Study on drug delivery for paclitaxel of the lactobacillus strains

This paper discussed the ability of minicells which were generated from Lactobacillus acidophilus VTCC-B-871 and Lactobacillus rhamnosus JCM 15113 for drug delivery of paclitaxel.. The p

VIETNAM NATIONAL UNIVERSITY – HOCHIMINH CITY

INTERNATIONAL UNIVERSITY

STUDY ON DRUG DELIVERY FOR PACLITAXEL

OF THE LACTOBACILLUS STRAINS

A Thesis submitted to the School of Biotechnology, International University

in partial fulfillment of the requirements for the degree of

Master of Science in Biotechnology

Student name: Doan Thi Thanh Vinh – MBT04015 Supervisor: Dr Nguyen Hoang Khue Tu

July 2013

Study on Drug Delivery for Paclitaxel of the

Lactobacillus Strains

By

DOAN THI THANH VINH

ABSTRACT

The practice of developing molecularly targeted drugs to achieve a higher degree

of cancer therapy and antibiotic resistance is indispensable Lactobacillus strains

participated in the anti-cancer effects and performed the high-level specificity for cancer cell lines This paper discussed the ability of minicells which were

generated from Lactobacillus acidophilus VTCC-B-871 and Lactobacillus rhamnosus JCM 15113 for drug delivery of paclitaxel L acidophilus VTCC-B-871 and L rhamnosus JCM 15113 formed minicells with highly significant number

(1,070,000 and 787,000 minicells, respectively) in modified MRS broth with the concentration of fructose 10 g/l and lactose 50 g/l, respectively Nanoparticle size of obtained minicells ( 400 nm in diameter) was determined using scanning electron microscopy The minicells packaged paclitaxel (10 µg/ml) and cephalosporin (10 µg/ml) for different times of incubation (10, 15 and 24 hours)

at 37°C Our results showed that drugs could be completely absorbed for 10 hours by detecting extracted solution from drug packaged minicells on antimicrobial activities The significant influence of different concentrations of loading paclitaxel (5, 10, 20 µg/ml) on drug packaging was studied The results indicated that after extraction of the paclitaxel packaged minicells and analysis with high performance liquid chromatography for determining the number of paclitaxel presented in a minicell, there was a huge amount of paclitaxel encapsulated in a minicell (31 million paclitaxel molecules per minicell with loading paclitaxel (20 µg/ml)) The present work was the first report on the

generation of minicells from Lactobacillus acidophilus VTCC-B-871 and Lactobacillus rhamnosus JCM 15113 and the drug delivery for paclitaxel of these

minicells

Key works: Lactobacillus acidophilus, Lactobacillus rhamnosus, minicells,

paclitaxel, cephalosporin, antimicrobial activities, high performance liquid chromatography

ACKNOWLEDGEMENTS

First of all, it is undeniable that this thesis has taken me a great deal of time and effort Moreover, I have to insist that this thesis cannot be well-completed without the encouragements, the nice ideas and helps from a lot of people For these reason, I would like to thank to many people that contributed one way or another to the realization of this paper

Remarkably, I would like to express my most sincere gratitude and appreciation

to Dr Nguyen Hoang Khue Tu for all her enthusiastic supervision, patience and encouragement of this work Her critical comments, constructive suggestions, professional directions also guided effectively my logical ideation and efficient communication skills throughout the whole research period

Furthermore, I would love to send my best wishes to all members of the School

of Biotechnology of International University for their tremendous enthusiasms as well as for their nice behavior towards me, and also for give me the good chance to conduct and have my research study completed

Definitely, it is certain that I really send my thanks to Dr Ha Dieu Ly and all the members at Department of Reference Substances, Institute of Drug Quality Controls and also all members at Scanning Electron Microscopy Laboratory Room, Vietnam Academy of Science and Technology for their assistance and technical support

Words cannot express my respect, profound gratitude for my parents who are always on me, encourage, and share their great love for me I love to send my sincere thanks to my good friends who are being with me during my bad and good time

Last but not least, I greatly thank you who spend precious time to read this document This actually has profound significance for me as the fruit of my labor

is also gotten your acceptance

TABLE OF CONTENTS

ABSTRACT i

ACKNOWLEDGEMENTS ii

LIST OF FIGURES v

LIST OF TABLES vi

ACRONYMS AND ABBREVIATIONS vii

CHAPTER 1 INTRODUCTION 1

CHAPTER 2 LITERATURE REVIEW 3

2.1 Cancer and tumor-targeted drug delivery systemsError! Bookmark not defined 2.1.1 Worldwide cancer burden 3

2.1.2 Cancer treatment and recent advances in cancer therapy 4

2.1.3 Obstacles in cancer therapy and targeted drug delivery systems 5

2.1.4 Approaches to improve the therapeutic index of anti-cancer drugs 8

2.2 Bacterially-derived minicells 10

2.2.1 Minicells 10

2.2.2 Bacterially-derived mincells as controlled drug delivery for cancer therapy 11

2.2.4 Current research of minicells in delivering anti-cancer drugs 12

2.3 An overview of Lactobacillus species 14

2.3.1 The Genus Lactobacillus 14

2.3.2 Lactobacillus acidophilus 15

2.3.3 Lactobacillus rhamnosus 15

2.4 Paclitaxel 16

2.5 Orientation of implementing to delivery paclitaxel using minicells derived from the lactobacillus strains 17

2.6 Process of project 18

CHAPTER 3 MATERIAL AND METHODS 19

3.1 Materials 19

3.1.1 Bacterial strains 19

3.1.2 Tools and equipments 19

3.1.3 Media and chemicals 19

3.2 Research methodology 21

3.2.2 Design condition for minicells production from Lactobacillus strains 21

3.2.2 Minicell isolation 21

3.2.3 Microscopic studies for characterization of minicell morphology 21

3.2.4 Packaging of drug into minicells 22

3.2.5 Drug extraction for measurement 23

3.2.6 Antimicrobial activity tests 23

3.2.7 Paclitaxel assay using HPLC-UV/Vis Spectroscopy 23

3.2.8 Calculation of the number of drug molecules 24

3.2.9 Data analysis 25

CHAPTER 4 RESULTS AND DISCUSSION 26

4.1 Screening the carbon sources for study on minicell generation from Lactobacillus strains 26

4.1.1 Minicells generation in Lactobacillus acidophilus VTCC-B-871 30

4.1.2 Minicells generation from Lactobacillus rhamnosus JCM 15113 34

4.2 Microscopic studies: characterization of minicell morphology 37

4.3 Package of drug into minicells generated from Lactobacillus strains 39

4.3.1 Antimicrobial activity tests 39

4.3.1.1 Drug packaging of minicells produced from L acidophilus 39 4.3.1.2 Drug packaging of minicells produced from L rhamnosus 41

4.3.2 Paclitaxel quantitation using HPLC-UV/Vis Spectroscopy 43

4.3.2.1 The minicell drug packaging in response to incubation times 44

4.3.2.2 The minicell drug packaging in response to concentrations 45

CHAPTER 5CONCLUSION AND RECOMMENDATION 49

REFERENCES 50

LIST OF FIGURES

Figure 2.1 Nanoparticle platforms for drug delivery 8

Figure 2.2 Schematic showing minicell formation and bispecific antibody-targeted, drug/siRNA-packaged minicells 11

