Thông tin tài liệu
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
chromatography.
i
activities,
high
performance
liquid
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.
ii
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
iii
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 5 CONCLUSION AND RECOMMENDATION ........................ 49
REFERENCES ................................................................................... 50
iv
LIST OF FIGURES
Figure 2.1 Nanoparticle platforms for drug delivery. ..................................... 8
Figure 2.2 Schematic showing minicell formation and bispecific antibodytargeted, 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-B871with 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
v
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
vi
ACRONYMS AND ABBREVIATIONS
ATCC
American Type Culture Collection
BsAbs
Bispecific Antibodies
BSG
Buffer Saline Gelatin
CSCs
Cancer Stem Cells
DDSs
Drug Delivery Systems
EGFR
Epidermal Growth Factor Receptor
EPS
Exoploysaccharide
FDA
Food And Drug Administration
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
LAB
Lactic Acid Bacteria
NCI
National Cancer Institute
PBS
Phosphate Buffer Saline
SEM
Scanning Electron Microscope
shRNA
Short Hairpin RNA
siRNA
Small interfering RNA
VTCC
Vietnamese Type Culture Collection
vii
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, doselimiting 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 Grampositive (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
1
(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 anti-oxidative, anticarcinogenic, 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.
2
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)
20301
2008
20302
Region
Cases
Deaths
Cases
Deaths
Cases
Deaths
World
12.4
7. 6
20.0
12.9
26.4
17.0
Africa (AFRO)
0.7
0.5
1.2
0.9
1.6
1.3
Europe (ERO)
3.4
1.8
4.1
2.6
5.5
3.4
East Mediterranean (EMRO)
0.5
0.3
0.9
0.6
1.2
0.9
Pan-America (PAHO)
2.6
1.3
4.8
2.3
6.4
3.1
South-East Asia (SEARO)
1.6
1.1
2.8
1.9
3.7
2.6
Western Pacific (WPRO)
3.7
2.6
6.1
4.4
8.1
5.9
¹ no change in current rates
² with 1% annual increase in rates
3
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
al., 2010a).
Vietnam
Male
Female
Both sexes
Population (thousands)
42973
44122
87095
Number of new cancer cases (thousands)
55.0
56.5
111.6
15.9
12.8
14.1
43.7
38.3
82.0
12.7
8.8
10.5
Risk of getting cancer before age 75 (%)
Number of cancer deaths (thousands)
Risk of dying from cancer before age 75 (%)
5 most frequent cancers (ranking defined by total number of cases)
Liver
Liver
Liver
Lung
Lung
Lung
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).
4
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 Obstacles in cancer therapy and tumor-targeted drug delivery
systems
There are several serious obstacles in cancer therapy including drug resistance,
high tumor interstitial fluid pressure (IFP), and cancer stem cells (CSCs).
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
5
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
6
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).
7
Polymeric
Nanoparticle
Dendrimer
Liposome
Polymeric
Micelle
Inorganic
Nanoparticle
Polymerosome
Protein
Carrier
Biological
Nanoparticle
Polymer-drug
Conjugate
Hybrid
Nanoparticle
Hydrophobic Polymer
Therapeutic load
Hydrophilic Polymer
Targeting Ligand
Lipid
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 anticancer 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 drug8
resistant and recurrent cancers. However, despite the significant progress made
in the development of drug delivery system (DDS) and other nanoplatformbased approaches, serious limitations have also been identified in applications of
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
Low solubility
index of drug (actinomycin-D)
2. Liposome increased stability via
Short half-life
encapsulation
3. Liposomes are non-toxic, flexible,
Sometimes phospholipid
biocompatible, completely biodegradable,
undergoes oxidation and
and non-immunogenic for systemic and non-
hydrolysis-like reaction
systemic administrations
4. Liposomes reduce the toxicity of the
Leakage and fusion of
encapsulated agent (amphotericin B, Taxol)
5. Liposomes help reduce the exposure of
encapsulated drug/molecules
Production cost is high
sensitive tissues to toxic drugs
6. Site avoidance effect
Fewer stables
7. Flexibility to couple with site-specific ligands
to achieve active targeting
9
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, nonliving 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.
10
Figure 2.22 Schematic showing minicell formation and bispecific antibodytargeted, 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
11
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 doublestranded 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.
12
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
13
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
14
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 nondigestible
food
fraction
or
because
of
their
immunomodulating or cholesterol lowering activity
anti-tumoral,
anti-ulcer,
(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
15
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
16
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
17
(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
strains
Minicells isolation
Packaging of paclitaxel into minicells
Drugs extraction
Antimicrobial activity tests of extracted
drugs
Confirmation the packaging ability with
cephalosporin
Drug (paclitaxel) quantification and data
analysis
18
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)
Neubauer hemocytometer
Syringe filters, sterilized filter membranes (pore size of 0.2 µm and
0.45 µm) (Whatman, England)
3.1.3 Media and chemicals
3.1.3.1 Chemicals
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)
19
Gram staining kit (Sigma Aldrich, USA)
3.1.3.2 Carbohydrate fermentation testing kit
API 50 CHL Medium (BioMérieux, USA)
3.1.3.3 Buffer preparation
Phosphate buffer saline (PBS, pH 7.4) and buffer saline gelatin (BSG, pH 7.0)
can be referred from Appendix A.
3.1.3.4 Drug preparation
Paclitaxel stock solution was prepared by dissolving completely in ethanol
absolute to give a 1 mg/ml stock solution, then diluted 1:10 (vol/vol) in PBS
buffer to give 100 µg/ml stock solution. Cephalosporin was dissolved totally in
distilled water to perform a 1 mg/ml stock solution, and then diluted 1:10
(vol/vol) in distilled water to give 100 µg/ml stock solution.
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)
Ingredients
Formula per Liter
Peptone
10.0
g
Meat extract
10.0
g
Yeast extract
5.0
g
20.0
g
Tri-ammonium citrate
2.0
g
Sodium acetate
5.0
g
Dipotassium hydrogen phosphate (K2HPO4)
2.0
g
Manganese sulfateTetrahydrate (MnSO4. 4H2O)
0.05
g
Magnesium sulfate Heptahydrate (MgSO4. 7H2O)
0.2
g
Tween 80
1.0
ml
D-Glucose
Modified culture for study
The modified MRS media were prepared by modifying carbon source in MRS
ingredients (detail in sub-section 3.2.2).
20
3.2 RESEARCH METHODOLOGY
3.2.1 Design condition for minicells production from
Lactobacillus
strains
In order to study the influence of various carbon sources on the minicell
formation, this study implemented experiments on different kinds of sugar with
different concentration in the culture medium. Lactobacillus acidophilus VTCC-B871 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.
Overnight
culture of
minicell
producing
bacterial cells
Differential
centrifugation
at 2000 rpm
for 20 min
First filtration
through 0.45
µm filter
Filtration
through 0.45
µm filter
Minicells
collected
Second
filtration
through 0.45
µm filter
Filtration
through 0.2
µm filter
Figure 31 Process of isolation of bacterially-derived minicells from the parent
cells culture.
3.2.3 Microscopic studies for characterization of minicell morphology
3.2.3.1 Light microscopy
Isolated
minicells
were
observed
for
scanning
the
microscopic
minicell
morphologies and cell counting using a Neubauer hemocytometer under a light
microscopy with a total magnification of 100 X. Minicells were counted in five
small squares (the four 1/25 sq. mm corners plus the middle square) in the
central area into focus at low power. The numbers of obtained minicells per unit
volume of a suspension was calculated as following equation (3.1).
21
(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.).
22
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,
aeruginosa (P.
Candida albicans (C.
albicans) ATCC 14053, Pseudomonas
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
23
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).
(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:
(
(eq. 3.3)
)
Where,
NPL is the number of paclitaxel molecules present in the external medium
IPW is the initial amount of paclitxel loaded
The number of Paclitaxel molecules present in the total minicells (NPT) is defined
as:
(
(eq. 3.4)
)
24
Where,
NPT is the number of Paclitaxel molecules present in the total minicells
EPW is the amount of Paclitaxel encapsulated in the minicells (determined
by HPLC analysis)
Therefore, the number of molecules of drug per minicell (NPM) can be calculated
as follows:
(eq. 3.5)
Where,
NPM is the number of paclitaxel molecules per minicells
Encapsulation efficiency (EE %) is defined as:
(eq. 3.6)
3.2.9
Data analysis
Results are expressed as means ± standard deviations of triple replicates for all
treatments. Data was analyzed using one-way ANOVA followed by post-hoc
Tukey‟s test for paired comparisons of means, with p < 0.05 being considered
statistically significant (SPSS 16, SPSS Inc., Chicago, USA). Data had also been
tested for homogeneity of variances using Levene statistic (with p > 0.05, the
Levene's test showed homogeneity of variance).
25
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.
26
Table
4.1
The
API
50CHL
biochemical
testing
Lactobacillus acidophilus VTCC-B-871
Strip No.
