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Determination of LAMP-2B from HEK293T cells transfected by pCMV6-lamp2b-Myc-DDK

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Determination of LAMP-2B from HEK293T cells transfected by pCMV6-lamp2b-Myc-DDK

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Content Determination of LAMP-2B from HEK293T cells transfected by pCMV6-lamp2b-Myc-DDK
by
Shiyu Bian
A Thesis Presented to the
FACULTY OF THE USC ALFRED E. MANN SCHOOL OF PHARMACY AND
PHARMACEUTICAL SCIENCES
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)
May 2024
Copyright 2024 Shiyu Bian

ii
Acknowledgments
Time flies by like a fleeting show. My journey through graduate life is nearing its end. These
past two years have been the most challenging and captivating of my life, and I am truly grateful
for all the experiences and people I have encountered during this time. I would like to express my sincere appreciation first and foremost to Professor Sarah
Hamm-Alvarez, my Principal Investigator. She has graciously allowed me to study in her
laboratory and has been a constant source of inspiration and guidance throughout my project and
thesis. I would also like to extend my heartfelt gratitude to all the members of the lab. Xiaoyang Li
(Eddie), my supervisor and senior schoolmate, taught me various experimental skills and assisted
my research and thesis preparation. Daniel guided me in initiating my project and taught me
about cell culture techniques. Cindy assisted me with immunofluorescence, Min supported me
with immunoprecipitation, and Maria provided valuable insights into the finer details of the
experiments. I am also grateful to Carlos, Stephen, Echo, and Sambid for creating such a friendly
atmosphere in our lab. This truly is an exceptional laboratory that I will cherish forever. I would also like to express my gratitude to Dr. Andrew Mackay and Dr. Jennica Zaro, who have
been incredibly helpful in reviewing and editing my thesis. At last, I am truly fortunate to have such amazing parents who have consistently provided me
with unwavering support throughout my life. Their constant encouragement and belief in my
abilities have been instrumental in shaping the person I am today.

Table of Contents
Chapter One: 1. Introduction ..........................................................................................................1
1.1. Exosome biogenesis ....................................................................................1
1.2. Exosome distribution ..................................................................................4
1.3. Exosome uptake ..........................................................................................5
1.4. Exosome characters as drug carriers ...........................................................8
1.5. Sjögren’s Syndrome ....................................................................................9
1.6. Lysosome-associated membrane protein 2B ............................................10
1.7. Exosome engineering strategies ................................................................10
1.8. Exosome extraction and characterization .................................................11
1.9. Cell transfection …………………............................................................12
Chapter Two: 2. Materials and Methods.......................................................................................16
2.1. Plasmid .......................................................................................................16
2.2. Plasmid resuspension .................................................................................17
2.3. Plasmid amplification ................................................................................17
2.4. Plasmid Purification ...................................................................................19
2.5. NanoDropTM Spectrophotometer..............................................................22
2.6. Gene sequencing ........................................................................................23
2.7. HEK 293T cell and cell culture .................................................................24
2.8. Transfection and collection of lysates ........................................................27
2.9. Bicinchoninic Acid Assay (BCA) ..............................................................28

2.10. Western blotting (WB) .............................................................................29
2.11. Immunoflourescence (IF) .........................................................................33
2.12. iZON Size Exclusion Column .................................................................36
2.13. ZetaView Nanoparticle Tracking Analysis ..............................................38
2.14. Immunoprecipitation (IP) .........................................................................38
Chapter Three: 3. Results...............................................................................................................41
3.1. The amplified plasmid is identical to the original plasmid ........................41
3.2. Verification of the concentration of the vector to transfect HEK293T
cells ...................................................................................................................................41
3.3. LAMP-2B intracellular and extracellular amount change with plasmid
concentrations ...................................................................................................................42
3.4. Determination of LAMP-2B extracellular amount ....................................44
3.5. Determination of the association between LAMP-2B and DDK ...............46
3.6. Immunofluorescence analysis of transfected cells .....................................47
3.7. Determination of exosomes .......................................................................48
Chapter Four: 4. Discussion ..........................................................................................................52
Chapter Five: 5. Conclusion .........................................................................................................55
References .....................................................................................................................................57

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List of Figures
Figure 1. Exosome biogenesis ........................................................................................................3
Figure 2. Direct interaction and membrane fusion..........................................................................5
Figure 3. Exosomes internalization..................................................................................................6
Figure 4. Stable transfection and transient transfection.................................................................13
Figure 5. Plasmid map...................................................................................................................16
Figure 6. QIAGEN Plasmid Kit procedure from QIAGEN® Plasmid Purification Handbook....19
Figure 7. Comparison of different plasmid transfection concentration.........................................42
Figure 8. Comparison of LAMP-2B intracellular and extracellular amount.................................44
Figure 9. The different signal of lysate and media with and without plasmid transfection...........45
Figure 10. Image of HEK293T cells with and without transfection and secondary
control............................................................................................................................................48
Figure 11. The median particle size of exosome samples from HEK293T cells with and without
transfection.....................................................................................................................................49
Figure 12. The particle number of exosome samples from HEK293T cells with and without
transfection.....................................................................................................................................49
Figure 13. The size distribution of exosomes in the cell culture media (CCM) from transfected
and non-transfected cells................................................................................................................50
Figure 14. The different signal of exosomes in media with and without plasmid transfection.....51

iv
List of Tables
Table 1. Extravesicle subtype characteristics ................................................................................1
Table 2. Flow chart of purifying plasmid......................................................................................20
Table 3. The tube labels and component in each tube...................................................................24
Table 4. The dosage of transfection solution.................................................................................28
Table 5. Dilution of standard solution in BCA..............................................................................29
Table 6. Dilution of samples in WB..............................................................................................30
Table 7. Antibodies and dilution for WB.......................................................................................32
Table 8. Dosage of the transfection solution.................................................................................34
Table 9. Dilution ratios of primary antibody in IF.........................................................................35
Table 10. Dilution ratios of secondary antibody in IF...................................................................35
Table 11. Typical concentrate volume vs. Spin time (Swinging-bucket rotor) ............................37