Figure 2.3 Chemical structure of paclitaxel 16

Figure 3.1 Process of isolation of bacterially-derived minicells from the parent cells culture 21

Figure 4.1 The API 50CHL biochemical testing of Lactobacillus acidophilus 26

Figure 4.2 The API 50CHL biochemical testing of Lactobacillus rhamnosus 28

Figure 4.3 The number of minicells (×104) produced from L acidophilus 32

Figure 4.4 Photomicrographs of Lactobacillus acidophilus VTCC-B-871 and its minicells (100X)l 33

Figure 4.5 The number of minicells (×104) produced from L rhamnosus 35

Figure 4.6 Photomicrographs of Lactobacillus rhamnosus JCM 15113 and its minicells (100X) 36

Figure 4.7 Photomicrograph showing the morphology of minicells from Lactobacillus acidophilus VTCC-B-871 under the light microscope after isolation procedure (100 X) 37

Figure 4.8 Representative SEM images showing the formation of minicells 38

Figure 4.9 Representative SEM images of minicells from L acidophilus VTCC-B-871with minicell size after isolation procedure 39

Figure 4.10 Drug quantitation in minicells when minicells incubated in the presence of different drug loading concentrations 46

LIST OF TABLES

Table 2.1 Estimated (2008) and projected numbers (millions) of cancer

cases and deaths, all cancers, both sexes, by development status

or WHO region 3

Table 2.2 The summary statistics of estimated incidences, mortality in men, women, and both sexes in Vietnam in 2008 4

Table 2.3 Advantages of using nanoparticles as a drug delivery system 9

Table 3.1 Lactobacilli MRS broth 20

Table 4.1 The API 50CHL biochemical testing of Lactobacillus acidophilus 27

Table 4.2 The API 50CHL biochemical testing of Lactobacillus rhamnosus 29

Table 4.3 The number of minicells produced from L acidophilus VTCC-B-871 in modified MRS medium 32

Table 4.4 The number of minicells produced from L rhamnosus JCM 15113 in modified MRS medium 35

Table 4.5 Antimicrobial spectrum of extracted drugs from drug-packaged minicells of L acidophilus VTCC-B-871 and L rhamnosus JCM 15113 against Gram-positive and Gram-negative bacteria 41

Table 4.6 Antibacterial activity of extracted paclitaxel and cephalosporin from drug-packaged minicells of L acidophilus against bacterial species 41

Table 4.7 Antibacterial activity of extracted paclitaxel and cephalosporin from drug-packaged minicells of L rhamnosus against bacterial species 42

Table 4.8 HPLC analysis of paclitaxel in the loading solution in response to varying times of incubation 44

Table 4.9 HPLC analysis of paclitaxel in minicells when incubated in the presence of different drug concentrations 46

Table 4.10 Paclitaxel quantification in minicells when incubated in the presence of different drug concentrations 47

ACRONYMS AND ABBREVIATIONS

ATCC American Type Culture Collection

BsAbs Bispecific Antibodies

EGFR Epidermal Growth Factor Receptor

GRAS Generally Recognized As Safe

HPLC High Performance Liquid Chromatography

IARC International Agency For Research On Cancer IFP Interstitial Fluid Pressure

JCM Japanese Collection Microorganism

SEM Scanning Electron Microscope

siRNA Small interfering RNA

VTCC Vietnamese Type Culture Collection

CHAPTER 1 INTRODUCTION

Cancer is the general name for a group of more than 100 diseases which is being one of the major health problems on over the world Despite the availability of several treatment modalities, mortality rates due to cancer are high Therefore, effective cancer therapy continues to be a daunting challenge due mainly to considerable tumor cell heterogeneity, drug resistance of cancer cells, dose-limiting toxicity of chemotherapeutics, and difficulties of targeted delivery to tumors (MacDiarmid et al., 2011)

A key obstacle in the use of current chemotherapeutic anticancer drugs is their lack of specificity for cancer cells, resulting in severe toxicity when they are administered systemically (Flemming, 2007) Consequently over the past decade

a significant global effort has focused on the discovery and development of molecularly targeted drug delivery systems (DDSs) (MacDiarmid et al., 2007a) DDSs are being developed to achieve a higher degree of tumor cell specificity and reduce toxic side effects, as well as overcome several challenges to the treatment of cancer (Chidambaram et al., 2011), including drug resistance and metastatic disease (Alexis et al., 2010) However, current strategies that use nanoparticles (Brannon et al., 2004), liposomes (Zheng et al., 2001); or polymer therapeutics (Duncan, 2003) are hampered by shortcomings such as drug

leakage in vivo, lack of versatility in terms of packaging a diverse range of

different drugs, thereby reducing drug potency, and difficulties in production scale-up (Ferrari et al., 2005), particularly for nanoparticles

Recently, a new promising technology for targeted and intracellular delivery of chemotherapeutic drugs relies on using bacterially derived nano-sized particles (termed as minicells) Minicells are originated from the normal cell deleting the

min gene (de Boer et al., 1989), but they can package a range of different

chemotherapeutic drugs and specifically targeting the minicells to tumor cell surface receptors via bispecific antibodies coating the minicells (Flemming, 2007) This technique has been experimented successfully for both Gram-

positive (Listeria monocytogenes) and Gram-negative bacteria (Salmonella typhimurium, Escherichia coli, Shigella flexneri, and Pseudomonas aeruginosa); drug-packaged nano-sized particles effected apoptosis of tumor cells both in vitro and in vivo; and they also targeted to cancer cells in vivo with high specificity and, thus, delivered in high concentration in vivo without toxicity

(MacDiarmid et al., 2007a) Although minicells currently generated from both Gram-positive and Gram-negative bacteria and also tested for encapsulating chemotherapeutic drugs and functioning as nanovectors for drug delivery in cancer therapy (MacDiarmid et al., 2007b) This mutation may effect on the growth of bacteria under their control so far (de Boer et al., 1989) Moreover, up

to now, there has not any research on generating bacterially-minicells from

Lactobacillus strains using other method without min gene deletion Especially, Lactobacillus strains are GRAS (Generally Recognized as Safe) microorganisms

used as the probiotics that are very important in foods, pharmaceuticals, and animal husband (Macfarlane and Cummings, 2002) The benefits of probiotics were found protection against gastrointestinal pathogens, enhancement of the immune system, reduction of lactose intolerance, reduction of serum cholesterol level and blood pressure, anti-carcinogenic activity, improved utilization of nutrients and improved nutritional value of food Members of the genus

Lactobacillus have also been reported on the significant oxidative,

anti-carcinogenic, and anti-bacterial activity, as well as the inhibitory effects on cancer cell growth besides being effect on human immune system (Choi et al.,

2006; Kim et al., 2002) Among the tested Lactobacillus species, Lactobacillus acidophilus (Choi et al., 2006) and Lactobacillus rhamnosus (Cenci et al., 2002)

strains participated in the anti-cancer effects, anti-carcinogenic ability and performed the high-level specificity for cancer cell lines

From those points, this paper presented the generation of bacterially derived

minicells from Lactobacillus strains for drug delivery and investigated if minicells

derived from these strains were able to package with chemotherapeutic drug

(Paclitaxel); in order to detectable a robust and versatile system for in vitro drug

delivery using minicell, a bacterially-derived lactic acid bacteria carrier Moreover, this study also confirmed the ability of encapsulation of minicells with other drug as cephalosporin This study was the primary research on the drug delivery system which was able to carry many drugs and necessary products for food and pharmaceutical fields

