity

PTX alonePTX + 20uM CUR

F

* *

46

Figure 12: Cytotoxicity of PTX and CUR (5, 10, and 20 uM) combinations on T98G cells, when administered in the form of solutions (A, B, C) and nanoemulsions (D, E, F).

Mean +/- S.D. (n=6) (* p<0.05 when compared with the PTX alone)

From these cell viability studies, it was finally found that at the fixed concentration of

CUR at 20 µM, there was a significant reduction in IC50 of PTX, when treated on the MRP-1

expressing resistant T98G cell line as shown in Figure 13. The efficiency of combination

therapy as found in the resistant cell line may be probably because of the down regulation of

MRP-1 gene by CUR.

Effect of PTX + 5 uM CUR solution on T98G cells

0

20

40

60

80

100

120

Blnk PEI 10nM 50nM 100nM

conc of PTX alone

% c

ell v

iabi

lity

PTX alonePTX + 5uM CUR

z

Effect of PTX + 10 uM CUR solution on T98G cells

0

20

40

60

80

100

12

Blnk PEI 10nM 50nM 100nM

conc of PTX in nM

% c

ell v

iabi

lity

0PTX alonePTX + 10 uM CUR

Effect of PTX + 20 uM CUR solution on T98G cells

0

20

40

60

80

100

120

Blnk PEI 10nM 50nM 100nM

conc of PTX in nM

% c

ell v

iabi

lity

PTX alonePTX + 20 uM CUR

Effect of PTX + 5 uM CUR emulsion on T98G cells

0

20

40

60

80

100

120

140

Blnk PEI 10nM 50nM 100nM

conc of PTX in nM

% c

ell v

iabi

lity

PTX alonePTX + 5 uM CUR

Effect of PTX + 10 uM CUR emulsion on T98G cells

0

20

40

60

80

100

120

140

Blnk PEI 10nM 50nM 100nM

conc of PTX in nM

% c

ell v

iabi

lity

PTX alonePTX + 10 uM

Effect of PTX + 20 uM CUR emulsion on T98G cells

0

20

40

60

80

100

120

140

Blnk PEI 10nM 50nM 100nM

conc of PTX in nM

% c

ell v

iabi

lity

PTX alonePTX + 20 uM CUR

A

* * *

B

* * *

C

* **

D

E

F

* * *

* * *

* * *

Synergism shown with NE on T98G cells

0

20

40

60

80

100

120

Blnk PEI 20uM 100nM 20uM+100nM

% c

ell v

iabi

lity

*

Figure 13: Comparison of Cytotoxicity of PTX and CUR combination, administered together in the form of nanoemulsion with PTX alone in MRP-1 gene

expressing T98G cells. Mean +/- S.D. (n=6) (*p<0.05 when compared with PTX alone)

4.6 Qualitative Apoptosis Study by TUNEL Staining

TUNEL assay is used to study apoptosis qualitatively. The assay is based on the

peroxidase catalyzed conversion of DAB and hydrogen peroxide to a brown precipitate, staining

apoptotic nuclei brown. rTdT enzyme, a component of the TUNEL kit end labels fragmented

DNA of apoptotic cells with biotinylated nucleotide at 3’OH end. Streptavidin-HRP bound to the

biotinylated nucleotide is detected by the peroxidase substrate, hydrogen peroxide and the

chromogen DAB, staining apoptotic nuclei brown. 100nM of PTX and 20µM of CUR were the

concentrations used to see the apoptotic activity in U87 and T98G cells. Untreated cells were

found to have no apoptotic activity as shown in Figure 14. Additionally, Figure 15 shows the

apoptotic activity of U87 cells upon different treatments and Figure 16 shows the apoptotic

activity of T98G cells upon different treatments. Both U87 and T98G cells treated with

nanoemulsions (PTX-NE, CUR-NE, PTX+CUR-NE) have shown more apoptotic activity when

47

compared to that of cells treated with aqueous solutions (PTX-S, CUR-S, PTX+CUR-S). The

most apoptotic activity was observed when 100nM of PTX was co-administered with 20µM of

CUR in the form of nanoemulsion in both sensitive U87 and resistant T98G human glioblastoma

cells. Figure 15 and Figure 16 also show that there were fewer cells when treated with

combination, which confirms that most of the cells were dead and got washed away. The

TUNEL results were found to be in correlation with Cytotoxicity results. TUNEL results suggest

that the apoptotic activity was enhanced with combination therapy and hence improved the

efficiency of glioblastoma chemotherapy.

U87 untreated cells T98G untreaetd cells

20X 20X

Figure 14: TUNEL staining images (20X) of untreated U87 and T98G cells

48

Qualitative Apoptosis in U87-MG cells by TUNEL staining

PTX-S

CUR-S

PTX+CUR-S

PTX-NE

CUR-NE

PTX+CUR-NE

Figure 15: TUNEL staining images (40 X) of apoptotic nuclei of U87 cells upon treatment with solutions (PTX-S, CUR-S, PTX+CUR-S) and nanoemulsions (PTX-NE, CUR-NE, PTX+CUR-

NE)

49

Qualitative Apoptosis in T98G cells by TUNEL staining

PTX-S

CUR-S

PTX+CUR-S

PTX-NE

CUR-NE

PTX+CUR-NE

Figure 16: TUNEL staining images (40 X) of apoptotic nuclei of T98G cells upon treatment with solutions (PTX-S, CUR-S, PTX+CUR-S) and nanoemulsions (PTX-NE, CUR-NE,

PTX+CUR-NE)

50

5. CONCLUSIONS

The result of this study has shown that PTX and CUR combination therapy in the form of

a nanoemulsion has significant potential for the treatment of human glioblastoma. Pine-nut oil-

containing nanoemulsions, which is rich in omega-3 PUFA, formulated using the ultrasound

method has proved to be a suitable carrier for both PTX and CUR. The results of

immunocytometric and Western blot analyses have shown the over-expression of MRP-1 in

resistant T98G cells when compared to that of sensitive U87 cells. The cytotoxicity results have

demonstrated that the nanoemulsions were more efficient in killing cells when compared to the

free drug in aqueous solution. Synergistic effect was observed especially in the resistant T98G

cells when 20 μM CUR was co-administered with 100 nM of PTX in the form of nanoemulsion,

making the system efficient for the treatment of glioblastoma. The results of the cellular uptake

and distribution study have shown that the designed nanoemulsion system can effectively deliver

PTX and CUR within the U87 and T98G cells. Western blot analysis has also shown the MRP-1

down regulation in both U87 and T98G cells and has also shown

the inhibition of NFκB pathway by CUR in U87 cells, but not to a great extent in T98G

cells which confirms that the synergism observed in the co-therapy is due to the MRP-1 down-

regulation by CUR which helps by increasing the accumulation of PTX within the tumor cell and

also overcoming the MDR phenomenon by inhibiting the NFκB pathway. Apoptosis induced by

PTX and CUR in U87 and T98G cells was well understood using the Qualtitative apoptosis

TUNEL assay.

In conclusion, it has been shown in this study that the nanotechnology based oil-in-water

pine-nut oil nanoemulsions improved the therapeutic efficacy by successfully delivering

combination therapeutic agents in GBM cells. It was found that CUR has the ability to sensitize

51

the glioblastoma cells to PTX-induced apoptosis. This dual therapeutic strategy has significant

potential in clinical management of GBM.

52

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