v
Abbreviations
LAMP-2B: Lysosome-associated membrane protein 2B
WB: Western blotting
IP: Immunoprecipitation
NTA: Nanoparticle tracking analysis
IF: immunofluorescence
EVs: Extracellular vesicles
ESCRT: Endosomal sorting complex required for transport
ILVs: Intraluminal vesicles
MVBs: Multivesicular bodies
SNARE: Sensitive factor attachment protein receptor
ER: Endoplasmic reticulum
CDCs: Cardiosphere-derived cells
CMP: Cardiomyocyte specific peptide
SS: Sjögren's Syndrome
LG: Lacrimal gland
SG: Salivary gland
NOD: Non-Obese Diabetic
ICAM-1: Intercellular adhesion molecule-1
IBP: ICAM-1 binding peptide

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Abstract
Exosomes are small vesicles secreted by cells that aid in cell-cell communication and have
received much attention as new carriers for drug delivery. It has been found that exosomes are
ideal drug delivery carriers since they have good histocompatibility and can protect the
encapsulated therapeutic agents from degradation. LAMP-2B, a member of the lysosome
associated membrane protein families, is an exosome surface protein, whose N-terminal
extracellular domain can be genetically fused with targeting peptides to achieve a targeting effect
for these modified exosomes. The plasmid, pCMV6-lamp2b-Myc-DDK, contains the gene
sequence of LAMP-2B fused to a FLAG epitope tag. The goal of this project was to isolate
exosomes enriched in epitope-tagged LAMP-2B through transfection of this plasmid into
HEK293T cells. In this project, the author developed a robust set of protocols capable of
transfecting cells, tested transfection efficiency and determined epitope-tagged LAMP-2B
intracellular and extracellular abundance. Western blotting and immunoprecipitation techniques
were optimized to quantify protein levels, while nanoparticle tracking analysis was employed for
determining the number of exosomes in the cell culture media. These protocols were adapted
from the existing literature and modified to work with our samples. Compared with the
LAMP-2B amount of HEK293T cells without transfection, the LAMP-2B amount with epitope
tag increased, but the endogenous LAMP-2B appeared decreased. The exosomes present in the
transfected cell media exhibit a statistically significant increase compared to those found in
non-transfected cell media (p=0.03).

1
Chapter One
1. Introduction
1.1. Exosome biogenesis
Exosomes represent a subset of Extracellular vehicles (EVs). EVs are a heterogeneous group of
vesicles that play a crucial role in intercellular communication, contributing to the maintenance
of cellular homeostasis and response to stimuli. EVs were initially identified as pro-coagulant
particles derived from blood platelets in 1967 [1]. EVs can be categorized into three distinct
subtypes, namely exosomes, microvesicles, and apoptotic bodies [2]. Exosomes are produced by
exocytosis, microvesicles are formed by budding and shedding from the plasma membrane. Additionally, apoptotic bodies emerge as blebs during cellular demise [3]. There are variations in
size, shape, and constituent proteins among them, as illustrated in Table 1. Table 1. Extravesicle subtype characteristics [4]
Exosomes Microvesicles Apoptotic bodies
Origin Exocytosis Plasma membrane
budding
Blebbing
Size 30-200 nm 100-1000nm >1000 nm
Shape Spheroid Irregular Variable
Typical constituent
proteins
Tetraspanins, ESCRT proteins
(Alix, TSG101), integrins, heat
shock proteins
Integrins, selectins, CD40
ligand, flotillin-2, adenosine
diphosphate
ribosylation factor
6, phosphatidylserine
Annexin V, phosphatidylserine

2
The term "exosomes" was initially introduced in 1981 to describe plasma membrane vesicles
released from cultured cell lines through exudation [5]. The identification of exosome biogenesis
was documented in a study of the downregulation of transferrin receptors in reticulocytes
maturation process in 1992. Exosomes play an important role in selectively transporting proteins
that are lost during cellular maturation, while retaining major transmembrane proteins, through a
highly specific mechanism [6]. Further research shows that endosomal sorting complex required
for transport (ESCRT) proteins, including Alix and TSG101, and tetraspanins play a pivotal role
in the process of exosome biogenesis and efficient sorting of diverse cargoes such as proteins, lipids, RNA molecules, among others [7]. Additionally, lipid-enriched regions within the
endosomal membrane, such as sphingomyelin and ceramide, have the capability to transport
cargos independently of ESCRT machinery [8]. After endocytosis and formation of early endosome, exosomes originate from intraluminal
vesicles (ILVs) that are enriched within multivesicular bodies (MVBs). These exosomes are then
released from cells through exocytosis, as illustrated in Figure 1 [4].

3
Figure 1. Exosome biogenesis (Created with BioRender.com) (1) The fusion of primary
endocytic vesicles (2) Sorting into early endosome by returning the remaining cargo to the
plasma membrane (3) The formation of MVB/ late endosome by inward membrane budding
leading to cargo sequestration and distribution into intraluminal vesicles (4) The internalization
of cargoes from Golgi and cytosol and the elimination of cargoes (5) Transportation to the
plasma membrane (PM) (6) The fusion with PM (7) The secretion of exosomes from the ILVs. In the context of exosome secretion, MVBs can undergo fusion with lysosomes leading to
degradation. Rab GTPases is in charge of modulating the exosomes heterogeneously toward
either lysosomal degradation or extracellular release, depending on the specific cell type and
cargo composition [9]. The soluble N-ethylmaleimide-sensitive factor attachment receptor
(SNARE) complex, which consists of synaptotagmin on MVBs and syntaxin on the plasmid
membrane, docks MVBs with the plasma membrane, drives membrane fusion and consequently