Therefore, the main aim of this study was to develop a new drug nano-sized

carrier derived from probiotic Lactobacillus for delivery of chemotherapeutic drug

with fewer side effects, and with further modification, to produce a systemic complex of molecular targeted drug delivery including probiotics properties From this aim, those specific objectives including, performing drug-packaged bacterially-derived minicells and determining the number of drug molecules presented in minicell

CHAPTER 2 LITERATURE REVIEW

2.1 CANCER AND TUMOR-TARGETED DRUG DELIVERY SYSTEMS

2.1.1 Worldwide cancer burden

Cancer is a group of diseases characterized by the uncontrolled growth of abnormal cells that disrupt body tissue, metabolism, etc and tend to spread locally and to distant parts of the body Life-threatening cancer develops gradually as a result of a complex mix of factors such as complex interactions of viruses, a person‟s genetic make-up, their immune response and their exposure

to other risk factors which may favor the cancer (Win, 2006) Based on the GLOBOCAN 2008 estimates (Ferlay et al., 2010a), the standard set of worldwide estimates of cancer incidence and mortality produced by the International Agency for Research on Cancer (IARC) for 2008, there were an estimated 12.4 million cases of cancer diagnosed and 7.6 million deaths from cancer and

28 million persons alive with cancer around the world in 2008 (Table 2.1); of these, 56% of the cases and 64% of the deaths occurred in the less developed regions of the world, many of which lack the medical resources and health systems to support the disease burden By 2030, it could be expected that there could be 27 million incident cases of cancer, 17 million cancer deaths annually and 75 million persons alive with cancer within five years of diagnosis (Table 2.1)

Table 2.1 Estimated (2008) and projected numbers (millions) of cancer cases

and deaths, all cancers, both sexes, by development status or WHO region (Boyle and Levin, 2008)

¹ no change in current rates

² with 1% annual increase in rates

Overall in 2008, based on the most recently available international data (GLOBOCAN 2008 estimates) produced by IARC, about 111,600 incident cases of cancer and 82,000 cancer deaths were estimated to have occurred in Vietnam (Table 2.2) The most commonly diagnosed cancers were liver (23,251 cases, 20.8% of the total new cancer cases), lung (20,659 cases, 18.5%) and stomach (15,068 cases, 9.7%) (Figure 2.1) Liver cancer is also the leading cause of cancer death for both sexes, accounting for 21,748 deaths, comprising 26.5% of the total cancer deaths (Ferlay et al., 2010a)

Table 2.2 The summary statistics of estimated incidences, mortality in men,

women, and both sexes in Vietnam in 2008; Source : GLOBOCAN 2008 (Ferlay et

Number of cancer deaths (thousands) 43.7 38.3 82.0

Risk of dying from cancer before age 75 (%) 12.7 8.8 10.5

5 most frequent cancers (ranking defined by total number of cases)

Liver Liver Liver

Stomach Breast Stomach Incidence and mortality data for all ages

Proportions per 100,000

2.1.2 Cancer treatment and recent advances in cancer therapy

Although great effort has been made in cancer research, no substantial progress can be observed in the past fifty years in the USA or almost twenty years in Vietnam in fighting against cancer The death rate in the USA was 193.9 per 100,000 in 1950 and remained as high as 194.0 per 100,000 in 2001 (Jemal et al., 2004) Based on the data from the Hanoi cancer registry, IARC estimated that in 1990 the mortality in Vietnam was about 82.2 per 100,000 (Pham and Nguyen, 2002) and actually stayed lower than that 94.1 per 100,000 in 2008 (Ferlay et al., 2010a) It is clear that the progress in cancer treatment has been slow and inefficient (Win, 2006) It is a multidisciplinary challenge needing more and closer collaboration between clinicians, medical and biomedical scientists and biomedical engineers to eventually find a satisfactory solution (Win, 2006)

The choice of treatment depends on the type and location of the cancer, whether the disease has spread, the patient's age and general health, and other factors Clinical treatment for cancer therapy is a multidisciplinary therapy consisting of surgery, radiation therapy, chemotherapy, biological therapy and other methods (e.g., biological therapy, targeted therapy, or gene therapy for cancer (ACS, 2013) Chemotherapy is most effective against cancers that divide rapidly and have a good blood supply (Win, 2006) Aims of chemotherapy treatments are to cure; to maintain long term remission (free of disease); to increase the effectiveness of surgery or radiotherapy; to help control pain or other symptoms However, research still continues in finding ways to make chemotherapy less toxic and also to minimize the side effects Molecularly targeted therapy – a new generation of cancer treatments has emerged as one approach to overcome the lack of tumor specificity of conventional cancer therapies (Gerber, 2008)

Currently, the pharmaceutical industry has been successful in discovering many new cytotoxic drugs that can potentially be used for the treatment of cancer, this life-threatening disease still causes over 7 million deaths every year worldwide and the number is growing (Ferlay et al., 2010a) Thus, the ongoing obligation to the design and discovery of new cancer therapy is urgent

2.1.3.1 Obstacles in cancer therapy

A vast array of resistance mechanisms, involving overexpression of drug transporters (which are the plasma membrane P-glycoprotein (P-gp) product of the multidrug resistance (MDR) gene as well as other associated proteins) (Gottesman, 2002), mutations or amplification of the target enzyme, decreased drug activation, increased drug degradation due to altered expression of drug metabolizing enzymes, diminished drug-target interaction, enhanced DNA repair,

or failure to apoptosis, can defeat single agents, no matter how well designed and targeted (Chorawala et al., 2012)

High tumor interstitial fluid pressure (IFP) is another barrier for efficient drug delivery (Heldin et al., 2004) Increased IFP contributes to a decreased transcapillary transport in tumors leads to a decreased uptake of drugs or

therapeutic antibodies Cancer cells are therefore exposed to a lower effective concentration of therapeutic agent than normal cells, lowering the therapeutic efficiency and increasing toxicity It is now well established that the IFP of most solid tumors is increased This increase makes the uptake of therapeutic agents less efficient in solid tumors (Wu et al., 2006) There are a number of factors that contribute to increase IFP in the tumour, such as vessel leakiness, lymph vessel abnormalities, fibrosis and contraction of the interstitial matrix

The discovery of cancer stem cells (CSCs) in solid tumors has changed our view

of carcinogenesis and chemotherapy Cancer stem cells are defined as those cells within a tumour that can self-renew and drive tumorigenesis (Dean et al., 2005) The CSCs, which are also accurately called „tumor-initiating cells‟, represent a small population of cancer cells, sharing common properties with normal stem cells (SCs), that can initiate new tumors following injection into animal models, while the majority of other cancer cells cannot (Vinogradov and Wei, 2012) Natural properties of the small group of cancer stem cells involved in drug resistance to standard chemotherapy agents, metastasis and relapse of cancers can significantly affect tumor (Dean et al., 2005)

In addition to the obstacles in cancer therapy, current chemotherapeutic drugs are constrained by severe systemic toxicity due to indiscriminate drug distribution and narrow therapeutic indices A key obstacle in the use of chemotherapeutic anticancer drugs is their lack of specificity for cancer cells, resulting in severe toxicity when they are administered systemically (Sarosy and Reed, 1993) This is exacerbated by the fact that systemically delivered cancer chemotherapy drugs often must be delivered at very high dosages to overcome poor bioavailability of the drugs and the large volume of distribution within a patient