Substrate
Isolate
0
Control
negative
1
Glycerol
negative
2
Erythritol
negative
3
D-Arabinose
negative
4
L- Arabinose
negative
5
Ribose
negative
6
D-Xylose
negative
7
L-Xylose
negative
8
Adonitol
negative
9
Β Methyl-D-Xyloside
negative
10
Galactose
positive
11
Glucose
positive
12
Fructose
positive
13
Mannose
positive
14
Sorbose
negative
15
Rhamnose
negative
16
Dulcitol
negative
17
Inositol
negative
18
Mannitol
negative
19
Sorbitol
negative
20
α-Methyl-D-Mannoside
negative
21
α-Methyl-D-Glucoside
negative
22
N-Acetyl-Glucosamine
positive
23
Amygdalin
positive
24
Arbutin
positive
25
Esculin
negative
26
Salicin
positive
27
Cellobiose
positive
28
Maltose
positive
29
Lactose
positive
30
Melibiose
negative
31
Sucrose
positive
27
of
32
Trehalose
positive
33
Inulin
negative
34
Melezitose
negative
35
Raffinose
negative
36
Starch
negative
37
Glycogen
negative
38
Xylitol
negative
39
Gentiobiose
positive
40
D-Turanose
negative
41
D-Lyxose
negative
42
D-Tagatose
negative
43
D-Fucose
negative
44
L-Fucose
negative
45
D-Arabitol
negative
46
L- Arabitol
negative
47
Gluconate
negative
48
2-Keto-Gluconate
negative
49
5-Keto-Gluconate
negative
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 28
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
Lactobacillus rhamnosus JCM 15113
testing
Strip No.
Substrate
Isolate
0
Control
negative
1
Glycerol
negative
2
Erythritol
negative
3
D-Arabinose
negative
4
L- Arabinose
negative
5
Ribose
positive
6
D-Xylose
negative
7
L-Xylose
negative
8
Adonitol
negative
9
Β Methyl-D-Xyloside
negative
10
Galactose
positive
11
Glucose
positive
12
Fructose
positive
13
Mannose
positive
14
Sorbose
positive
15
Rhamnose
positive
16
Dulcitol
negative
17
Inositol
negative
18
Mannitol
positive
19
Sorbitol
positive
20
α-Methyl-D-Mannoside
negative
21
α-Methyl-D-Glucoside
negative
22
N-Acetyl-Glucosamine
positive
23
Amygdalin
positive
24
Arbutin
positive
25
Esculin
positive
26
Salicin
positive
27
Cellobiose
positive
28
Maltose
positive
29
Lactose
positive
30
Melibiose
negative
31
Sucrose
positive
29
of
Strip No.
Substrate
Isolate
32
Trehalose
positive
33
Inulin
negative
34
Melezitose
positive
35
Raffinose
negative
36
Starch
negative
37
Glycogen
negative
38
Xylitol
negative
39
Gentiobiose
positive
40
D-Turanose
negative
41
D-Lyxose
positive
42
D-Tagatose
positive
43
D-Fucose
negative
44
L-Fucose
negative
45
D-Arabitol
negative
46
L- Arabitol
negative
47
Gluconate
positive
48
2-Keto-Gluconate
negative
49
5-Keto-Gluconate
negative
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
30
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.
31
Table 4.3 The number of minicells produced from Lactobacillus acidophilus VTCC-B-871 in modified MRS medium
containing each kind of selected sugar separately in different concentrations.
Sugar concentrations (g/l)
Selected
sugars
0a, b
5a, b
10a, b
15a, b
20a, b
30a, b
40a, b
50a, b
Glucose
6.03±0.25
41.1±2.71
66.2±3.35
38.8±2.55
35.4±4.78
33.6±4.28
31.2±4.42
27.3±2.95
Lactose
6.04±0.23
48.2±3.22
78.3±3.17
44.8±3.55
36.7±2.98
35.8±3.24
34.5±2.76
31.0±3.00
Fructose
6.07±0.20
81.0±2.59
107±6.03
69.0±3.74
56.3±2.60
41.9±4.19
38.7±2.52
37.8±3.36
Maltose
6.04±0.20
64.6±3.00
88.5±2.90
49.4±4.40
42.1±3.23
38.7±3.15
36.1±3.47
35.2±3.45
Saccharose
6.05±0.16
36.6±3.96
41.7±2.81
23.4±2.80
17.3±2.22
14.5±3.02
13.2±2.46
12.8±2.21
b
Data are means ± standard deviations of triplicates for all treatments
Expressed ×104
The number of minicells
(×104 particles)
a
Glucose
Lactose
Fructose
Maltose
Saccharose
120
100
80
60
40
20
0
0
5
10
15
20
30
Sugar concentrations (g/l)
40
50
Figure 4.3 The number of minicells (×104) produced from Lactobacillus acidophilus VTCC-B-871 in
modified MRS medium with differently 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).
Data represented means ± SD, n=3.
32
The number of obtained minicells after filtration was 1.07×106 particles from 100
ml starting culture with the density of cells before filtration about 2.57×1011
cells. It meant that every four million of parent cells, there was one minicell. In
comparison with previous studies on other bacteria, a yield of approximately 1011
minicells from a 6 L starting culture being routinely obtained (MacDiarmid et al.,
2007b), these showed that the number of obtained minicells in this study was
not as high as expected. However, the techniques were quite simple in
comparison with other studies on the world about filter system. Moreover, the
minicells were usually formed by mutation during cell division whereas this study
utilized carbon sources (sugars) as factors that effect on minicell generation in
order to produce minicells with the normal properties of L. acidophilus.
Remarkably, this study was the first research on minicell production from
Lactobacillus strains using modified culture medium. In conclusion, the obtained
minicells in this study were checked for the packaging of drug paclitaxel and
confirmation by packaging other drug as cephalosporin in the next sections of
this study.
The Figure 4.4 illustrated the minicell formation at the end of L. acidophilus. As
above analysis, this phenomenon was not caused by cell division. The reason
was the effect of fructose on the cell development and differentiation. Moreover,
the practice of formation of single or pair in rod of L. acidophilus (Figure 4.4A),
gave advantages for minicell producing at the pole end and that was convenient
for purification (Figure 4.4B).
A
B
Figure 4.4 Photomicrographs of Lactobacillus acidophilus VTCC-B-871 and its
minicells (100X): A) the morphology of L. acidophilus in basic MRS medium;
B) the formation of minicells (black arrow) in the modified MRS medium with
fructose 10 g/l.
33
4.1.2 Minicells generation from Lactobacillus rhamnosus JCM 15113
According to the results of biochemical tests of Lactobacillus rhamnosus JCM
15113 (Table 4.2 and Figure 4.2), the different sugars (glucose, lactose,
sucrose, maltose, and fructose) were also used to study for testing the effect on
minicell generation from L. rhamnosus (Appendix C). All analyzed data was
shown in Table 4.4 and Figure 4.5. The statistical analysis identified that carbon
sources influenced significantly on the minicell formation and the minicells were
generated from L. rhamnosus depending on the sugar concentrations (p < 0.05).
As presented in Table 4.4, the lowest level of producing minicells was found
about 34,000 particles when L. rhamnosus was grown in the modified MRS
media without containing sugar. The data presented that the number of obtained
minicells increased gradually since raising the concentration of sugars (glucose,
lactose, and maltose) from 5 g/l to 50 g/l for all tested sugars. On the other
hand, in the fructose and saccharose conditions, production of minicells
increased at low concentration of these sugars (from 5 g/l to 10 g/l) and then
reduced at high concentration (from 15 g/l to 50 g/l).
According to Table 4.4, the
number
of
obtained minicells
reached
their
maximum when using lactose as carbon source (from 146,000 to 787,000
particles). In the maltose medium, the quantity of minicells was lower than a
half by comparison with lactose condition (from 98,700 to 351,000 particles).
Through the usage of glucose, the number of minicells was a half of those
numbers in maltose condition. Fructose and saccharose had similar levels of
production of minicells such as between 48,200 to 151,000 particles each. At
level of factor treatment of 50 g/l, the generated minicells were the highest
number at each of kind of sugars (glucose, maltose, and lactose). In modified
MRS media with these sugars (50 g/l), the minicells obtained were about
164,000; 351,000 and 787,000 particles, respectively. Meanwhile, the minicells
obtained from L. rhamnosus in fructose and saccharose media got the highest
values at concentration of 10 g/l (129,000 particles and 151,000 particles for
saccharose and fructose, respectively) (Table 4.4). In summary, lactose was
carbon source with a concentration of 50 g/l by means of which the highest
number of minicells is generated from L. rhamnosus (around 800,000 particles).
Consequently, the minicells produced from L. rhamnosus in modified MRS with
lactose 50 g/l and applied to drug delivery of the study.
34
Table 4.4 The number of minicells produced from Lactobacillus rhamnosus JCM 15113 in modified MRS medium
containing each kind of selected sugar separately in different concentrations.
Sugar concentrations (g/l)
Selected
sugars
0a, b
5 a, b
10 a, b
15 a, b
20 a, b
30 a, b
40 a, b
50 a, b
Glucose
3.39±0.72
3.46±0.75
6.87±1.59
7.48±2.15
8.13±2.03
9.38±1.97
11.7±1.85
16.4±1.97
Lactose
3.46±0.39
14.6±2.15
25.8±2.72
32.3±2.69
36.7±2.11
52.9±3.31
63.5±2.86
78.7±2.55
Fructose
3.41±0.36
9.61±2.77
15.1±1.40
13.9±1.76
12.6±2.11
11.9±1.93
10.5±1.57
9.68±2.54
Maltose
3.43±0.66
9.87±1.36
18.6±2.16
19.2±2.28
20.4±2.43
23.9±2.35
30.7±2.92
35.1±2.75
Saccharose
3.35±0.55
4.82±0.30
12.9±2.86
8.86±0.75
7.85±0.76
6.24±0.34
7.39±1.07
5.91±0.38
b
Data are means ± standard deviations of triplicates for all treatments
Expressed ×104
The number of minicells
(×104 particles)
a
Glucose
90
Lactose
80
Fructose
70
Maltose
60
Saccharose
50
40
30
20
10
0
0
5
10
15
20
30
Sugar concentrations (g/l)
40
50
Figure 4.5 The number of minicells (×104) produced from Lactobacillus rhamnosus JCM 15113 in
modified MRS medium with differently 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).