4
promotes exosome secretion [10]. Understanding the process of exosome formation and release
is crucial as it plays a pivotal role in the development of novel therapeutic approaches. 1.2. Exosome distribution
After being released from cells, the localization of exosomes is determined by the proteins
present on their plasma membrane [4]. Exosomes are secreted by a wide range of cell types and
circulate within various biological fluids, including tears, blood, breast milk, saliva, and synovial
fluids [11]. Upon reaching target cell sites, exosomes are internalized through specific targeting
mechanisms involving ligand-receptor interactions or recognition of recipient cell-specific
membrane constituents. For instance, ovarian cancer cells possess exosomes with an abundance
of glycoproteins containing mannose and sialic acid, enabling them to selectively target specific
cell types [12]. Furthermore, exosomes can be engineered to incorporate diverse components to
achieve distinct functionalities, such as avoiding immune system clearance. for example, The
presence of CD47 on the surface of modified exosomes aids in evading recognition by immune
cells within the host's bloodstream [13]. Heterologous exosomes are commonly employed in research and pharmaceutical applications, with their distribution being influenced by the routes of administration [14]. For instance, oral
administration facilitates widespread distribution to various organs, including the brain [14]. Intravenous administration primarily targets the liver for distribution, while intratumoral
injection prolongs exosome survival within tumors [15-17]. Furthermore, intranasal
administration predominantly directs exosomes towards the brain [18].

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1.3. Exosome uptake
Reaching the target cells, exosomes can trigger downstream signaling by interacting with
receptors on the plasma membrane or merging with it to release their contents, as illustrated in
Figure 2 [4]. For instance, exosomes derived from umbilical cord blood, which contain tumor
antigens like MHC-I, MHC-II, and tetraspanins (CD34, CD80) on the membrane, induce T cell
proliferation to generate anti-tumor responses [19].
Figure 2. Direct interaction and membrane fusion (Created with BioRender.com)
a. Receptors located on the surface of exosomes and target cell plasma membranes have the
ability to interact, leading to the initiation of downstream signaling pathways in the recipient cell. b. Exosome membranes possess the capability to merge with the plasma membrane and release
their contents directly into the cytosol. Additionally, exosomes are mainly internalized through receptor-mediated endocytosis to release
cargoes [20]. The internalization processes encompass clathrin-mediated endocytosis, lipid
raft-mediated endocytosis, phagocytosis, caveolin-mediated endocytosis, and micropinocytosis

6
(Figure 3). These mechanisms are distinguished by their involvement of distinct elements [4]. Different mechanisms may be involved in one cargo uptake. For example, the process of
exosome uptake by bone marrow stem cells can also be partially facilitated by macropinocytosis
and membrane fusion, in addition to caveolin-dependent uptake [21]. The cargoes can be
subsequently released via exosomes and re-uploaded multiple times until they fuse with
lysosomes for degradation [22]. As previously mentioned, exosomes derived from CDCs have
demonstrated their ability to provide protection for the heart. This is due to the presence of
numerous anti-apoptotic microRNAs that are highly expressed within these exosomes [23, 24]. Once taken up by cells, exosomes transport their cargo of mRNA and miRNAs to the
Endoplasmic reticulum (ER), where they facilitate rapid translation and regulate gene expression
changes [25].

7
Figure 3. Exosomes internalization (Created with BioRender.com) a. Clathrin-mediated
endocytosis: clathrin (triskelion scaffold) and various transmembrane receptors and ligands form
clathrin-coated vesicles to entry cells. b. Lipid raft mediated endocytosis: Lipid rafts exhibit a
higher concentration of cholesterol, sphingolipids, and proteins anchored by
glycosylphosphatidylinositol (GPI). Changes in any of these factors may potentially impact the
process of exosome uptake. c. Caveolin-mediated endocytosis: caveolins, integral membrane
proteins, facilitate the formation of caveolae structures that mediate the internalization process of
caveosomes. d. Phagocytosis: Phagosomes encompass various entities, including bacteria, deceased cells, and even minute substances such as exosomes. Ultimately, they guide the
internalized contents towards lysosomes. e. Macropinocytosis: Macropinosomes are generated
through actin-driven lamellopodia, which facilitate the inward invagination of the plasma
membrane.

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1.4. Exosome characters as drug carriers
Exosomes, the carrier the project uses, are lipid bilayer nanoparticles secreted by almost all cells
[14]. Exosomes are also found in all body fluids, including tears and saliva, and are important
modes of cell-cell communication and substance exchange [11]. Exosomes selectively envelop
proteins, lipids, miRNA, mRNA, DNA, lncRNA, and other nucleic acids in the cytoplasm [10]. When exosomes bind to the recipient cells, they transport their contents to the recipient cells and
then affect the physiological and pathological processes of the recipient cells [4]. In addition, since the membrane of exosomes is derived from two successive invaginations of the cell
membrane, the spatial conformation of exosome membrane proteins is consistent with that of the
cell membrane proteins [26]. Therefore, the cell membrane proteins can be genetically
engineered to display homing peptides (ligands) on the surface of the exosomes and engender the
targeting of the exosomes. It has been found that exosomes have good biodistribution and
histocompatibility, are natural and stable, protect the encapsulated molecules from degradation, can penetrate biological barriers, have low immunogenicity/toxicity, and can carry a variety of
therapeutic agents at the same time, so they are ideal drug delivery carriers [27, 28]. There are many papers on the targeting properties of engineering exosomes as drug carriers, for
example, Kyle l. Mentkowski and Jennifer K. Lang engineered cardiosphere-derived cells (CDCs)
to express LAMP-2B, an exosomal membrane protein, fused to a cardiomyocyte specific peptide
(CMP), to generate an efficient exosomal delivery system that can improve cardiac tropism [29]. The author extends this idea to the treatment of Sjögren's Syndrome and plans to design