In general, cancer chemotherapy is usually accompanied by severe side effects and acquired drug resistance Therefore, we anxiously await the development of molecularly targeted therapy that will allow greater tumor specificity and less toxicity Over these years, cancer targeting treatment has been greatly improved by new tools and approaches based on the development

of nanotechnology Nanotechnology is the creation and utilization of materials, devices, and systems through the control of matter on the nanometer scale (Jain, 2005) Nanocarrier systems can be designed to interact with target cells and tissues or respond to stimuli in well-controlled ways to induce desired

physiological responses They represent new directions for more effective diagnosis and therapy of cancer (Alexis et al., 2010)

2.1.3.2 Tumor-targeted drug delivery nanoparticles

In recent years, the rapid advent of nanotechnology has stimulated the development of many novel drug delivery strategies (Wang et al., 2007) Nanoparticles applied as nanoscale drug delivery vehicles have shown the ability

to encapsulate a variety of therapeutic agents such as small molecules (hydrophilic and/or hydrophobic), peptides, protein-based drugs, and nucleic acids, and protect them against enzymatic and hydrolytic degradation (Mohanraj and Chen, 2006) By encapsulating these molecules inside a nanocarrier, the known shortcomings of many anticancer drugs can be potentially overcome, such as low aqueous solubility, stability, high nonspecific toxicity or lack of selectivity of anticancer drugs (Chidambaram et al., 2011), while at the same time increasing the circulation time and bioavailability of encapsulated drugs (Langer, 1998)

Through encapsulation of drugs in a macromolecular carrier, such as a liposome, the volume of distribution is significantly reduced and the concentration of drug

in a tumor is increased This causes a decrease in the amounts and types of nonspecific toxicities, and an increase in the amounts of drug that can be effectively delivery to a tumor (Moghimi, 2006) The surface of the nanocarrier can be engineered to increase the blood circulation half-life and influence the biodistribution, while attachment of targeting ligands to the surface can result in enhanced uptake by target tissues (Gref et al., 1994; Moghimi et al., 2001) The small size allows nanocarriers to overcome biological barriers and achieve cellular uptake (Brigger et al., 2002) The net result of these properties is to lower the systemic toxicity of the therapeutic agent while increasing the concentration of the agent in the area of interest, resulting in a higher therapeutic index for the therapeutic agent (Gilstrap et al., 2011) against the most difficult cancer challenges (Chidambaram et al., 2011), including drug resistance and metastatic disease (Alexis et al., 2010)

Figure 2.1 Nanoparticle platforms for drug delivery Nanoparticle platforms are

characterized by their physicochemical structures, including polymer-drug

conjugates, lipid-based nanoparticles, polymeric nanoparticles, protein-based nanoparticles, biological nanoparticles, and hybrid nanoparticles (Alexis et al., 2010)

Nanoparticles applied as drug delivery systems are submicronsized particles (10

to 1000 nm) (Shim and Turos, 2007), devices, or systems that can be made using a variety of materials including polymers (polymeric nanoparticles, micelles, or dendrimers), lipids (liposomes), magnetic, even inorganic/metallic compounds (iron, silica) and bacteria (bacterial nanoparticles or “minicells”) (MacDiarmid and Brahmbhatt, 2011; MacDiarmid et al., 2007b, 2009) (Figure 2.2)

2.1.4 Principle approaches to improve the therapeutic index of

anti-cancer drugs

Nanoparticle drug delivery systems are being studied to overcome limitation of conventional therapeutic areas particularly in cancer chemotherapy As mentioned in subheading 2.1.3.2, nanomedicine performed a strong potential to accelerate the development of effective approaches to the treatment of drug-

Biological Nanoparticle

Hybrid Nanoparticle

Hydrophobic Polymer Therapeutic load Hydrophilic Polymer Targeting Ligand Lipid

resistant and recurrent cancers However, despite the significant progress made

in the development of drug delivery system (DDS) and other based approaches, serious limitations have also been identified in applications of

nanoplatform-these therapies in vivo (Vinogradov and Wei, 2012) Several important

limitations of nanoparticles are highlighted (mostly liposomes (Table 2.4)), such

as ineffective uptake and distribution in tumor tissue, retention in bypassing organs and by macrophages of the reticuloendothelial system after systemic administration, and limited oral availability (Yun et al., 2012) Other nanovector systems such as synthetic biodegradable nanoparticles, polymer micelles, and

several others, are also hampered by drug leakage in vivo, lack of versatility in

terms packaging a diverse range of different drugs, thereby reducing drug potency, and difficulties in production scale-up (Ferrari, 2005) Despite the enhanced efficacy demonstrated by many targeted nanoparticles, they also face three major limitations: immunogenicity or non-specificity of the targeting ligand leading to accelerated blood clearance; further impaired tumor penetration compared to the nontargeted nanoparticles; and receptor-mediated endocytosis and subsequent lysosomal digestion resulting in a major dose loss by the lysosomal digestion (Chen et al., 2012)

Table 2.3 Advantages and disadvantages of liposome (Anwekar et al., 2011) Advantages of liposome Disadvantages of liposome

1 Liposomes increased efficacy and therapeutic

index of drug (actinomycin-D)

Low solubility

2 Liposome increased stability via

encapsulation

Short half-life

3 Liposomes are non-toxic, flexible,

biocompatible, completely biodegradable,

and immunogenic for systemic and

non-systemic administrations

Sometimes phospholipid undergoes oxidation and hydrolysis-like reaction

4 Liposomes reduce the toxicity of the

encapsulated agent (amphotericin B, Taxol)

Leakage and fusion of encapsulated drug/molecules

5 Liposomes help reduce the exposure of

sensitive tissues to toxic drugs

Production cost is high

7 Flexibility to couple with site-specific ligands

to achieve active targeting

Because problems continue to hamper significantly the success of cancer therapeutics, an urgent need exists for new targeted drug delivery strategies that will either selectively deliver drugs to tumor cells and target organs, protect normal tissues from administered antineoplastic agents, or prevent existing problems in cancer therapies The present invention relates to ongoing efforts to achieve a targeted drug delivery by means of intact bacterial minicells, which are

able to delivery drugs intracellular, within desired target cells in vivo and in vitro

(MacDiarmid et al., 2007a, 2007b, 2009) Minicells containing chemical or biochemical drugs constitute novel delivery vehicles, capable of being targeted to specific cells Because of the benefits of delivering chemotherapeutics drugs for cancer treatment, the practice of synthesizing and packagage of cytotoxic drug into minicells is the key point of this study The present study builds on these recent discoveries relating to minicells, and addresses the continuing needs for improved drug delivery strategies, especially in the context of cancer chemotherapy