35
Data represented means ± SD, n=3.
In the Figure 4.6, the minicells were formed at one end of L. rhamnosus. The
reason was the effect of lactose on the cell development and cell differentiation.
Moreover, the practice of formation of rod chain of L. rhamnosus (Figure 4.6A),
the minicells were produced in chain (Figure 4.6B). The cell culture of L.
rhamnosus after cultivating in sugar was filtered through 0.45 µm; the minicells
were collected and calculated. However, the yield of obtained minicells was too
low (Table 4.4). Those meant that even morphological differentiation, the cell to
cell connection was so rigid or the size of minicells was bigger than 0.45µm filter.
Those caused the decrease of filtration efficiency.
A
B
Figure 4.6 Photomicrographs of Lactobacillus rhamnosus JCM 15113 and its
minicells (100X): A) the morphology of L. rhamnosus basic MRS culture medium;
B) the formation of minicells (black arrow) in the modified MRS medium with
lactose 50 g/l.
Moreover, it was clear that there was difference in the influence of sugars on
minicell formation between L. acidophilus and L. rhamnosus by comparison the
levels of factor (sugar concentration) treatment of both of strains. The number of
obtained minicells from L. acidophilus tended to decrease significantly whereas
the production of minicells from L. rhamnosus rose at the high concentration of
some of sugars (such as glucose, maltose and lactose). For this situation, more
study should be done so far. According to the results of the yield of minicell
generated from both L. acidophilus and L. rhamnosus, the quantity of minicell
production from L. acidophilus was larger than that from L. rhamnosus. L.
acidophilus was grown in MRS modified containing only 10 g/l of fructose, there
36
were 1,070,000 minicells obtained from culture with the density of about
2.57×1011 cells. Whereas, L. rhamnosus utilized 50 g/l lactose for generating
and isolated only around 787,000 particles from the same cell density.
4.2 Microscopic studies for characterization of minicells morphology
Minicells were achieved by using a procedure to homogenize and eliminated
contaminants such as parent bacterial cells, cellular debris (Figure 4.7). By
filtration through the filter membranes of 0.45 m and 0.2 m, the size of
resultant minicells were less than 450 nm.
Figure 4.27 Photomicrograph showing the morphology of minicells from
Lactobacillus acidophilus VTCC-B-871 under the light microscope after
isolation procedure (100 X).
According to the obtained results in sub-headings 4.1.2 and 4.1.3, minicells
generated from L. acidophilus in modified MRS with fructose 10 g/l and their
isolated minicells that were selected as the representative samples for scanning
the microscopic minicell morphologies under the scanning electron microscopy
(SEM) (Figure 4.8 and 4.9). The Figure 4.8 represented the minicells formation
at the end of one polar of L. acidophilus. This study used SEM for confirmation
the minicell size; the minicell shape was captured, recorded and determined the
nanoparticle size.
37
A
B
Figure 4.2 Representative SEM images showing the formation of minicells (red
arrow) of Lactobacillus acidophilus VTCC-B-871. A) and B) SEM images observed
with zoom out 20,000 times.
As can be seen from the Figure 4.9, the SEM images of minicells with their
diameter ranged from 290 nm to 410 nm. As the results, minicells were
successfully generated as nano-cells. In the practice, nanoparticles were useful
for drug delivery with the sizes up to 1000 nm (Shim and Turos, 2007).
Therefore, they are continued to apply in drug delivery. The final minicell
preparation was suggested to demonstrate the absence of bacterial colonies by
plating the entire preparation on MRS agar plates and incubating it overnight at
37ºC. The result regarding minicell preparation did not contain filamentous,
gram positive bacteria (such as Lactococcus, Staphylococcus genus) from
environment.
38
A
B
C
D
Figure 4.29 Representative SEM images of minicells from Lactobacillus
acidophilus VTCC-B-871with minicell size after isolation procedure. A) minicells
observed with the zoom out 3,000 times; B) minicells with the zoom out 10,000
times; C) minicell with the zoom out 20,000 times; and D) minicell with the
zoom out 50,000 times.
4.3 Package of drug into minicells generated from Lactobacillus strains
4.3.1 Antimicrobial activity tests
The experiments of testing antimicrobial activity supported for determining the
ability of packaging drug paclitaxel into minicells, because paclitaxel is not only
anti-tumor activity but also antimicrobial activity. To test the ability and potency
of drug delivery of our minicells, the minicells encapsulated with two different
drugs (paclitaxel, and cephalosporin), and incubated at different time (10, 15
and 24 hours). The extracted solutions from drug-packaged minicells were
centrifuged and used to test the antibacterial activities on the pathogens.
39
4.3.1.1 Drug packaging of minicells produced from Lactobacillus
acidophilus
The extracted solution from drug-packaged minicells was centrifuged and used
to test the activities on the above mentioned pathogens. Results obtained in the
present study relieved that extracted solution possess potential antibacterial
activity against both of gram negative (S.
9637, P .
aureus ATCC 25923, E. coli ATCC
aeruginosa ATCC 27853), positive bacteria (S. typhi ATCC 19430),
and yeast (C. albicans ATCC 14053) (Table 4.5). Meanwhile, extracted solution
from control minicells that were not incubated with the drug did not show any
antibacterial activity (Appendix D). Images showed off the antimicrobial activities
of extracted drugs from drug-packaged minicells on bacteria that could be
referred in Appendix D.1. The inhibition zone diameters were from around 4.6 to
23.0 mm and from 4.0 to 19.0 mm for extracted paclitaxel and cephalosporin,
respectively (Table 4.6). The results of statistical analysis also showed that there
were significantly different in antibacterial ability of extracted solutions when
minicells from L. acidophilus loaded with drugs at different incubation times (10,
15, 24 hours) at 37°C with rotation (p < 0.05). The data concluded that the zone
inhibition showed the highest activity at 10 hours of incubation with the tested
drugs (table 4.6). For this condition, the highest antibacterial activity of 23.0 ±
1.5 mm for paclitaxel and 19.3 ± 0.5 mm for cephalosporin in against P.
aeruginosa and least activity recorded in C. albicans measured 16.0 ± 1.0 mm
and 13.0 ± 1.0 mm for paclitaxel and cephalosporin, respectively. After 24 hours
of incubation of minicells with drugs, these numbers were significantly reduced
to 7.6 ± 0.5 mm for paclitaxel in P.
aeruginosa, and to 6.0 ± 1.0 mm for
cephalosporin in P. aeruginosa as well (Table 4.6). The antibacterial activities of
extracted paclitaxel and cephalosporin against the other bacteria had also the
similar characteristics in P. aeruginosa. Those meant that antibacterial activity
of drug extracts decreased since increasing the time of incubation for minicells
loading drugs. These results indicated that time of incubation have certain effect
on the drug loading into minicells. Consequently, the generated minicells could
package with many drugs.
40
Table 4.5 Antimicrobial spectrum of extracted drugs from drug-packaged
minicells
of
Lactobacillus
acidophilus
VTCC-B-871
and
Lactobacillus
rhamnosus JCM 15113 against Gram-positive and Gram-negative bacteria.
Inhibition1
Test organism
Gram-positive
Staphylococcus aureus ATCC 25923
(+)
Gram-negative
Escherichia coli ATCC 9637
(+)
Salmonella typhi ATCC 19430
(+)
Pseudomonas aeruginosa ATCC 27853
(+)
Yeast
Candida albicans ATCC 14053
1
(+)
(+) Inhibited by extracted drugs from drug-packaged minicells, (-) not inhibited.
Table 4.6 Antibacterial activity of extracted paclitaxel and cephalosporin from
drug-packaged minicells of Lactobacillus acidophilus against bacterial species
Zone of inhibition (mm)
Indicator
Bacteria
10 hours
15 hours
Pac
Cel
Pac
S. typhi
18.0 ± 1.0
14.3 ± 0.5
11.0 ± 1.0
E. coli
19.3 ± 1.5
15.3 ± 0.5
C. albicans
16.0 ± 1.0
P. aeruginosa
S. aureus
24 hours
Cel
Pac
Cel
8.6 ± 0.5
5.0 ± 1.0
5.0 ± 1.0
12.3 ± 0.5
9.3 ± 0.5
5.3 ± 1.5
5.0 ± 1.0
13.0 ± 1.0
9.3 ± 0.5
7.3 ± 0.5
4.6 ± 0.5
4.0 ± 0.0
23.0 ± 1.5
19.3 ± 0.5
15.0 ± 1.0
13.3 ± 0.5
7.6 ± 0.5
6.0 ± 1.0
22.0 ± 1.0
18.3 ± 0.5
14.3 ± 0.5
12.0 ± 1.0
7.0 ± 0.0
6.3 ± 0.5
Measurements are means ± standard deviations of 3 replicates for all treatments
Pac: paclitaxel; Cel: cephalosporin
4.3.1.2 Drug packaging
rhamnosus
of
minicells
produced
from
Lactobacillus
Even though the number of obtained minicells from L. rhamnosus after filtration
was less than that from L. acidophilus, this study also tried to test the packaging
drug into minicells. In the practice, L. acidophilus and L. rhamnosus also had the
same in applied properties as probiotics. This was clear that using L. acidophilus
or L. rhamnosus as the bacteria source for producing minicells. Moreover, L.
acidophilus was applied more popular than L. rhamnosus in pharmaceutical field.