9
experiments to prove the HEK 293T cells can be permanently transduced to express the
reengineered LAMP-2B with tropism for markers of inflammation, namely ICAM-1. 1.5. Sjögren’s Syndrome
Sjögren's Syndrome (SS) is a multifaceted systemic autoimmune disorder that impacts
approximately 1% of individuals and results in the lymphocytic infiltration of the fluid-secreting
glands by B-, T- and antigen presenting cells, such as lacrimal glands (LG) and salivary glands
(SG) [30]. This infiltration leads to symptoms like reduced saliva production and insufficient tear
production [31]. The clinical manifestation also encompasses different sicca symptoms, such as
dryness in the vaginal area and urinary tract, tracheobronchitis characterized by dryness, xerosis
cutis and a painful throat. It also impacts the regular operation of bodily organs like the liver, joints, nervous system, and cardiovascular system [32]. These symptoms significantly impact an
individual's quality of life. Currently, symptomatic management of sicca symptoms and
immunosuppressive therapy for organ involvement are frequently employed in clinical practice
[32]. The occurrence of LG inflammation and dysfunction in male Non-Obese Diabetic (NOD) mice
is like that observed in SS, appearing at around 3 to 4 months before the onset of diabetes
[33-35]. Intercellular adhesion molecule-1 (ICAM-1), commonly overexpressed common in
inflammation and immune cells, is increased in the inflammatory lacrimal gland of male NOD
mice and can be targeted by ICAM-1 binding peptide (IBP) [36]. As we mentions above, Kyle l. Mentkowski and Jennifer K. Lang engineered CDCs to express LAMP-2B fused to CMP to

10
target cardiomyocytes [29]. We can theoretically fuse IBP with LAMP-2B to target diseased LG
in Sjögren's Syndrome in the future after we confirm the expression of LAMP-2B after
transfection in HEK 293T cells. More efficient and less toxic drugs can be created then. 1.6. Lysosome-associated membrane protein 2B
Lysosome-associated membrane protein 2B (LAMP-2B) is a member of the LAMP family of
lysosome-associated membrane proteins, which are mainly localized in lysosomes and
endophytes, with a small amount distributed on the surface of cells. In addition, a large number
of LAMP-2B is also expressed on exosomes and is used to prove targeting. For instance, Lydia
Alvarez-Erviti et al employ a fusion of LAMP-2B with the neuron-specific RVG peptide to
precisely target the brain in murine models [37]. Human LAMP-2B contains a 39-amino acid
signaling peptide, a large N-terminal extracellular domain, and a C-terminal transmembrane
region, containing a very short cytoplasmic tail region. The three LAMP-2 isoforms, LAMP-2A, LAMP-2B, and LAMP-2C, share an identical lysosomal domain at their N terminus but have
distinct transmembrane and C-terminal cytosolic domains composed of 11 amino acids [38]. Therefore, targeting peptides can fuse with the N-terminal extracellular domain of LAMP-2B to
achieve a targeting effect. 1.7. Exosome engineering strategies
Most exosomes exhibit limited affinity for specific cells, leading to the development of various
targeting techniques to enhance the therapeutic potential of systemically administered exosomes

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[39]. The current strategies can be broadly categorized into two main groups: approaches that
focus on modifying cells and those that involve altering exosomes directly. Modifying cells
enables the manipulation of exosome surface proteins to express and present target proteins. This
is achieved by incorporating the coding sequence of the desired ligand into a transmembrane
protein's coding sequence, specifically positioned between the signal peptide and the N-terminus
of its mature peptide. Subsequently, gene transfer vectors are employed to fuse the modified
gene with the cell, facilitating efficient expression. Commonly altered transmembrane proteins
encompass tetraspanins such as CD63, CD9, and CD81[40], LAMP-2B[37], glycosylphosphatidylinositol (GPI) [41], platelet-derived growth-factor receptors (PDGFRs) [42], and lactadhein within the C1C2 domain [43, 44]. Alternatively, exosome modification can be categorized into two approaches: passive-loading
techniques that rely on natural membrane interactions, such as encapsulation of hydrophobic
drugs by physical capture, and active-loading methods that involve temporary disruption of the
EV membrane to facilitate cargo influx, such as electroporation [39]. Furthermore, there is
continuous expansion in methodologies for loading non-native cargo into EVs, thereby further
broadening their therapeutic capabilities. 1.8. Exosome extraction and characterization
As the potential application value of exosomes has been continuously explored, the extraction
process of exosomes has made corresponding progress. Most of the current isolation and
extraction techniques cannot separate exosomes from lipoproteins with similar biophysical

12
properties and extracellular vesicles from non-endosomal pathways, resulting in low purity of
exosomes. Ultracentrifugation (UC), size-based separation, polymer precipitation, and
immunoaffinity capture are more commonly used. Exosomes of superior purity are typically
acquired through the implementation of multiple isolation techniques [26]. In addition, we generally identify whether the extracted components are exosomes from three
levels, including electron microscopy (EM) to identify exosome morphology, nanoparticle
tracking analysis (NTA) to identify exosome size, and Western Blot (WB) to identify exosome
surface protein markers [39]. Additionally, the storage methods of exosomes mainly include freezing, freeze-drying, and spray
drying, but long-term storage will affect their biological activity [26]. 1.9. Cell transfection
Cell transfection refers to the process of introducing foreign nucleic acids into cells [45]. The
primary objective of transfection is to investigate the role of genes or gene products by
manipulating specific gene expression in cells, either increasing or decreasing it, and to generate
recombinant proteins in mammalian cells [46]. In the process of cell transfection, many factors
need to be considered, such as target cell type, transfection efficiency, cytotoxicity, and stability
of exogenous genes, in order to select the appropriate transfection method and conditions. For target cell type, HEK293T cells are highly transfectable. HEK293 cells are often selected for
the expression of recombinant proteins in research due to their rapid timelines, usually 5-7 days, and comparable or high titers [47].