2.2 BACTERIALLY-DERIVED MINICELLS

2.2.1 Minicells

Minicells were first observed and described by Howard Adler and colleagues in

1967, who also coined the term “minicell” with the first description of a mutation

that led to the minicell phenotype in Escherichia coli (Adler et al., 1967), and to

more accurately describe the particle, people propose the new term “nanocell‟ instead of “minicell” since the scale size of the vector is nanometer and is not in

the mini- or micro-range (MacDiarmid et al., 2007a) They are anucleate,

non-living nano-sized cells (100 – 400 nm in diameter) and are produced as a result

of mutations in genes that control normal bacterial cell division (de Boer et al., 1989; Lutkenhaus, 2007; Ma et al., 2004) there-by depressing polar sties of cell fission (Figure 2.3) The resultant minicells do not contain any of the original DNA and the chromosome present in its larger sister, but may contain all of the molecular components of the parent cell The minicells are capable of protein synthesis and normal metabolic functions but are incapable of undergoing further rounds of cell division Minicell formation has since been described in a number

of other Gram-positive and Gram-negative species First discovered over 70 years ago, minicells are becoming of interest to researchers in their potential as anti-tumor agents

Figure 2.22 Schematic showing minicell formation and bispecific

antibody-targeted, drug/siRNA-packaged minicells Minicells can be loaded with siRNAs (purple), shRNA (green) or chemotherapeutics (black) Loaded minicells are then functionalized via bispecific antibody conjugates, with one arm specific for minicell-surface O-polysaccharide (red) and the other specific for the tumor cell-surface receptor (blue) (Karagiannis and Anderson, 2009)

2.2.2 Bacterially-derived mincells as controlled drug delivery for cancer

therapy

Minicells are small, semi-spherical, bacterial nano-size particles that contain all

of the components of the parental bacteria, except chromosomes Without chromosomes, they cannot divide and are non-infectious, making them highly

suitable for development as in vivo delivery products So far, clever attempts at

delivering potent drugs straight to the cancer cells using techniques such as conjugating them to antibodies specific to those cells, have been inconclusive at best

The use of molecularly targeted minicell nanovectors affords multiple potential advantages over conventional cancer therapy (MacDiarmid et al., 2007a) Firstly, minicells possess the ability to easily package therapeutically significant concentrations of different cytotoxic or molecularly targeted drugs into the minicell, ability to encapsulate both hydrophilic and hydrophobic drugs Secondly, minicells have the ability to readily attach different bispecific antibodies (BsAbs) on the minicell surface in order to target a receptor found on the surface of a tumor cell Thirdly, minicells are able to deliver the drug intracellularly within a tumor cell and without leakage of drug/siRNA/shRNA from the vector during systemic circulation In addition, minicells also provide a dramatic increase in the therapeutic index with minimal to no toxic side effects This also enables the use of potent cytotoxics that have failed toxicity trails but have the potential to be highly potent anti-cancer drugs Moreover, minicells are

easily purified to homogeneity and the long standing pharmaceutical industry experience in bacterial fermentation and production of bacterial vaccines shows that such processes are relatively cheap The minicells nanovector has the potential to significantly reduce cost of goods particularly since a minicell-based anti-cancer therapeutic would carry tiny fractions of the drug and the targeting antibody compared to free drug or free antibody therapy Finally, obstacles in anticancer therapy such as multi-drug resistance of tumor can also be overcome via receptor-mediated endocytosis which triggered by the adhesion of the minicells to tumor-surface receptors, or via sequential minicell-mediated delivery

of siRNA followed by drugs (MacDiarmid et al., 2009)

2.2.4 Current research situation of minicells in delivering anti-cancer

drugs

Minicell has been observed and described in the studies on the bacteria cell division from long time ago However, it is actually noticeable in recent years as Himanshu Brahmbhatt, Jennifer MacDiarmid and colleagues firstly showed that report promising results using bacterial minicells as the drug delivery system in

2007 (MacDiarmid et al., 2007a) The unusual drug delivery vehicle was generated by inactivating genes that control normal cell division in bacteria This led to the formation of anucleate particles that have a uniform diameter of

400 nm, and high yields are readily produced from Gram-positive (Listeria monocytogenes (L monocytogenes)) and Gram-negative bacteria (Salmonella enterica serovar Typhimurium (S typhimurium), Escherichia coli (E coli), Shigella flexneri (S flesneri), and Pseudomonas aeruginosa (P aeruginosa))

These minicells were loaded with a range of therapeutically significant concentrations of chemotherapeutics (such as doxorubicin, paclitaxel, irinotecan, 5-flourouracil, cisplatin, carboplatin, and vinblastine) with differing charge, hydrophobicity and solubility by simple co-incubation in few hours Minicell has also been demonstrated that a minicell can be efficiently loaded with si/shRNA (MacDiarmid et al., 2009) The ability of minicells encapsulated a large number

of drug molecules (1- 10 million per minicell), and they loaded with stranded siRNA to an estimated density of nearly 12,000 molecules per minicell These minicells selectively targeted to cancer cells via BsAbs Cancer-cell targeting was achieved by coupling minicells to bispecific antibodies, in which one arm recognizes surface lipopolysaccharide (LPS), and the other a surface receptor on the targeted cell

double-In vivo experiments with targeted doxorubicin-loaded minicells led to the

dramatic inhibition and regression of tumour growth in mice that had human breast, ovarian, leukaemia or lung cancer xenografts Furthermore, the drug was undetectable in the plasma of minicell-treated animals, and none of the animals developed any signs of toxicity The anticancer efficacy of the minicells was further evaluated in dogs with advanced T-cell non Hodgkin‟s lymphoma; treatment led to marked tumour regression and tumour lysis Importantly, the treatment was tolerated without adverse side effects despite repeat dosing, and there was no increase in pro-inflammatory cytokines Further experiments in pigs confirmed no adverse reactions in terms of haematological indices, serum chemistries, food intake or growth, and surprisingly anti-LPS titers remained at background levels The team tested this using a form of siRNA designed to prevent the production of a protein that causes multi-drug resistance in cancer cells

Recently, an early phase clinical trial using the platform of minicell nanoparticle for drug delivery has been tested for the first time on patients with advanced cancer and found to be safe, well-tolerated and even induced stable disease in patients with advanced, incurable cancers with no treatment options remaining This clinical trial phase I was implemented by Associate Professor Benjamin Solomon at the Peter MaCallum Cancer Centre in Melbourne, Australia with colleagues In a Phase I trial, minicells were loaded with with a cytotoxic chemotherapy drug called paclitaxel and coated with cetuximab, antibodies that target the epidermal growth factor receptor (EGFR) which is often overexpressed

in a number of cancers, as a „homing‟ device to the tumor cells The treatment, code-named EGFRminicellsPac, was well tolerated, and of the 28 people treated,

10 had stable disease at 6 weeks, and one patient safely received 45 doses over

15 months (ECCO, 2012) This important study shows for the first time that these bacterially-derived minicells can be given safely to patients with cancer It thereby allows further clinical exploration of a completely new paradigm of targeted drug delivery using this platform coupled with different concentration of cell-killing drugs or other treatments such as RNA interference, and with different targeting antibodies

After all, the minicell technology is actually a platform for the targeted delivery

of many different molecules, including drugs and molecules for silencing rogue genes which cause drug resistance in late stage cancer The technology can also

be viewed as a powerful antibody drug conjugate where up to a million

molecules of drug can be attached to targeting antibodies and delivered to the body in a safe way In the future this will enable a truly personalized medicine approach to cancer treatment, since the minicell payload can be adjusted depending on the genetic profile of the patient Approaches resulting in selective delivery of anti-cancer drugs to tumour cells are highly interesting as it may lead

to a reduction in adverse side-effects and improved anti-tumour activity In this respect, the use of minicells is a novel and promising technique (ECCO, 2012)