From those points, this study also implemented the research on the paclitaxel
packaging into minicells from L. rhamnosus. To determine the ability of paclitaxel
41
packaged minicells by using antimicrobial activity testing because paclitaxel is
not only anti-tumor activity but also antimicrobial activity. The study also tested
the cephalosporin packaging of this kind of minicells. The minicells were
incubated with two different drugs (paclitaxel, and cephalosporin) at different
time (10, 15 and 24 hours). The extracted solutions from drug-packaged
minicells were centrifuged and used to test the antibacterial activities on the
mentioned pathogens.
Table 4.7 Antibacterial activity of extracted paclitaxel and cephalosporin from
drug-packaged minicells of Lactobacillus rhamnosus against bacterial species
Zone of inhibition (mm)
Indicator
Bacteria
10 hours
Pac
15 hours
Cel
Pac
24 hours
Cel
Pac
Cel
S. typhi
13.6 ± 0.5
11.0 ± 1.0
8.3 ± 0.5
5.3 ± 0.5
2.3 ± 0.5
3.3 ± 0.5
E. coli
14.6 ± 0.5
12.0 ± 1.0
8.0 ± 1.0
6.3 ± 0.5
4.3 ± 0.5
3.6 ± 0.5
C. albicans
12.0 ± 1.0
8.3 ± 0.5
6.0 ± 0.0
4.3 ± 0.5
4.0 ± 1.0
2.0 ± 1.0
P. aeruginosa
19.3 ± 1.0
16.6 ± 0.5
11.0 ± 1.0
8.3 ± 0.5
6.6 ± 0.5
5.0 ± 1.0
S. aureus
18.0 ± 1.0
14.6 ± 0.5
11.0 ± 1.0
8.0 ± 0.0
4.0 ± 0.0
5.0 ± 1.0
Measurements are means ± standard deviations of triplicates for all treatments
Pac: paclitaxel; Cel: cephalosporin
As was illustrated by the Table 4.5, the extracted solution from drug-packaging
minicells of L. rhamnosus showed antimicrobial activities against both of gram
negative and gram positive bacteria. Inhibited activity of the growth of tested
bacteria S. aureus ATCC 25923, E. coli ATCC 9637, S. typhi ATCC 19430, C.
albicans ATCC 14053 and P. aeruginosa ATCC 27853 were presented by the
action
of
the
supernatants (Appendix E). Moreover, extracted solution from
control minicells that were not incubated with the drug did not show any
antibacterial activity (Appendix E). As the information provided by Table 4.7,
extracted solutions of drug-loading minicells showed varied in the zone of
inhibition from about 2.0 - 19.3 mm against all the tested bacteria. It was similar
in L. acidophilus, there were also significantly different in antibacterial ability of
extracted solution when minicells from L. rhamnosus incubated with drugs at
different incubation times (10, 15, 24 hours) at 37°C with rotation (P < 0.05).
When minicells from L. rhamnosus had been incubated with drugs at 10 hr, the
highest antibacterial activity of their drug extracts were observed in all indicator
bacteria. The inhibition zone diameters of extracted solution of paclitaxel were
from around 13.6 mm to 19.3 mm and from about 8.3 mm to 16.6 mm of
42
cephalosporin (Table 4.7). After 24 hours of incubation of minicells with drugs,
the antibacterial activity of extracted solutions was significantly reduced from
about 19.3 mm to 6.6 mm for paclitaxel in P. aeruginosa, and from 16.6 mm to
5.0 mm for cephalosporin in P. aeruginosa as well (Table 4.7). The antibacterial
activities of extracted paclitaxel and cephalosporin against other bacteria had
also the same characteristics as in P.
aeruginosa. It was clearly that
antibacterial activities vary with the extracted solutions from drug-packaged
minicells which were incubated with drugs at different time.
The results of present investigation about the antibacterial activity of extracted
solutions
clearly
cephalosporin
in
indicated
the
that
extracted
there
were
solutions
presence
of
of
paclitaxel
drug-packaging
and
minicells.
Consequently, the generated minicells from Lactobacillus strains could package
with many drugs. Moreover, the incubation time of minicells with drugs also
affected on the drug loading into minicells. To test how the minicells could
package with the drugs, more studies should be done so far.
From the results in the Table 4.6 and 4.7 it was interesting that the drug
extracts from L. acidophilus drug-packaged minicells were almost higher
antibacterial activities when compared with the extracted solution from minicells
of L. rhamnosus. This meant that the concentration of drugs in extracted
solutions from drug-packaged minicells of L. acidophilus were higher than those
of L. rhamnosus. In general, the efficient packaging of many drugs into minicells
generated from L. acidophilus was better than that into minicells produced from
L. rhamnosus. Therefore, isolated minicells from L. acidophilus in this study were
selected as the representative samples for certain confirmation of packaging of
chemotherapeutic drug paclitaxel using a high sensitive and accurate technique,
HPLC-UV/Vis Spectroscopy. From that, the packaged paclitaxel concentration
and the number of paclitaxel molecules presented per minicell were determined.
4.3.2 Paclitaxel quantitation using HPLC-UV/Vis Spectroscopy
To confirm certainly 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.
HPLC analysis was applied to detect the existence of paclitaxel and determined
the amount of packaged paclitaxel as well as the number of paclitaxel molecules
presented per minicell.
43
4.3.2.1 The minicell drug packaging in response to varying times of
incubation
For minicells depending upon the times of incubation in the loading solution,
minicells were separately 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.
Paclitaxel
supernatants
were
introduced
to
High
Performance
Liquid
Chromatography (HPLC) for paclitaxel detection and quantification whose results
were showed in Appendix G. The data obtained from HPLC analysis was analyzed
by comparison with the data of standard paclitaxel. With this method, the
remaining concentration of paclitaxel in the loading solution could be measured
as following the linear calibration equation of paclitaxel (Appendix K) in order to
determine whether paclitaxel could be absorbed by minicells in the loading
solution or not. According to the HPLC chromatogram analysis (detailed in
Appendix G), the retention time and the remaining paclitaxel concentration were
summarized in Table 4.8.
Table 4.8 HPLC analysis of paclitaxel in the loading solution in response to
varying times of incubation
Incubation
time (hr)
Retention time
(minutes)
Variation
(%)
Remaining paclitaxel
concentration (µg/ml)
0
0
0
0
10
5.846 ± 0.006
2.24 ± 0.097
8.56 ± 0.025
15
5.807 ± 0.006
1.56 ± 0.105
9.16 ± 0.015
24
5.826 ± 0.006
1.88 ± 0.096
9.71 ± 0.03
Data are means ± standard deviations of triplicates for all treatments
The retention time of paclitaxel standard was 5.718 min (Appendix F). As Table
4.8 underlined that the paclitaxel still remained in the supernatants after 10 hr,
15 hr to 24 hr with the retention time about at 5.846 ± 0.006, 5.807 ± 0.006,
and 5.826 ± 0.006 min, respectively. The remaining paclitaxel concentration was
inferred from the linear standard graph of peak area which was drawn in the
appendix K. Comparing with the initial amount of paclitaxel loaded (10 (µg/ml)),
data showed that the drug concentration decreased in all loading solutions after
incubating time (Table 4.8). This finding therefore proved that chemotherapeutic
drug (paclitaxel) was absorbed by minicells which were presented in the
44
incubated solution. This totally matched with the result of testing antimicrobial
activity which indicated there were presence of paclitaxel the extracted solutions
of drug-packaging minicells (sub-heading 4.3.1.1). The results of statistical
analysis also showed that drug-incubating time affected significantly on the
efficient packaging paclitaxel activity in minicells (p < 0.05). There was gradual
raise of leftover drug concentration in the loading solutions as increasing the
incubation time of minicells with drug. The data in Table 4.8 revealed increase in
concentration of paclitaxel remaining according to the incubation time, from 8.56
± 0.025 (µg/ml) with 10
hr of incubation time go up to 9.71 ± 0.03 (µg/ml)
with 24 hr. Indeed, the peak area of samples after 15 hr and 24 hr of incubation
were higher than that from 10 hr (Appendix G). With these points, minicells
should be soaked in paclitaxel loading solution for 10 hr in order to get the
highest yield of encapsulated activity.
4.3.2.2 The
minicell
drug
packaging
in
response
to
varying
concentrations
Purified minicells were separately loaded in solution of the paclitaxel drug, at
different final concentrations of 5 µg/ml, 10 µg/ml, and 20 µg/ml and then
incubated at 37°C with rotation for 10 hr. In order to confirm certainly the drugpackaging in minicells, the extracted solutions from drug-packaged minicells
were centrifuged and used to determine the concentration of drug in minicells
using HPLC. This experiment also revealed quantitation of minicells loading when
incubated in the presence of different paclitaxel concentrations.