13
To achieve stable transfection, introduced genetic material, which often has marker genes for
selection, is integrated into the host genome and maintains transgene expression even after host
cell replication (Figure 4a) [48]. In contrast to stably transfected genes, transiently transfected
genes are only expressed for a limited time and do not integrate into the genome, and transiently
transfected genetic material may be lost due to environmental factors and cell division (Figure
4b) [49]. The lipofectamine 3000 Transfection Kit utilized in this research employs cationic
liposome-mediated transfection, a kind of transient transfection, allowing for high transfection
efficiency with minimal toxicity.
Figure 4. Stable transfection and transient transfection (Created with PowerPoint) a. Stable
transfection: Recombinant DNA (red wave) is transported into the nucleus by passing through
the cell and nuclear membranes. The recombinant DNA becomes integrated into the host genome
(black wave) and is expressed continuously. b. Transient transfection: Some of the foreign DNA
enters the nucleus but does not integrate into the genome. (Red circles represent proteins
expressed from transfected nucleic acids. Black arrows indicate delivery of foreign nucleic
acids.)

14
The common transfection methods include chemical method, physical method, and viral vector
method [45]. Chemical transfection is the predominant method utilized in research [50]. Chemical methods typically involve the use of cationic polymers, calcium phosphates, cationic
lipids, and cationic amino acids [50]. The fundamental principles of the chemical approach are
similar in that positively charged chemicals form complexes with negatively charged nucleic
acids [45]. These positively charged complexes are attracted to the negatively charged cell
membrane and enter the cell membrane through endocytosis and phagocytosis. The transfected
DNA must be delivered to the nucleus for expression. The transfection efficiency of chemical
methods is influenced by factors such as the nucleic acid to chemistry ratio, solution pH, and cell
membrane conditions. The efficiency of these methods is lower compared to other methods, especially in vivo. However, they offer the advantage of relatively low cytotoxicity, absence of
mutagenesis, no additional carried DNA, and no size limitation on the packaged nucleic acids
[45]. Physical transfection is a technique that utilizes physical methods to transport nucleic acids, such
as microinjection, biological particle delivery, electroporation, laser transfection, ultrasound
delivery and magnetic field delivery [51-53]. These techniques involve the direct delivery of
nucleic acids into the cytoplasm or nucleus, or the creation of pores in the cell membrane to
facilitate nucleic acid passage. Virus-mediated transfection, also known as transduction, involves the integration of the viral
vector and target DNA into the host genome, allowing for stable transgene expression through

15
replication as part of the host genome [54]. The primary drawback of virus-mediated transfection
is the potential for inflammatory responses and insertional mutations caused by the introduction
of viral vectors [55]. The random integration of viral vectors into the host genome may lead to
disruption of tumor suppressor genes, activation of oncogenes, or interruption of essential genes. Additionally, this approach is limited in terms of space for maintaining infectiousness of
virus-loaded foreign genes [45]. This study aims to investigate the impact of transfection on cellular behavior and the expression
of recombinant proteins, specifically focusing on cytotoxicity and the stability of exogenous
genes.

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Chapter Two
2. Materials and Methods
2.1. Plasmid
The plasmid utilized in this project is the Lamp2 (NM_001290485, LAMP-2B) Mouse Tagged
ORF Clone obtained from Origene (CAT#: MR229796, Figure 5). It functions as an expression
plasmid harboring a Myc-DDK tag. The vector employed is pCMV6-Entry (PS100001). Kanamycin serves as the selection agent for E. coli. The origin reading frame (ORF) clone
undergoes ion-exchange column purification and is shipped within a 2D barcoded Matrix tube
containing 10ug of transfection-ready, desiccated plasmid DNA.
Figure 5. Plasmid map

17
2.2. Plasmid resuspension
After centrifuging the sample at 5,000xg for 5 minutes, the tube was carefully opened and 100ul
of sterile water was added to dissolve the DNA. This step ensures that the DNA is fully
solubilized and ready for downstream applications. Once the water has been added, the tube was
closed and incubated for an additional 10 minutes at room temperature. After incubation, a brief
vortexing of the tube followed by a quick spin (less than 5000xg) will help concentrate any
remaining liquid at the bottom of the tube. It is important not to exceed this speed as higher
speeds can cause damage to delicate plasmid structures. It's important to note that storing suspended plasmid DNA at -20°C will help maintain its
stability over time. In fact, when stored properly, this type of DNA can remain stable for up to
one year from date of shipping. 2.3. Plasmid amplification
To prepare the culture medium, Microbiology LB broth (MILLER) weighing 6.25g was
dissolved in 250ml of water that has been filtered using a Vent filter MPK01 from Millipore
(Catalogue No TANKMPK01). This filtered water was used throughout the experiment unless
otherwise specified. When using this culture medium to culture E.coli transfected with the
plasmid, kanamycin at 25 ug/mL was added. In addition to the LB broth, LB agar weighing
3.75g was dissolved in 250ml of water. To ensure sterility, these mixtures were autoclaved for 20
minutes. Once the LB agar solution has cooled down and was no longer too hot, kanamycin at 25
ug/mL was added in LB agar medium and ten dishes were prepared aseptically with each

18
containing approximately 25ml of the prepared LB agar solution. After preparing the selective
plates, they were left open with their caps slightly lifted and allowed to cool at room temperature
for about one hour. Finally, these prepared petri dishes were incubated at a temperature of 4℃. If
they are intended for long-term storage, they are sealed with parafilm. To transform the plasmid to E. coli, one vial of One ShotTM TOP10 chemically competent cells
were thawed on ice. 1μl of the plasmid (10ng/μl) was added into a vial of cells and mixed gently. The vials were incubated on ice for 30 min and then the cells were heated for 30 seconds at 42℃
without agitation. The vials were removed from the 42℃ bath and placed on ice for 2 min. 250
μl of pre-warmed S.O.C. Medium was added aseptically to each vial. The vials were capped
tightly and shaken horizontally at 37℃ for 1 h at 225 rpm in a shaking incubator. The remaining
transformation mix was stored at 4℃ if using the next day. To amplify the plasmid in the cells, 250μl of the transformed cells was spread on a pre-warmed
selective plate and incubated at 37℃ for 18 hours. A single colony was picked from a freshly
streaked selective plate and inoculated into a starter culture of 3 ml LB broth medium
supplemented with kanamycin (25μg/ml). 4 groups of LB broth medium were created: A, B, C, D. They were incubated at 37°C with vigorous shaking (250 rpm) for approximately 8 hours or
overnight. The tube or flask used had a volume at least four times greater than that of the culture. The most turbid starter cultures (B and D) were diluted by a factor of 1/1000 in 250 ml selective
LB medium containing kanamycin (25μg/ml). These diluted cultures were cultivated at 37°C
with vigorous shaking (250 rpm) for 12–16 hours. The transformed cells exhibited proficient