2.3 AN OVERVIEW OF Lactobacillus SPECIES

2.3.1 The Genus Lactobacillus

2.3.1.1 General description of the genus

Taxonomically, the genus Lactobacillus belongs to the phylum Firmicutes, class Bacilli, order II Lactobacillales, and family Lactobacillaceae and its closest

relatives, being grouped within the same family, are the genera

Paralactobacillus and Pediococcus (Felis et al., 2009) This genus includes a high

number of GRAS species (Generally Recognized as Safe)

Species of genus Lactobacillus are some of the most important taxa involved in food microbiology and human nutrition: several Lactobacillus species are

remarkably essential in fermented food production and are used as starter cultures or food preservatives Moreover, certain strains of human origins are being exploited as probiotics or vaccine carriers (Goh and Klaenhammer, 2009) The lactobacilli strains can be used as potential candidates for cancer

prevention (Choi et al., 2006; Liu and Pan, 2010) A few particular strains of L acidophilus, L casei, L paracasei, L johnsonii, L reuterui, L salivarius and L rhamnosus have been extensively studied as candidates of probiotics and their

functional properties and safety have been well-documented

2.3.1.2 The important role of Lactobacillus species in human life

Many Lactobacillus species are associated with food production, because of

preservative action due to acidification, and/or enhancement of flavour, texture

and nutrition (Jay, 1996; Stiles, 1996) Members of the genus Lactobacillus are

commonly present as members of probiotics (termed as living microorganisms that are associated with the beneficial effects for humans and animals)

Lactobacilli strains have been used to study the anti-oxidative activity,

antibacterial and anti-cancer discovery besides being effect on human immune system This genus evaluated the inhibitory effects of on various human cancer

cell lines and attempted to demonstrate whether such effects were cancer cell

selective (Choi et al., 2006) The inhibition of cancer cell growth by Lactobacillus

has also been reported in other studies (Kim et al., 2002) Antioxidant activities

and antiproliferative activities against breast and colon cancer cell lines in vitro

gave a strong evidence of possessing significant anti-cancer activities of several local lactobacilli strains (Liu and Pan, 2010) Short chain fatty acids produced by

L acidophilus, L rhamnosus are reported to inhibit the generation of

carcinogenic products by reducing enzyme activities (Cenci et al., 2002) These

results suggest that Lactobacillus can be used as adjuncts in fermentation of

food and are potential candidates for cancer prevention

2.3.2 Lactobacillus acidophilus

Among many Lactobacillus species, L acidophilus is likely the most common

probiotics for dietary use (Parvez, 2006) In addition to its gram positive rod

shape with rounded ends, the typical size of L acidophilus is 1.5 - 6.0 µm in length L acidophilus is an obligately homofermentative LAB that growth in

anaerobic condition Because they utilize sugars (e.g glucose, aesculin, cellobiose, galactose, lactose, maltose, salicin, and sucrose) as their substrates for fermentation, they inhabit environments with high sugar abundance, such as the GI tract in humans and animals (Vijayakumar et al., 2008)

Health benefits of L acidophilus include providing immune support for infections

or cancer, providing a healthy replacement of good bacteria in the intestinal tract following antibiotic therapy, reducing occurrence of diarrhea in humans, aiding in lowering cholesterol and improving the symptoms of lactose intolerance

Moreover, some of L acidophilus cell components have potential use in many

different areas of biotechnology such as vaccine development (Jafarei and Ebrahimi, 2011) It is demonstrated that exoploysaccharide (EPS) from bacteria

specially Lactobacillus sp may contribute to human health, either as

non-digestible food fraction or because of their anti-tumoral, anti-ulcer, immunomodulating or cholesterol lowering activity (Ganesh, 2006; Vuyst and Degeest, 1999) EPS has anti-carcinogenic ability mediated by the stimulation of the mitogenic activity of B lymphocytes (Ruas-Madiedo et al., 2006; Xu et al., 2010)

2.3.3 Lactobacillus rhamnosus

Lactobacillus rhamnosus GG is a clinically documented bacterial strain which is

used in many countries as a probiotic culture in different dairy products or in

pharmaceutical diet supplements (Korpela et al 1997) L rhamnosus GG is a rod

shaped, Gram-positive, with 2.0-4.0 µm in length, and often with square ends, and occur singly or in chains This bacterium is considered safe (GRAS)

microorganism L rhamnosus GG is a homofermentative LAB (Berry et al 1997)

Lactobacillus rhamnosus has shown antimicrobial activity against Escherichia coli, Enterobacter aerogenes, Salmonella typhi, Shigella sp., Proteus vulgaris, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Bacillus subtilis, Bacillus megaterium, Bacillus cereus, Helicobacter pylori, Campylobacter jejuni, and Listeria monocytogenes (Ambalam et al., 2009) L rhamnosus are

reported to inhibit the generation of carcinogenic products by reducing enzyme activities (Cenci et al., 2002)

2.4 PACLITAXEL

Among the available drugs for chemotherapy, paclitaxel (Taxol®) is one of the best anti-cancer drugs and also reported to possess radio-sensitizer properties Paclitaxel is a white to off-white crystalline powder with empirical formula of

C47H51NO14 and a molecular weight of 853.91 It is highly lipophilic, insoluble in water, and melts at around 216-217°C It is a complex, oxygen-rich diterpenoid (Rowinsky and Donehower, 1995; Rowinsky et al., 1992) and its chemical structure has been elucidated by chemists as in Figure 2.5 It consists of some benzene rings and other hydrophobic structures, which lead to its high water insolubility of paclitaxel

Figure 2.43 Chemical structure of paclitaxel

The major limitation of paclitaxel is also the obstacle of chemotherapy, drug resistance in the mucosa of the small and large intestine which limits the oral uptake of paclitaxel and mediates direct excretion of the drug in the intestinal lumen (Adams et al., 1993) Paclitaxel has been recognized as the most potent anticancer agent for the past few decades However, its use as an anti-cancer drug is compromised by its intrinsically poor water solubility The effective chemotherapy using paclitaxel is relying on the development of new delivery systems which attracted a substantial number of studies investigated to deliver paclitaxel by new formulations

2.5 ORIENTATION OF IMPLEMENTING TO DELIVERY PACLITAXEL

USING MINICELLS DERIVED FROM THE LACTOBACILLUS STRAINS

It is clear that bacterially-derived minicells proved successfully their ability to encapsulate a range of different chemotherapeutic drugs, target to the specific tumor cells, and inhibit the growth of tumour in animal and human in the first clinical trial Thanks to their proven benefits and the minicells applications as nanovectors for drug delivery in cancer therapy, it is necessary to develop new molecularly targeted drug delivery systems from bacteria Currently, although minicells generated from both Gram-positive and Gram-negative bacteria and also tested for encapsulating chemotherapeutic drugs and functioning as nanovectors for drug delivery in cancer therapy, the minicells were prepared from genetically defined minCED(-) chromosomal deletion bacteria and then the subsequent minicells were purified (MacDiarmid et al., 2007b) This deletion of minCED(-) out of the bacteria cell may affect on their growth under their control

so far (de Boer et al., 1989) Up to now, there has not any study on generating

bacterially-minicells from Lactobacillus strains, or not any research on the drug

delivery of this genus, one kind of largest genus within the group of lactic acid bacteria (LAB) with their properties as probiotics