As mentioned in Appendix F, retention time of standard was 5.718 min whereas
others were slightly varied. To identify the presence of paclitaxel, the variation
between paclitaxel peaks of samples and standard were calculated as equation
3.2 (see sub-heading 3.2.8). These variations could due to noise and
disturbance. However, the measured variations of retention time should be in
the acceptable range (less than 5%). Since all of experiments had retention time
variation less than 5% which were recognized as peak of standard paclitaxel. In
addition, extracted solution from control minicells that were not incubated with
the drug did not show paclitaxel peak (Appendix F). As comparing with the
retention time of standard paclitaxel (5.718 min), the retention time 5.444 ±
0.007, 5.454 ± 0.006 and 5.671 ± 0.004 min for samples of 5, 10 and 20 µg/ml
initial paclitaxel concentration, respectively, pointed that the existence of
paclitaxel in the minicells (Table 4.9, Appendix H).
45
Table 4.9 HPLC analysis of paclitaxel loading in minicells when incubated in
the presence of different drug concentrations
Initial Paclitaxel
Concentration
(µg/ml)
Retention time
(minutes)
Variation
(%)
Concentration of
extracted paclitaxel
(µg/ml)
0
0
0
0
5
5.444 ± 0.007
4.80 ± 0.124
0.35 ± 0.025
10
5.454 ± 0.006
4.61 ± 0.105
1.07 ± 0.026
20
5.671 ± 0.004
0.83 ± 0.071
2.22 ± 0.015
Data are means ± standard deviations of triplicates for all treatments
According to the linear calibration graph of area peak as a function of
concentration for paclitaxel standard solutions assayed by HPLC (Appendix K),
the amount of paclitaxel packaged into the total minicells was determined. As
the information provided by Table 4.9 and Figure 4.10, it was clear that
paclitaxel packaging was dependent on, and directly related to the external
loading concentration of paclitaxel. Indeed, the results of statistical analysis also
showed that the input drug concentration in the loading solution influenced
significantly on the encapsulation efficiency paclitaxel of minicells (p < 0.05)
(Table 4.10). There was the raise of packaged drug concentrations in minicells
from 0.35 to 2.22 µg/ml as initial drug concentrations were increased from 5 to
20 µg/ml, respectively (Table 4.9). It meant that the amount of packaged drug
increased six fold as raising the input drug quantity to four times.
Drug (µg) in 2.12 ×106
minicells
Figure 4.10 Drug quantitation in minicells when minicells incubated in the
2.50
2.00
paclitaxel
packaging
1.50
1.00
0.50
0.00
5
10
20
Initial drug concentration (µg/ml)
presence of different drug loading concentrations. Data are means ± standard
deviations of triplicates for all treatments.
46
The number of drug molecules per minicell was calculated according to equations
in sub-heading 3.2.9. The number of drug molecules in the incubation solution,
in the total of minicells, and in per minicell; the encapsulation efficiency of
packaging paclitaxel into minicells were summarized in Table 4.10. For the
incubation of 5×107 minicells for 10 hr with different paclitaxel concentrations of
5 µg/ml, 10 µg/ml and 20 µg/ml, the amount paclitaxel packaged was 0.35 ±
0.025 µg, 1.07 ± 0.026 µg, and 2.22 ± 0.015 µg, respectively (Table 4.9). This
equated to about 5 million, 15 million, and 31 million drug molecules packaged
per minicell, respectively (Table 4.10). When the initial paclitaxel concentration
increased to 20 µg/ml, the encapsulated efficiency achieved almost 2 times
higher than at 5 µg/ml (Table 4.10). At input paclitaxel concentration of 20
µg/ml, the highest number of paclitaxel molecules (about 31 million molecules)
was packaged per minicell. Table 4.10 also indicated that at paclitaxel
concentration of 20 µg/ml, there were the total number of 14.1×10 15 paclitaxel
molecules presented in the loading solution, and 1.57×1015 paclitaxel molecules
entrapped in 5×107 minicells.
Table 4.10 Paclitaxel quantification in minicells when incubated in the presence of
different drug concentrations
Initial Paclitaxel
Concentration
(µg/ml)
NPL
(molecules) b
NPT
(molecules)a, b
NPM
(molecules)a, c
EE (%)a
0
0
0
0
0
5
352
24.9±1.77
49.8±3.55
6.6±0.503
10
705
75.4±1.87
151±3.73
10.7±0.265
20
1410
157±1.08
313±2.15
11.1±0.076
a
Data are means ± standard deviations of triplicates for all treatments;
Expressed ×1013 ; c Expressed ×105;
NPL: the number of paclitaxel molecules present in the loading solution; NPT: the number
of paclitaxel molecules present in the total minicells; NPM: the number of paclitaxel
molecules per minicells; EE%: is encapsulation efficiency.
b
In general, this study was discovered that an unprecedented concentration of 5
million to 31 million paclitaxel molecules can be packaged within a minicell. In
contrast, other bacterially deriver minicells have been shown to package 300,000
(MacDiarmid et al., 2007b) molecules of paclitaxel. This was clear that minicells
generated from this study which was able to encapsulate a huge amount of drug
(more than 100 times in comparison with previous study about packaging
paclitaxel in minicells). Although minicells were produced in this study at low
47
yield (about 106 in comparison with 1011 minicells generated from other bacteria
in previous study (MacDiarmid et al., 2007b)), the number of paclitaxel drug
molecules packaged by a minicell was very large.
48
CHAPTER 5
CONCLUSION AND RECOMMENDATION
The study was the first report that L. acidophilus VTCC-B-871 and L. rhamnosus
JCM 15113 in the modified MRS broth with carbon source as fructose (10 g/l)
and lactose (50 g/l), respectively, gave the significant minicell quantity.
The
number of minicells was generated from L. acidophilus VTCC-B-871 and L.
rhamnosus JCM 15113 about 1,070,000 and 787,000 minicells, respectively. The
result of this study revealed that carbon sources (sugars) actually affected on
the minicell production or cell division of L. acidophilus VTCC-B-871 and L.
rhamnosus JCM 15113 strains.
This study captured and determined the
nanoparticle size of obtained minicells (with their diameter ranged from 290 nm
to 410 nm) using scanning electron microscopy. Minicells were performed the
ability of packaging paclitaxel. The study also tested the packaging of
cephalosporin to confirm the packaging ability. Moreover, the study also
suggested the easy, fast and cheap way to test the paclitaxel and cephalosporin
using antimicrobial activity. However, the higher technique (using HPLC) was
applied in this study to measure the paclitaxel packaging in the small amount.
Also, the study was optimized the minicell generation from L. rhamnosus that
was able to package paclitaxel and cephalosporin by testing on antimicrobial
activity. The input drug concentration in the loading solution and the incubation
time of drug with minicells influenced significantly on the encapsulation efficiency
paclitaxel of minicells. Surprisingly, the minicell generated from L. acidophilus
could package with the drug better than that from L. rhamnosus. In addition, the
ability of minicells to encapsulate a large number of drug molecules (5 million to
31 million paclitaxel molecules packaged per minicell), as well as a convenient
drug-loading process that required only one-step co-incubation of a solution of
the drug with the vehicle, made minicells the potentially attractive alternative to
other macromolecule based drug formulations. Frequently, the following minicell
production was due to the min deletion. The practice of modifying carbon
sources to produce minicell had the potential role in drug delivery. This really
helped significantly to the combination of probiotics which contain useful bacteria
(including L. acidophilus and L. rhamnosus strains), with different kinds of
antibiotics and some chemotherapeutic drugs. From those points, the drug
absorption by human body might also be better. More studies should be done so
far for the mechanisms and drug packaging ability in L. acidophilus and L.
rhamnosus strains. In conclusion, this study contributed to drug formulation for
pharmaceutical area and the related fields.
49
REFERENCES
ACS. 2013. Chemotherapy. American Cancer Society. Available at:
http://www.cancer.org/treatment/treatmentsandsideeffects/treatmenttypes/c
hemotherapy/index.
Adams JM and Strasser A. 2008. Is Tumor Growth Sustained by Rare Cancer
Stem Cells or Dominant Clones? Cancer Res. 68: 4018–4021.
Adler HI, Fisher WD, Cohen A, and Hardigree AA. 1967. Miniature Escherichia
coli cells deficient in DNA. Proc. Natl. Acad. Sci. U. S. A. 57: 321–326.
Akbarzadeh A, Rezaei-Sadabady R, Davaran S, Joo SW, Zarghami N,
Hanifehpour Y, Samiei M, Kouhi M, and Nejati-Koshki K. 2013. Liposome:
classification, preparation, and applications. Nanoscale Res. Lett. 8: 102.
Alexis F, Pridgen EM, Langer R, and Farokhzad OC. 2010. Nanoparticle
technologies for cancer therapy. Handb. Exp. Pharmacol. 55–86.
Allen, T.M. 1997. Liposomes. Opportunities in drug delivery. Drugs. 54: 8–14.
Angelis MD, and Gobbetti M. 2011. Stress Responses of Lactobacilli. In: Stress
Responses of Lactic Acid Bacteria, Tsakalidou E, and PapadimitrioU K, eds.
(Springer US), pp. 219–249.
Anwekar H, Patel S, and Singhai A. 2011. Liposomes as drug carriers. Int. J.
Pharm. Life Sci. 2: 945–951.
Arnold J, and Bailey G. 2000. Surface finishes on stainless steel reduce bacterial
attachment and early biofilm formation: scanning electron and atomic force
microscopy study. Poult. Sci. 79: 1839–1845.
Bilensoy E. 2010. Cancer Nanotechnology: Targeting Tumors with Nanoparticles.
In: Handbook of Nanophysics: Nanomedicine and Nanorobotics, (CRC Press),
pp. 35–1.