19
proliferation in medium while effectively replicating and amplifying the plasmid within their
cellular environment. 2.4. Plasmid Purification
Bacterial lysates were clarified by centrifugation using QIAGEN Plasmid Kits. Clarified lysates
were loaded onto anion exchange columns, and plasmid DNA was subjected to selective binding
to resin under appropriate low salt conditions and pH. RNA, proteins, metabolites, and other
low-molecular-weight impurities were removed with a medium-salt wash, and pure Plasmid
DNA was eluted with a high-salt buffer (Figure 6). The resulting DNA was concentrated and
desalted by isopropanol precipitation and collected by centrifugation. The specific process is
shown in the flow chart (Table 2).
Figure 6. QIAGEN Plasmid Kit procedure from QIAGEN® Plasmid Purification
Handbook

20
Table 2. Flow chart of purifying plasmid

21

22
2.5. NanoDrop
TM Spectrophotometer
The NanoDrop
TM Spectrophotometer is utilized for quantifying the concentration of DNA, RNA, and protein. Following instrument initialization, dsDNA was selected as the measurement mode. The detection area was rinsed with RNase-free water and subsequently wiped clean using a

23
Kimwipe. Once the instrument was prepared for measurements, 2 μl of RNase-free water was
loaded to establish a blank reading. Subsequently, 2 μl of the sample was loaded onto the
detection spot to measure plasmid concentration while evaluating DNA and RNA purity through
A260/A280 and A260/A230 ratios respectively. The 260/280 value of around 1.8 is considered
pure for DNA, while a ratio of around 2.0 is considered pure for RNA. This ratio can indicate the
presence of contaminants such as proteins or phenols if it is significantly lower than expected
[56]. The 260/230 value represents another measure of nucleic acid purity, expected values for
which range from 2.0 to 2.2. If this ratio is considerably lower than expected, it may suggest the
presence of contaminants that absorb at 230 nm. After each measurement, the instrument was cleaned with RNase-free water and Kimwipe was
used for further cleaning purposes. After measuring the amplified plasmid concentration, the plasmid was centrifuged at 14000rpm
for 10 min at room temperature. The plasmid was changed into a new tube and stored at –20°C
as DNA may degrade in the absence of buffering and chelating agents. 2.6. Gene sequencing
After performing purification on the amplified plasmid, the sequence accuracy was verified
through utilization of Forward sequencing primer VP1.5 and Reverse sequencing primer XL39
(Origene). The specific steps involved adding 10 ul of dH2O to generate a 10-uM stock in the
tube. The tube was incubated for 10 minutes at room temperature or overnight at 4℃. The tube
was vortexed briefly and then spun quickly to concentrate the liquid at the bottom. The primer

24
stock was prepared for addition to a DNA sequencing reaction (1ul=10pmol). The samples (B1, B2, D1, D2, O1, O2) were submitted to the GENEWIZ from Azenta Life Sciences and the
sequencing result was came out after one week. (Table 3)
Table 3. The tube labels and component in each tube
Label B D O F R
Component
6.55 μl
B0
4.72μl
D0
12.36 μl
O0
10μl
F 0
10μl
R0
93.45μl
dH2O
95.28μl
dH2O
7.64μl
dH2O
10μl
dH2O
10μl
dH2O
Label B1 B2 D1 D2 O1 O2
Plasmid Amount:600ng
Plasmid Volume:10μl
Primer Amount: 25pmol
(pmol=μM*μL)
Primer Volume:5μl
10μl
B
5μl
F
10μl
B
5μl
R
10μl
D
5μl
F
10μl
D
5μl
R
10μl
O
5μl
F
10μl
O
5μl
R
B0: 0.916 μg/μl cloning plasmid
D0: 1.270 μg/μl cloning plasmid
O0: 97 ng/μl original plamid
F0: Forward sequencing primer VP1.5 (1μl=10pmol)
R0: Reverse sequencing primer XL39 (1μl=10pmol)
2.7. HEK 293T cell and cell culture
Human embryonic kidney 293 cells (HEK 293T cells), a derivative human cell line expressing a
mutant variant of the SV40 large T antigen, have been extensively employed in cell biology
research for protein synthesis and recombinant retrovirus production due to their consistent
growth and transfection efficiency [57].

25
To thaw the frozen HEK 293T cells, warm DMEM with 10% FBS was prepared and a water bath
at 37℃ was set up prior to retrieving the cells from liquid nitrogen. The cryotube
TM vial
containing the cells was immediately transferred to dry ice after removal from liquid nitrogen. Subsequently, the cryotube
TM vial was warmed in a 37℃ water bath for precisely 90 seconds, ensuring that the cap remained above the water surface. To maintain sterility, the vial's exterior
surface was meticulously cleaned by spraying it with 70% ethanol. 0.5 mL of DMEM with 10%
FBS was aseptically added into the vial and performed gentle aspiration and expulsion
movements to mix thoroughly before this solution was transferred into a sterile 15 mL tube
containing an additional volume of 10 mL DMEM with 10% FBS. This tube was centrifuged at
2000 rpm for five minutes. Supernatant was carefully removed and discarded, then the cells were
resuspended in precisely 4 mL of DMEM with 10% FBS through gentle aspiration and expulsion
motions repeated five to six times. Finally, these resuspended cells were transferred into a
suitable cell culture flask containing sufficient volume of DMEM with 10% FBS based on flask
capacity requirements. To passage the cells, the DMEM with 10% FBS was carefully removed from the side wall of the
culture flask without disturbing the cells at the bottom. Taking a 75 cm3 flask as an example, it
was gently washed with 10 mL of PBS by allowing the liquid to flow through. The volume of
PBS was adjusted accordingly for other sizes of flasks. For a 1:10 split, 2 mL of 0.05%
Trypsin-EDTA was added and ensured that it covered the entire bottom surface. The flask was
placed in a Series II Water Jacketed CO2 Incubator (ThermoForma) set at 5.0% CO2, 37.0℃, and