According to the principle of bacteria cell division, the normal generation of two equally sized daughter cells are maintained by the regulatory protein system

(Min system) which encoded by the min locus (consists of three genes, minC, minD, minE that encode three proteins, designated MinC, MinD, and MinE, respectively in E coli) Abnormalities this regulatory system for cytokinesis

results in the aberrant division near a cell pole, leading to the formation of small spherical minicells and short filaments Recently, there were studies on detection

the overexpression and characterization of minC homolog from Lactobacillus acidophilus (Nguyen et al., 2012), minD from L acidophilus and L rhamnosus

(Nguyen et al., 2013a, 2013b) These studies showed the role and function of

min genes from Lactobacillus strains in cell division and minicell formation The results of these studies also reported an interesting overexpression of E.coli carrying min genes of Lactobacillus strains showed the phenomenon of cell

differentiation under different sugar stresses

From above stated points, this paper presented the investigation about the

generation minicells from the rod Lactobacillus strains under different sugar

stresses Then, minicells were used for drug delivery by packaging drugs

2.6 PROCESS OF PROJECT

Screening the carbon source for culturing

minicell – generating Lactobacillus strains

Minicell production from Lactobacillus

CHAPTER 3 MATERIAL AND METHODS

3.1 MATERIALS

3.1.1 Bacterial strains

Lactobacillus acidophilus VTCC-B-871 obtained from stock cultures maintained

by the Vietnamese Type Culture Collection (Hanoi, Vietnam)

Lactobacillus rhamnosus JCM 15113 was kindly provided by the Japanese

Collection Microorganism (Japan)

Indicator microorganisms for antimicrobial activity tests were supported from

American Type Culture Collection (Manassas, USA), including: Staphylococcus aureus (S aureus) ATCC 25923, Escherichia coli (E coli) ATCC 9637, Salmonella typhimurium (S typhi) ATCC 19430, Pseudomonas aeruginosa (P aeruginosa) ATCC 27853, and Candida albicans (C albicans) ATCC 14053

3.1.2 Tools and equipments

 Light Microscope

 Filter vacuum system

 Scanning Electron Microscope (SEM) JSM-7401F (Jeol, USA)

 The High Performance Liquid Chromatography (HPLC) system (Shimadzu, Japan)

 Chemotherapeutic drug – Paclitaxel T7402 (microcrystalline powder,

>99.5% purity) (Sigma Aldrich, USA)

 D-Glucose (Merck, Germany)

 D-Fructose (Merck, Germany)

 Lactose (Merck, Germany)

 Maltose (Merck, Germany)

 Saccharose (Merck, Germany)

 Antibiotic – Cephalosporin C3270 (Sigma Aldrich, USA)

 Gram staining kit (Sigma Aldrich, USA)

3.1.3.2 Carbohydrate fermentation testing kit

 API 50 CHL Medium (BioMérieux, USA)

3.1.3.5 Culture media

Basic culture for bacteria growth

Both strains of Lactobacillus were grown in Lactobacilli MRS broth (De Man et al.,

1960)

Table 3.1 Lactobacilli MRS broth (De Man et al., 1960)

Dipotassium hydrogen phosphate (K2HPO4) 2.0 g

Manganese sulfateTetrahydrate (MnSO4 4H2O) 0.05 g

Magnesium sulfate Heptahydrate (MgSO4 7H2O) 0.2 g

Modified culture for study

The modified MRS media were prepared by modifying carbon source in MRS ingredients (detail in sub-section 3.2.2)

different concentration in the culture medium Lactobacillus acidophilus

VTCC-B-871 and Lactobacillus rhamnosus JCM 15113 were inoculated into modified

Lactobacilli MRS broth which containing each of carbon sources separately: glucose, lactose, sucrose, maltose, fructose, in different concentration (0 g/l, 5 g/l, 10 g/l, 15 g/l, 20 g/l, 30 g/l, 40 g/l, 50 g/l) and incubated at 37ºC for 48 hr

in order to produce minicells

3.2.2 Minicell isolation (Brahmbhatt and MacDiarmid, 2004)

Minicell-producing bacteria were subjected of the minicell isolation for free of contaminating parent bacterial cells, cellular debris

Figure 31 Process of isolation of bacterially-derived minicells from the parent

at 2000 rpm for 20 min

First filtration through 0.45

µm filter

Second filtration through 0.45

µm filter

Filtration through 0.2

µm filter

Filtration through 0.45

µm filter Minicells

collected

(eq 3.1)

3.2.3.2 Scanning electron microscopy

Morphology of the isolated minicells were observed by scanning electron

microscopy (SEM, Jeol JSM-7401F) at 10-15kV Samples were examined at

Scanning Electron Microscopy Laboratory Room, Vietnam Academy of Science

and Technology, 1 Mac Dinh Chi Street, 1 District, Ho Chi Minh City to

investigate morphological minicells

3.2.4 Packaging of drug into minicells

3.2.4.1 Packaging of the cancer chemotherapeutic drug (Paclitaxel) into

minicells (MacDiarmid et al., 2007b)

To determine if minicells can be packaged with chemotherapeutic drug

(Paclitaxel) and the kinetics of minicell drug packaging in response to varying

concentrations of drug in the loading solution or varying times of incubation, the

following methods were adopted Preparations of purified minicells derived from

LAB strains were separately incubated in a solution of the paclitaxel drug, at

different final paclitaxel concentrations in minicell extracellular environment of 5,

10 and 20 µg/ml The mixtures were incubated at 37°C with rotation Minicells

had been centrifuged at 13000 rpm for 15 min before were washed thoroughly

with ten exchanges of buffered saline gelatin (BSG) solution Drug was then

extracted from packaged minicells to prepare for drug qualification and

quantitation using HPLC-UV/Vis Spectroscopy system (detailed in sub-section

3.2.8)

For minicells depending upon the times of incubation in the loading solution,

minicells were seperately incubated with paclitaxel (10 µg/ml) for the time was

shown 10, 15 and 24 hours with rotation The minicell suspensions incubated

with paclitaxel were then centrifuged at 13000 rpm for 15 min The supernatants

were used to analyze for the paclitaxel remaining using HPLC-UV/Vis

Spectroscopy system (detailed in sub-section 3.2.8) Drug was extracted from

packaged minicells to prepare for testing antimicrobial acitivity (in sub-section

3.2.7.)

3.2.4.2 Packaging of antibiotic (cephalosporin) into minicells

To assist in determining the ability of the drug-packaged minicell, this study conducted on an experiment to test the encapsulation of antibiotic drug Cephalosporin into minicells Minicells were seperately incubated with cephalosporin (10 µg/ml) at 10, 15 and 24 hours with rotation The minicell suspensions incubated with cephalosporin was then centrifuged at 13000 rpm for

15 min Drug was extracted from packaged minicells to prepare for testing antimicrobial acitivity (detailed in sub-section 3.2.7.)