De Boer PA, Crossley RE, and Rothfield LI. 1989. A division inhibitor and a
topological specificity factor coded for by the minicell locus determine proper
placement of the division septum in E. coli. Cell 56: 641–649.
Bogdanov IG, Velichkov VT, Gurevich AI, Dalev PG, and Kolosov MN. 1977.
Antitumor effect of glycopeptides from the cell wall of Lactobacillus bulgaricus.
Biulleten Eksp. Biol. Meditsiny 84: 709–712.
Boyle P, and Levin B. 2008. World cancer report (Lyon, France: International
Agency for Research on Cancer).
Brahmbhatt H, and MacDiarmid J. 2004. Pharmaceutically compatible method for
purifying intact bacterial minicells.
Brannon-Peppas L, and Blanchette JO. 2004. Nanoparticle and targeted systems
for cancer therapy. Adv. Drug Deliv. Rev. 56: 1649–1659.
Brigger I, Dubernet C, and Couvreur P. 2002. Nanoparticles in cancer therapy
and diagnosis. Adv. Drug Deliv. Rev. 54: 631–651.
50
Cai Y, Pang H, Kitahara M, and Ohkuma M. 2012. Lactobacillus nasuensis sp.
nov., a lactic acid bacterium isolated from silage, and emended description of
the genus Lactobacillus. Int. J. Syst. Evol. Microbiol. 62: 1140–1144.
Carr FJ, Chill D, and Maida N. 2002. The lactic acid bacteria: a literature survey.
Crit. Rev. Microbiol. 28: 281–370.
Cenci G, Rossi J, Trotta F, and Caldini G. 2002. Lactic acid bacteria isolated from
dairy products inhibit genotoxic effect of 4-nitroquinoline-1-oxide in SOSchromotest. Syst. Appl. Microbiol. 25: 483–490.
Chabner BA, and Roberts TG. 2005. Chemotherapy and the war on cancer. Nat.
Rev. Cancer 5: 65–72.
Chen WC, Zhang AX, and Li SD. 2012. Limitations and niches of the active
targeting approach for nanoparticle drug delivery. Eur. J. Nanomedicine 4:
89–93.
Chidambaram M, Manavalan R, and Kathiresan K. 2011. Nanotherapeutics to
overcome conventional cancer chemotherapy limitations. J. Pharm. Pharm.
Sci. Publ. Can. Soc. 14: 67–77.
Cho K, Wang X, Nie S, Chen Z. (Georgia), and Shin DM. 2008. Therapeutic
Nanoparticles for Drug Delivery in Cancer. Clin. Cancer Res. 14: 1310–1316.
Choi SS, Kim Y, Han KS, You S, Oh S, and Kim SH. 2006. Effects of Lactobacillus
strains on cancer cell proliferation and oxidative stress in vitro. Lett. Appl.
Microbiol. 42: 452–458.
Chorawala MR, Oza PM, and Shah GB. 2012. Mechanisms of Anticancer Drugs
Resistance: An Overview. Int. J. Pharm. Sci. Drug Res. 4: 1–9.
Curti BD, Urba WJ, Alvord WG, Janik JE, Smith JW, Madara K, and Longo DL.
1993. Interstitial pressure of subcutaneous nodules in melanoma and
lymphoma patients: changes during treatment. Cancer Res. 53: 2204–2207.
Dean M, Fojo T, and Bates S. 2005. Tumour stem cells and drug resistance. Nat.
Rev. Cancer. 5: 275–284.
Duncan R. 2003. The dawning era of polymer therapeutics. Nat. Rev. Drug
Discov. 2: 347–360.
ECCO, E.C.O. 2012. First trial in humans of “minicells”: a completely new way of
delivering anti-cancer drugs.
Fan H. 2008. Nanocrystal-micelle: synthesis, self-assembly and application.
Chem. Commun. Camb. Engl. 1383–1394.
Fan Y, Du W, He B, Fu F, Yuan L, Wu H, Dai W, Zhang H, Wang X, Wang J, et al.
2013. The reduction of tumor interstitial fluid pressure by liposomal imatinib
and its effect on combination therapy with liposomal doxorubicin. Biomaterials
34: 2277–2288.
Felis GE, Dellaglio F, and Torriani S. 2009. Taxonomy of Probiotic
Microorganisms. In: Prebiotics and Probiotics Science and Technology,
Charalampopoulos D, and Rastall RA. (New York, NY: Springer New York), pp.
591–637.
51
Ferlay J, Shin
GLOBOCAN
CancerBase
Research on
HR, Bray F, Forman D, Mathers C, and Parkin DM. 2010a.
2008, Cancer Incidence and Mortality Worldwide: IARC
No. 10 [Internet]. Lyon, France: International Agency for
Cancer.
Ferlay J, Shin HR, Bray F, Forman D, Mathers C, and Parkin DM. 2010b.
Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int. J.
Cancer 127: 2893–2917.
Ferrari M. 2005. Cancer nanotechnology: opportunities and challenges. Nat. Rev.
Cancer 5: 161–171.
Fichera GA, and Giese G. 1994. Non-immunologically-mediated cytotoxicity of
Lactobacillus casei and its derivative peptidoglycan against tumor cell lines.
Cancer Lett. 85: 93–103.
Flemming A. 2007. Minicells deliver lethal load to tumours. Nat. Rev. Drug
Discov. 6: 519–519.
Gabizon A, Shmeeda H, and Barenholz Y. 2003. Pharmacokinetics of pegylated
liposomal Doxorubicin: review of animal and human studies. Clin.
Pharmacokinet. 42: 419–436.
el-Gawad IAA, el-Sayed EM, Hafez SA, el-Zeini HM, and Saleh FA. 2004.
Inhibitory effect of yoghurt and soya yoghurt containing bifidobacteria on the
proliferation of Ehrlich ascites tumour cells in vitro and in vivo in a mouse
tumour model. Br. J. Nutr. 92: 81–86.
Gerber DE. 2008. Targeted therapies: a new generation of cancer treatments.
Am. Fam. Physician 77: 311–319.
Gibson GR, Saavedra JM, Macfarlane S, and Macfarlane GT. 1997. Probiotics and
Intestinal Infections. In: Probiotics 2, Fuller R, ed. Dordrecht: Springer
Netherlands, pp. 10–39.
Gilstrap K, Hu X, Lu X, and He X. 2011. Nanotechnology for energy-based cancer
therapies. Am. J. Cancer Res. 1: 508–520.
Goh YJ, and Klaenhammer TR. 2009. Genomic features of Lactobacillus species.
Front. Biosci. J. Virtual Libr. 14: 1362–1386.
Gottesman MM. 2002. Mechanisms of cancer drug resistance. Annu. Rev. Med.
53: 615–627.
Greco F, and Vicent MJ. 2008. Polymer-drug conjugates: current status and
future trends. Front. Biosci. J. Virtual Libr. 13: 2744–2756.
Gref R, Minamitake Y, Peracchia MT, Trubetskoy V, Torchilin V, and Langer R.
1994. Biodegradable long-circulating polymeric nanospheres. Science 263:
1600–1603.
Gregson N, Sparrowhawk K, Mauskopf J, and Paul J. 2005. Pricing medicines:
theory and practice, challenges and opportunities. Nat. Rev. Drug Discov. 4:
121–130.
52
Gutmann R, Leunig M, Feyh J, Goetz AE, Messmer K, Kastenbauer E, and Jain
RK. 1992. Interstitial hypertension in head and neck tumors in patients:
correlation with tumor size. Cancer Res. 52: 1993–1995.
Hawkins MJ, Soon-Shiong P, and Desai N. 2008. Protein nanoparticles as drug
carriers in clinical medicine. Adv. Drug Deliv. Rev. 60: 876–885.
Heldin C-H, Rubin K, Pietras K, and Ostman A. 2004. High interstitial fluid
pressure - an obstacle in cancer therapy. Nat. Rev. Cancer 4: 806–813.
Hepner G, Fried R, St Jeor S, Fusetti L, and Morin R. 1979. Hypocholesterolemic
effect of yogurt and milk. Am. J. Clin. Nutr. 32: 19–24.
Himanshu Brahmbhatt, and MacDiarmid J. 2009. Pharmaceutically compatible
method for purifying intact bacterial minicells.
Hofheinz R.-D, Gnad-Vogt SU, Beyer U, and Hochhaus A. 2005. Liposomal
encapsulated anti-cancer drugs. Anticancer. Drugs 16: 691–707.
Hosono A, Kashina T, and Kada T. 1986. Antimutagenic Properties of Lactic AcidCultured Milk on Chemical and Fecal Mutagens. J. Dairy Sci. 69: 2237–2242.
Jafarei P, and Ebrahimi MT. 2011. Lactobacillus acidophiluscell structure and
application. Afr. J. Microbiol. Res. 5: 4033–4042.
Jain KK. 2005. Targeted drug delivery for cancer. Technol. Cancer Res. Treat. 4:
311–314.
Jain KK. 2008. Drug Delivery Systems. USA: Humana Press.
Jemal A, Tiwari RC, Murray T, Ghafoor A, Samuels A, Ward E, Feuer EJ, and
Thun MJ. 2004. Cancer J. Clin. 54: 8–29.
Jemal A, Bray F, Center MM, Ferlay J, Ward E, and Forman D. 2011. Global
cancer statistics. CA. Cancer J. Clin. 61: 69–90.
Kaláb M, Yang A-F, and Chabot D. 2008. Conventional Scanning Electron
Microscopy of Bacteria.