26
98% RH for one minute until all cells detached from the bottom of the flask. Once detached, 8
mL of DMEM with 10% FBS was added to disperse cells without introducing any bubbles while
gently pipetting up and down. 1 mL of cell solution was taken out and transferred into a new
flask containing 11 mL of DMEM with 10% FBS. The cell type, date, the ratio of split and
passage number were written on the flask. Cell condition was verified under a microscope to
ensure their viability. Subsequently, this new flask was placed in an incubator and cell growth
was monitored daily to prevent contamination or overgrowth before performing another round of
splitting when necessary. To enumerate the cells, a mixture was prepared comprising 15 μL of Trypan blue and 15 μL of
thoroughly mixed cell suspension in cell culture media. Subsequently, 10 μL of this mixture was
added to each well on the counting chip and the TC20TM Automated Cell Counter (BIO-RAD)
was employed for quantification. Two consecutive counts did not exhibit a discrepancy
exceeding 1*10^6. To freeze the cells for future use, they were collected prior to cell counting and determination of
total cell number. The cells were then centrifuged at 2000rpm for 5 minutes and the supernatant
was discarded. Cryotube
TM vials were prepared and labeled with pertinent information including
cell type, date, cell count, and passage number. A frozen media solution consisting of 50% FBS, 40% DMEM with 10% FBS, and 10%DMSO was prepared by vortexing. Next, a volume of 5
mL of the frozen media was added to the cells followed by gentle resuspension via pipetting up
and down. Additional frozen media was added until desired dilution was achieved before

27
transferring a volume of 1 mL of the cell suspension into each vial while ensuring that caps were
not closed too tightly. Finally, these vials were placed in a freezing container containing 70%
ethanol which facilitated slow solidification at -80℃ over 24 hours before being transferred to
liquid nitrogen storage for long-term preservation purposes. The addition of DMSO enabled a
controlled and gradual freezing process for cells, effectively preventing the formation of ice
crystals that can be detrimental to cell viability. However, DMSO possessed inherent toxicity;
therefore, swift execution of subsequent steps was crucial upon introducing the frozen media into
the cells and promptly removing any residual DMSO following cell thawing. 2.8. Transfection and collection of lysates
Pre-experimental validation was required to determine the optimal vector concentration for
transfection, ranging from 0.5 μg/well to 5 μg/well (including concentrations of 0.5, 1.5, 2.5, 3.5
and 4.5 μg/well). The HEK 293T cells were seeded in a 6-well plate one day prior to the
experiment, with a cell concentration of 6.5 × 10^5 cells per well and a plating medium volume
of 2 mL. Solution B was prepared first by using the lipofectamine 3000 Transfection Kit
(Thermo Fisher Scientific, CAT.# L3000015) and OPT-MEM, followed by preparing solution A
(by adding plasmid into OPT-MEM initially). The dosage was shown in the Table 4. After
keeping them separately for five minutes, solution B was added to solution A followed by
thorough mixing and incubation for thirty minutes.

28
Table 4. The dosage of transfection solution
Original
0.916μg/μl
Solution A
P3000 7.5μl
Plasmid(μl) OPT-MEM(μl)
control 0 180
0.5μg/well 0.81 179.19
1.5μg/well 2.46 177.54
2.5μg/well 4.1 175.9
3.5μg/well 5.73 174.27
4.5μg/well 7.36 172.64
Slution B
Lipofectamine™3000
45μl
187.5 for each Solution A
OPT-MEM 1080μl
OPT-MEM 1080μl
250μl mixture was added symmetrically to each well and incubate at 37℃ overnight. The media
was removed and the cells were gently washed with 2ml/well of PBS, and subsequently fresh
2ml/well of DMEM supplemented with 10% FBS was added. The cells were incubated overnight. Following this, the media was removed, the cells were gently washed twice with ice-cold PBS, and then 300μl of RIPA buffer (150mM NaCl, 50mM Tris-HCl, 0.5% Sodium deoxycholate, 0.5mM EDTA, 0.1%Tx-100, 1%NP-40) containing a protease/phosphatase inhibitor at a dilution
ratio of 1/200 was added. All cells were aspirated from the dish using a syringe, performing 10
cycles of up and down movements to disrupt cells. Subsequently, the cell suspension was
carefully transferred into a pre-cooled microcentrifuge tube. The whole cell extract (WCE) was
centrifuged at 14000rpm for 10 minutes at 4 ℃. Finally, the supernatant was transferred into
new microtubes. 2.9. Bicinchoninic Acid Assay (BCA)
Using the Micro BCA™ Protein Assay Kit (Thermo Fisher Scientific, Cat.# 23235), BCA