3.2.5 Drug extraction for measurement (MacDiarmid et al., 2007b)

The minicells which incubated with drugs (paclitaxel, celphalosporin) were harvested by centrifugation at 13000 rpm for 15 min and re-suspended in sterile BSG and washed 10 times with BSG The minicells were collected and prepared for drug extraction

The minicells were centrifuged at 13000 rpm for 15 min, and supernatant was discarded The phosphate-buffered saline (PBS) was added to re-suspend pellet, followed by five cycles of 1 minute vortexing and 1 minute sonicating The samples were diluted with an equal volume of ultrapure water and the five cycles were repeated The extracts were finally centrifuged for 5 min at 13000 rpm to pellet debris, thus yielding cell-free filtrates

3.2.6 Antimicrobial activity tests

Antimicrobial effects were tested on Staphylococcus aureus (S aureus) ATCC

25923, Escherichia coli (E coli) ATCC 9637, Salmonella typhimurium (S typhi) ATCC 19430, Candida albicans (C albicans) ATCC 14053, Pseudomonas aeruginosa (P aeruginosa) ATCC 27853 by the agar diffusion method The

tested microorganisms were propagated twice and then grown for 18 to 24 hr in

10 ml of appropriate growth media Sterile paper discs (5 mm of diameter) were then prepared and dropped on using 20 μl of cell-free filtrate The extracted solution from minicell suspension without drugs incubation was used as the control The inoculated plates were incubated for 18 to 24 hr at appropriate temperatures, and the diameter of the inhibition zone was measured in millimeters with calipers

3.2.7 Paclitaxel assay using HPLC-UV/Vis Spectroscopy

Extracted paclitaxel from packaged minicells (incubated in different final concentrations of 5, 10 and 20 µg/ml of paclitaxel) and supernatants from

paclitaxel-incubated minicell suspensions (shown at different time of incubation

10, 15 and 24 hr) were quantified and qualified by HPLC-UV/Vis Spectroscopy analyses Samples were practiced at Department of Reference Substances, Institute of Drug Quality Controls, 200 Co Bac, Co Giang ward, 1 District, Ho Chi Minh City, Vietnam

According to the linear standard equation, the concentrations of paclitaxel in the minicells and in supernatants were measured (see Appendix K)

In order to identify the presence of paclitaxel, the variation between paclitaxel peaks of samples and standard were calculated as following equation (eq 3.2)

Where,

V (%) is the variation

Rp is the retention time of sample paclitaxel

Rst is the retention time of standard

The acceptable range for the variation of retention time was less than 5%

3.2.8 Calculation of the number of drug molecules (MacDiarmid et al.,

2007b)

The number of molecules of drug (paclitaxel) packaged per minicell is calculated

as follows

Where,

MW is a molecular weight of paclitaxel

Avogadro number of molecules is 6.02 x 1023

The number of paclitaxel molecules present in the loading solution (NPL) is expressed as:

NPM is the number of paclitaxel molecules per minicells

Encapsulation efficiency (EE %) is defined as:

(eq 3.5)

(eq 3.6)

CHAPTER 4

RESULTS AND DISCUSSION

4.1 Screening the carbon sources for study on minicell generation from

Lactobacillus strains

In order to decide which sugars would be related to the investigation of the

effect of carbon sources on the minicell formation, Lactobacillus acidophilus and Lactobacillus rhamnosus were used to test the carbohydrate fermentation using

API 50CHL kit (BioMerieux) During fermentation of carbohydrates, the acid generated and decreased the pH by the change in color of the indicator; the positive tests corresponded to acidification revealed by the indicator contained in the medium changing to yellow

Figure 4.1, 4.2 and Table 4.1, 4.2: sugar (glucose, fructose, maltose, lactose, saccharose) fermentations showed the yellow change It meant that those

sugars had high impact on L acidophilus, and L rhamnosus In order to define the effect of sugars as carbon sources on cell differentiation of L acidophilus and

L rhamnosus, the research on screening the carbon sources for minicell generation from Lactobacillus strains was implemented

Figure 4.1 The API 50CHL biochemical testing of Lactobacillus acidophilus

VTCC-B-871; Positive tests corresponded to acidification revealed by the purple indicator contained in the medium changing to yellow

Table 4.1 The API 50CHL biochemical testing of

Figure 4.2 The API 50CHL biochemical testing of Lactobacillus rhamnosus

JCM 15113; Positive tests corresponded to acidification revealed by the purple indicator contained in the medium changing to yellow; For the Esculin test (tube no 25), a change in color from purple to black is observed

Table 4.2 The API 50CHL biochemical testing of

Strip No Substrate Isolate

4.1.1 Minicells generation in Lactobacillus acidophilus VTCC-B-871

Lactobacillus acidophilus VTCC-B-871was cultured in selected sugars, as glucose,

lactose, sucrose, maltose, fructose, with different concentrations (0 g/l, 5 g/l, 10 g/l, 15 g/l, 20 g/l, 30 g/l, 40 g/l, 50 g/l) The minicells from each culture were filtered through 0.45 µm The minicells were collected and calculated The results were summarized in Table 4.3 and Figure 4.3 The results of statistical analysis also identified that carbon sources influenced significantly on the minicell formation and the minicells were generated depending on the sugar concentrations (p < 0.05) It was clear from Table 4.3 that minicell generation

was not changed when L acidophilus was grown in the modified MRS media

without containing sugar The number of minicells obtained was always about 60,000 particles This was the lowest level of producing minicells The data presented that at all levels of factor treatment with sugars (from 5 g/l to 50 g/l), fructose was the carbon source by means of which the maximum number of minicells was obtained It was followed by the quantity of minicells which were generated in the maltose medium Lactose and glucose had similar levels of

production of minicells, such as between 270,000 to 780,000 particles each Through the usage of saccharose, the minimum number of minicells was obtained Since the fructose concentration changed in the culture medium from 5 g/l to 50 g/l, the number of obtained minicells in the medium containing fructose fluctuated between 378,000 particles and 1,070,000 particles These numbers were two and a half times as much as the number of obtained minicells in the medium containing saccharose (fluctuated between 128,000 and 417,000 particles) at the same range of sugar concentration As presented in Table 4.3,

the number of obtained minicells was increased considerably when L acidophilus

was grown in the modified MRS media containing concentrations from 5 g/l to 10 g/l for all tested sugars At level of factor treatment of 10 g/l, the generated minicells were the highest number at each of kind of sugar In modified MRS medium with glucose, lactose, fructose, maltose, saccharose (10 g/l) the minicells obtained were about 662,000; 783,000; 1,070,000; 885,000, and 417,000 particles, respectively The minicells obtained were decreased at high concentrations of sugars These numbers were reduced (2.5 times) to 273,000; 310,000; 378,000, 352,000, and 128,000 particles when the concentration of glucose, lactose, fructose, maltose, saccharose increased (10 times) to 50 g/l, respectively (Table 4.3) At the concentration of 20 g/l for each of sugars (including glucose in the standard MRS medium), the number of minicells was only a half of minicells producing in the culture media with 10 g/l of each

As a result, the generation of minicells reached their maximum (over one million particles) when using fructose as carbon source in a concentration of 10 g/l Consequently, fructose may not be suitable for the cell growth However, this was the conventional condition in order to produce minicells Saccharose effects

on the minicell phenotype of the bacteria were the least (about 417,000 particles

at concentration of 10 g/l in culture medium) In fact, during minD expression,

saccharose led the filamentation in cells (Nguyen et al., 2013a) By statistical analysis, the minicell in fructose condition showed highly significant than the other sugars Consequently, fructose (10 g/l) was used for minicell production in drug delivery of the study