Kamila Myszka, and Katarzyna Czaczyk. 2011. Effect of starvation stress on
morphological changes and production of adhesive exopolysaccharide (EPS)
by Proteus vulgaris. Acta Sci. Pol., Technol. Aliment. 10: 303–312.
Kaneda Y. 2000. Virosomes: evolution of the liposome as a targeted drug
delivery system. Adv. Drug Deliv. Rev. 43: 197–205.
Karagiannis ED, and Anderson DG. 2009. Minicells overcome tumor drugresistance. Nat. Biotechnol. 27: 620–621.
Kim JY, Woo HJ, Kim Y-S, and Lee HJ. 2002. Screening for antiproliferative
effects of cellular components from lactic acid bacteria against human cancer
cell lines. Biotechnol. Lett. 24: 1431–1436.
Klein G, Pack A, Bonaparte C, and Reuter G. 1998. Taxonomy and physiology of
probiotic lactic acid bacteria. Int. J. Food Microbiol. 41: 103–125.
Langer R. 1998. Drug delivery and targeting. Nature 392: 5–10.
53
LeBlanc JG, Matar C, Valdéz JC, LeBlanc J, and Perdigon G. 2002.
Immunomodulating Effects of Peptidic Fractions Issued from Milk Fermented
with Lactobacillus helveticus. J. Dairy Sci. 85: 2733–2742.
Less JR, Posner MC, Boucher Y, Borochovitz D, Wolmark N, and Jain RK. 1992.
Interstitial hypertension in human breast and colorectal tumors. Cancer Res.
52: 6371–6374.
Li C, and Wallace S. 2008. Polymer-Drug Conjugates: Recent Development in
Clinical Oncology. Adv. Drug Deliv. Rev. 60: 886–898.
Lidbeck A, Nord CE, Gustafsson JA, and Rafter J. 1992. Lactobacilli,
anticarcinogenic activities and human intestinal microflora. Eur. J. Cancer
Prev. Off. J. Eur. Cancer Prev. Organ. Ecp 1: 341–353.
Liu C-F, and Pan T-M. 2010. In Vitro Effects of Lactic Acid Bacteria on Cancer
Cell Viability and Antioxidant Activity. J. Food Drug Anal. 18: 77–86.
Ljungh A, and Wadström T. 2006. Lactic acid bacteria as probiotics. Curr. Issues
Intest. Microbiol. 7: 73–89.
Lutkenhaus J. 2007. Assembly dynamics of the bacterial MinCDE system and
spatial regulation of the Z ring. Annu. Rev. Biochem. 76: 539–562.
Ma L, King GF, and Rothfield L. 2004. Positioning of the MinE binding site on the
MinD surface suggests a plausible mechanism for activation of the Escherichia
coli MinD ATPase during division site selection. Mol. Microbiol. 54: 99–108.
MacDiarmid JA, and Brahmbhatt H. 2011. Minicells: Versatile vectors for targeted
drug or si/shRNA cancer therapy. Curr. Opin. Biotechnol. 22: 909–916.
MacDiarmid JA, Madrid-Weiss J, Amaro-Mugridge NB, Phillips L, and Brahmbhatt
H. 2007a. Bacterially-derived nanocells for tumor-targeted delivery of
chemotherapeutics and cell cycle inhibitors. Cell Cycle Georget. Tex 6: 2099–
2105.
MacDiarmid JA, Mugridge NB, Weiss JC, Phillips L, Burn AL, Paulin RP, Haasdyk
JE, Dickson K-A, Brahmbhatt H, Pattison ST, et al. 2007b. Bacterially derived
400 nm particles for encapsulation and cancer cell targeting of
chemotherapeutics. Cancer Cell 11: 431–445.
MacDiarmid JA, Amaro-Mugridge NB, Madrid-Weiss J, Sedliarou I, Wetzel S,
Kochar K, Brahmbhatt H Phillips L, Pattison ST, Pett C, et al. 2009. Sequential
treatment of drug-resistant tumors with targeted minicells containing siRNA or
a cytotoxic drug. Nat. Biotechnol. 27: 643–651.
Macfarlane GT, and Cummings JH. 2002. Probiotics, infection and immunity.
Curr. Opin. Infect. Dis. 15: 501–506.
De Man JC, Rogosa M, and Sharpe ME. 1960. A Medium for the Cultivation of
Lactobacilli. J. Appl. Microbiol. 23: 130–135.
Matsumura Y. 2008. Poly (amino acid) micelle nanocarriers in preclinical and
clinical studies. Adv. Drug Deliv. Rev. 60: 899–914.
54
Moghimi SM. 2006. Recent developments in polymeric nanoparticle engineering
and their applications in experimental and clinical oncology. Anticancer Agents
Med. Chem. 6: 553–561.
Moghimi SM, Hunter AC, and Murray JC. 2001. Long-circulating and targetspecific nanoparticles: theory to practice. Pharmacol. Rev. 53: 283–318.
Mohanraj V, and Chen Y. 2006. Nanoparticles – A Review. Trop. J. Pharm. Res.
5: 561–573.
Morgillo F, and Lee H-Y 2005. Resistance to epidermal growth factor receptortargeted therapy. Drug Resist. Updat. 8: 298–310.
Mozzi F Raya R, and Vignolo GM. 2010. Biotechnology of Lactic Acid Bacteria:
Novel Applications.
Murakami T, and Tsuchida K. 2008. Recent advances in inorganic nanoparticlebased drug delivery systems. Mini Rev. Med. Chem. 8: 175–183.
Najlah M, and D‟Emanuele A. 2007. Synthesis of dendrimers and drug-dendrimer
conjugates for drug delivery. Curr. Opin. Drug Discov. Devel. 10: 756–767.
Nasongkla N, Bey, Ren J, Ai H, Khemtong, C, Guthi JS, Chin S-F, Sherry AD,
Boothman DA, and Gao J. 2006. Multifunctional Polymeric Micelles as CancerTargeted, MRI-Ultrasensitive Drug Delivery Systems. Nano Lett. 6: 2427–
2430.
Nathanson SD, and Nelson L. 1994. Interstitial fluid pressure in breast cancer,
benign breast conditions, and breast parenchyma. Ann. Surg. Oncol. 1: 333–
338.
Nguyen THK, Doan VTT, Ha LD, and Nguyen HN. 2012. Cell division inhibitor
minC from Lactobacillus Acidophilus VTCC-B-871 and detection of
antimicrobial activities in functional study. Afr. J. Pharm. Pharmacol. 6: 3293–
3298.
Nguyen THK, Doan VTT, Ha LD, and Nguyen HN. 2013a. Studies of the Paclitaxel
Delivery and Antimicrobial Activities Generated by MinD from Lactobacillus
acidophilus VTCC-B-871. J. Pure Appl. Microbiol. 7: 379 – 384.
Nguyen THK, Doan VTT, Ha LD, and Nguyen HN. 2013b. Molecular Cloning,
Expression of minD Gene from Lactobacillus acidophilus VTCC-B-871 and
Analyses to Identify Lactobacillus rhamnosus PN04 from Vietnam Hottuynia
cordata Thunb. Indian J. Microbiol. 1–6.
Omri A, Suntres ZE, and Shek PN. 2002. Enhanced activity of liposomal
polymyxin B against Pseudomonas aeruginosa in a rat model of lung infection.
Biochem. Pharmacol. 64: 1407–1413.
Park JW. 2002. Liposome-based drug delivery in breast cancer treatment. Breast
Cancer Res. 4, 95–99.
Pham THA, and Nguyen BD. 2002. The Situation with Cancer Control in Vietnam.
Jpn. J. Clin. Oncol. 32: S92–S97.
Pridgen EM, Langer R, and Farokhzad OC. 2007. Biodegradable, polymeric
nanoparticle delivery systems for cancer therapy. Nanomed. 2: 669–680.
55
Salminen S, Wright AV, and Ouwehand AC. 2004. Lactic Acid Bacteria:
Microbiological And Functional Aspects, Third Edition, Revised And Expanded
(Marcel Dekker Inc.).
Salvetti E, Torriani S, and Felis GE. 2012. The Genus Lactobacillus: A Taxonomic
Update. Probiotics Antimicrob. Proteins. 4: 217–226.
Sarosy G, and Reed E. 1993. Taxol dose intensification and its clinical
implications. J. Natl. Med. Assoc. 85: 427–431.
Schiffelers RM, Storm G, and Bakker-Woudenberg JM. 2001. Host Factors
Influencing the Preferential Localization of Sterically Stabilized Liposomes in
Klebsiella Pneumoniae-Infected Rat Lung Tissue. Pharm. Res. 18: 780–787.
Senesi S, Celandroni F, Tavanti A, and Ghelardi E. 2001. Molecular
Characterization and Identification of Bacillus clausii Strains Marketed for Use
in Oral Bacteriotherapy. Appl. Environ. Microbiol. 67: 834–839.
Sharpe ME. 1979. Identification of the lactic acid bacteria. In Identification
Methods for Microbiologists. London, UK: Academic Press, pp. 246 – 255.
Shim J-Y, and Turos E. 2007. Nanoparticles for drug-delivery.
Soccol CR, Vandenberghe LP de S, Spier MR, Medeiros ABP., Yamaguishi CT,
Lindner JDD, Pandey A, and Thomaz-Soccol V. 2010. The Potential of
Probiotics: A Review. Food Technol. Biotechnol. 48: 413–434.
Stano P, Bufali S, Pisano C, Bucci F, Barbarino M, Santaniello M, Carminati P, and
Luisi PL. 2004. Novel camptothecin analogue (gimatecan)-containing
liposomes prepared by the ethanol injection method. J. Liposome Res. 14:
87–109.
Torchilin VP. 2005. Recent advances with liposomes as pharmaceutical carriers.
Nat. Rev. Drug Discov. 4: 145–160.
Urruticoechea A, Alemany R, Balart J, Villanueva A, Viñals F, and Capellá G.
2010. Recent advances in cancer therapy: an overview. Curr. Pharm. Des. 16:
3–10.
Veiga H, and Pinho MG. 2012. Bacterial cell division: what it takes to divide a
prokaryotic cell. 18–26.
Vinogradov S, and Wei X. 2012. Cancer stem cells and drug resistance: the
potential of nanomedicine. Nanomed. 7: 597–615.
Wang G, and Uludag H. 2008. Recent developments in nanoparticle-based drug
delivery and targeting systems with emphasis on protein-based nanoparticles.
Expert Opin. Drug Deliv. 5: 499–515.
Wang MD, Shin, DM, Simons JW, and Nie S. 2007. Nanotechnology for targeted
cancer therapy. Expert Rev. Anticancer Ther. 7: 833–837.
Win KY. 2006. Paclitaxel loaded nanoparticles of biodegradable polymers for
cancer chemotherapy. PhD thesis. National University of Singapore.
Wong HL, Bendayan R, Rauth AM, Xue HY, Babakhanian K, and Wu XY. 2006. A
mechanistic study of enhanced doxorubicin uptake and retention in multidrug
56
resistant breast cancer cells using a polymer-lipid hybrid nanoparticle system.
J. Pharmacol. Exp. Ther. 317: 1372–1381.
Wu H-C, Chang D-K, and Huang C-T. 2006. Targeted Therapy for Cancer. J.
Cancer Mol. 2: 57–66.
Yoon H, Benamouzig R, Little J, François-Collange M, and Tomé D. 2000.
Systematic review of epidemiological studies on meat, dairy products and egg
consumption and risk of colorectal adenomas. Eur. J. Cancer Prev. Off. J. Eur.
Cancer Prev. Organ. Ecp 9: 151–164.
Yuksekdag ZN, and Aslim B. 2008. Influence of Different Carbon Sources on
Exopolysaccharide Production by Lactobacillus delbrueckii Subsp. bulgaricus
(B3, G12) and Streptococcus thermophilus(W22). Braz. arch. biol. technol.
51: 581–587.
Yun Y, Cho YW, and Park K. 2012. Nanoparticles for oral delivery: Targeted
nanoparticles with peptidic ligands for oral protein delivery. Adv. Drug Deliv.
Rev.
Zheng JH, Chen CT, Au JL, and Wientjes MG. 2001. Time and concentration
dependent penetration of doxorubicin in prostate tumors. Aaps Pharmsci 3:
E15.
57
APPENDIX A
Buffers preparation
Table A.1 Buffered saline gelatin (BSG) preparation
Ingredients
Formula per Liter
NaCl
8.5 g
KH2PO4
0.3 g
Na2HPO4. 12H2O
0.6 g
Gelatin 1%
10 ml
pH 7.0, autoclaved at 121°C for 15 min
Table A.2 Phosphate buffered saline (PBS) preparation
Ingredients
Formula per Liter
NaCl
8.01 g
KH2PO4
0.24 g
Na2HPO4. 12H2O
3.63 g
KCl
0.2 g
pH 7.4, autoclaved at 121°C for 15 min
58
APPENDIX B
Morphology of Lactobacilus acidophilus in modified MRS broth with
different concentrations of sugar (0, 5, 10, 15, 20, 30, 40, 50 (g/l))
Figure B.1 Morphology of L.acidophilus in modified MRS broth containing different
concentrations of glucose (0, 5, 10, 15, 20, 30, 40, 50 g/L (w/v))
Figure B.2 Morphology of L.acidophilus in modified MRS broth containing different
concentrations of lactose (0, 5, 10, 15, 20, 30, 40, 50 g/L (w/v))
59
Figure B.3 Morphology of L.acidophilus in modified MRS broth containing different
concentrations of frucose (0, 5, 10, 15, 20, 30, 40, 50 g/L (w/v))
Figure B.4 Morphology of L.acidophilus in modified MRS broth containing different
concentrations of maltose (0, 5, 10, 15, 20, 30, 40, 50 g/L (w/v))
Figure B.5 Morphology of L.acidophilus in modified MRS broth containing different
concentrations of sacchrose (0, 5, 10, 15, 20, 30, 40, 50 g/L (w/v))
60
APPENDIX C
Morphology of Lactobacilus rhamnosus in modified MRS broth with
different concentrations of sugar (0, 5, 10, 15, 20, 30, 40, 50 (g/l))
Figure C.1 Morphology of L. rhamnosus in modified MRS broth containing different
concentrations of glucose (0, 5, 10, 15, 20, 30, 40, 50 g/L (w/v))
Figure C.2 Morphology of L. rhamnosus in modified MRS broth containing different
concentrations of lactose (0, 5, 10, 15, 20, 30, 40, 50 g/L (w/v))
61
Figure C.3 Morphology of L. rhamnosus in modified MRS broth containing different
concentrations of fructose (0, 5, 10, 15, 20, 30, 40, 50 g/L (w/v))
Figure C.4 Morphology of L. rhamnosus in modified MRS broth containing different
concentrations of maltose (0, 5, 10, 15, 20, 30, 40, 50 g/L (w/v))
Figure C.5 Morphology of L. rhamnosus in modified MRS broth containing different
concentrations of saccharose (0, 5, 10, 15, 20, 30, 40, 50 g/L (w/v))
62
APPENDIX D
Antimicrobial activity tests on bacteria of drug extracts of minicells from
Lactobacillus acidophilus at different drug incubation time (10, 15, 24hr)
A
B
C
D
E
Figure D.1 Extracted cephalosporin showing antibacterial activity against: A)
Salmonella typhi; B) Escherichia coli; C) Candida albicans; D) Pseudomonas
aeruginosa; E) Staphylococcus aureus. N is negative control (minicells without
drug) ; P is positive control (cephalosporin)
63
A
B
D
C
E
Figure D.2 Extracted paclitaxel showing antibacterial activity against: A) Salmonella
typhi; B) Escherichia coli; C) Candida albicans; D) Pseudomonas aeruginosa; E)
Staphylococcus aureus. N is negative control (minicells without drug) ; P is positive
control (paclitaxel)
64
APPENDIX E
Antimicrobial activity tests on bacteria of drug extracts of minicells from
Lactobacillus rhamnosus at different drug incubation time (10, 15, 24hr)
A
B
C
D
E
Figure E.1 Extracted cephalosporin showing antibacterial activity against: A)
Salmonella typhi; B) Escherichia coli; C) Candida albicans; D) Pseudomonas
aeruginosa; E) Staphylococcus aureus.
N is negative control (minicells without
drug) ; P is positive control (cephalosporin)
65
A
B
C
D
E
Figure E.2 Extracted paclitaxel showing antibacterial activity against: A) Salmonella
typhi; B) Escherichia coli; C) Candida albicans; D) Pseudomonas aeruginosa; E)
Staphylococcus aureus. N is negative control (minicells without drug) ; P is positive
control (paclitaxel)
66
APPENDIX F
HPLC
analysis
for
paclitaxel
standard,
incubated with paclitaxel), and blank.
67
control
(minicells
without
APPENDIX G
G.1. HPLC analysis for minicell loading paclitaxel when incubated at 10hr
68
APPENDIX G (cont.)
G.2. HPLC analysis for minicell loading paclitaxel when incubated at 15hr
69
APPENDIX G (cont.)
G.3. HPLC analysis for minicell loading paclitaxel when incubated at 24hr
70
APPENDIX H
H.1. HPLC assay for minicell loading when incubated in the presence of 5
(µg/ml) drug concentrations
71
H.2. HPLC assay for minicell loading when incubated in the presence of
10 (µg/ml) drug concentrations
72
H.3. HPLC assay for minicell loading when incubated in the presence of
20 (µg/ml) drug concentrations
73
APPENDIX K
Standard paclitaxel linear calibration graph of peak area
Table K.1 Concentration of standard paclitaxel and peak areas corresponding
Concentration of standard paclitaxel (µg/ml)
Peak area
0
0
5
68908
10
179816
15
294356
20
381602
30
583890
50
938518
1000000
y = 19120x - 5500
R² = 0.9983
800000
Peak area
600000
400000
Paclitaxel standard
200000
Linear (Paclitaxel standard)
0
0
-200000
10
20
30
40
50
Paclitaxel Concentration (µg/ml)
Figure B.1 Standard paclitaxel linear calibration graph of peak area
74
60
[...]... insolubility of paclitaxel Figure 2.43 Chemical structure of paclitaxel 16 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. .. 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. .. standard were calculated as following equation (eq 3.2) (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... 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. .. for study The modified MRS media were prepared by modifying carbon source in MRS ingredients (detail in sub-section 3.2.2) 20 3.2 RESEARCH METHODOLOGY 3.2.1 Design condition for minicells production from Lactobacillus strains In order to study the influence of various carbon sources on the minicell formation, this study implemented experiments on different kinds of sugar with different concentration... 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... 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... improve the therapeutic index of anticancer 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 drug8 resistant and recurrent cancers However, despite the. .. 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... 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 .. .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... 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... 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
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