29
solution was prepared with the ratio of A:B:C as 25:24:1. For each cell, 75μl of solution A, 72μl
of solution B, and 3μl of solution C were mixed. Standard solutions were prepared using a stock
solution of BSA at a concentration of 200μg/ml as Table 5 showed. The samples were diluted by
adding 5μl to 245μl (1:50) solution. 20μl of the sample/standard solutions and 150μl of ABC
solution were added to each well. The plate was incubated for 1.5 hours at a temperature of 37℃
while wrapping in tinfoil. Then the solution was cooled down to room temperature for
approximately seven minutes. The plate export was read at an absorbance wavelength of 562nm
and the volume required for Western blotting was calculated accordingly. Table 5. Dilution of standard solution in BCA
2 fold serial dilution
Tube # BSA(μl) Water(μl) Conc.(μg/ml)
1 100 200
2 50 50 100
3 50 50 50
4 50 50 25
5 50 50 12.5
6 50 50 6.25
7(Blank) 50 0
2.10. Western blotting (WB)
The BCA result was utilized for determining the appropriate dilution ratio of the sample to
ensure a consistent protein content of 25-30 μg per lane on the gels. The samples, water, and dye, 6X Laemmli SDS sample buffer (Thermo Fisher Scientific, Cat.# J60660.AC), were combined in
tubes according to the Table 6, vigorously mixed by vertexing, and subsequently centrifuged

30
briefly to ensure complete solution settling at the bottom. Table 6. Dilution of samples in WB
The sample tubes were heated at a 98-100℃ for 8-10 minutes. After allowing the tubes to cool
down to room temperature and then centrifuging them briefly. A 10% Mini-Protein TGX Gels
with 10 wells (BIO-RAD, Cat.#4561034) by moistening the Electrophoresis Chamber
(BIO-RAD, Cat.#1658004) was prepared by removing the comb and strip in the gel, assembling
the gels in the chambers, and adding the appropriate amount of 1x Tris-Glycine-SDS buffer. Next, 12μl of Full Range RainbowTM Recombinant Protein Molecular Weight Marker (CAT#
RPN800E) along with 26μl of room temperature samples was added into lanes on the gel. The
voltage was set at an initial value of 90v for the first 25 minutes followed by increasing it to
150v for the remaining time, totaling approximately 1.5 hours. Complete dye was migrated from
the gel during electrophoresis process.
Trans Conc.μg/well Conc.μg/ml *50 /1000 195μg/ H2O
Control 114.34 5717 5.717 34.1 115.9
0.5 100.208 5010.4 5.0104 38.9 111.1
1.5 93.844 4692.2 4.6922 41.6 108.4
2.5 117.466 5873.3 5.8733 33.2 116.8
3.5 112.244 5612.2 5.6122 34.7 115.3
4.5 102.136 5106.8 5.1068 38.2 111.8
Dye 50μl
Total 200μl
Concentration 0.975μg/μl

31
To transfer the gels onto the membrane, the blocking buffer was prepared by dissolving 5%
Blotting-Grade Blocker (BIO-RAD, Cat.#1706404) in 1x TBS (e.g., mix 2g of Blocker with
40ml of TBS). The iBlot™ 2 Gel Transfer Device (ThermoFisher Scientific, Cat.# IB21001) was
preheated for a few minutes. The gel was carefully removed from the chamber while ensuring it
remains moist. iBlot™ 2 PVDF Regular Stacks (Invitrogen by Thermo Fisher Scientific, REF
IB24001) were utilized and the gel was placed onto the transfer membrane. A filter paper was
placed that has been pre-soaked in deionized water onto the gel and the Blotting Roller was used
to eliminate any air bubbles. The white separator and position the Top Stack over the soaked
filter paper were taken off, using the Blotting Roller again to remove any remaining air bubbles. The electrical contacts of the Absorbent Pad with those on top of the Top Stack were aligned
before placing it on top. Some deionized water was introduced into the stack's center and the
device was closed. Transfer was initiated by selecting Point Template—>P0 option. Once
completed, the membrane was promptly immersed in the blocking buffer and incubated at room
temperature on the shaker for one hour. To incubate the primary antibody, 10 mL of the primary antibody solution was prepared in the
blocking buffer containing 0.1% Tween-20, as indicated by the ratios provided in Table 7. Subsequently, the membrane was gently shaken and incubated overnight at 4 ℃ in this mixture. For the secondary control membrane, it was incubated in a solution consisting of 10 mL of
blocking buffer with an addition of 0.1% Tween-20. After a brief wash with 1x TBS containing 0.05% Tween-20, the membrane was washed four

32
times for 10 minutes each using 1x TBS supplemented with 0.05% Tween-20. To incubate the secondary antibody, membranes were treated with 15mL of a diluted solution
containing the secondary antibody in the blocking buffer supplemented with 0.1% Tween-20 at
room temperature while gently shaking for 1 hour. After a brief wash with 1x TBS containing 0.05% Tween-20, four subsequent washes were
performed with 1x TBS supplemented with 0.05% Tween-20 for a duration of 10 minutes each. Finally, the washing steps were concluded by rinsing with 1x TBS solution. Table 7. Antibodies and dilution for WB
Primary antibody Dilution for
WB
Corresponding
secondary
antibody
Dilution for
WB
Anti-LAMP2B
abcam
CAT# ab18529
1:1000 Anti-Rabbit lgG
HRP
Promega
CAT#HRPW401B
1:10000
Anti-DDK
ORIGENE
CAT#TA50011-30
1:2000 Anti-mouse lgG
HRP
Promega
CAT#HRPW402B
1:20000
GAPDH
Biochain Institute
CAT#Y3322
1:5000 Anti-mouse lgG
HRP
Promega
CAT#HRPW402B
1:50000
To visualize the membranes, they were immersed in a solution of ECL western blot substrate (1:1
peroxide solution:luminol enhancer solution) for 1 minute. Excess solution was carefully
removed, and the blot was placed between plastic sheets without any bubbles before being
exposed to X-ray film. The resulting images were captured using ChemiDoc Imaging Systems
from BIO-RAD.

33
To visualize other proteins or optimize protein detection, the blot membrane was washed with
TBS and immersed in a striping buffer (Restore™ Western Blot Stripping Buffer, Thermo
Scientific™, Cat#21059) at room temperature for 15 minutes. Afterward, the striping buffer was
removed and the blot was rinsed with TBS. Subsequently, the membrane was re-blocked for 1
hour. The steps were repeated to incubate with the primary antibody as mentioned above. 2.11. Immunofluorescence (IF)
Immunofluorescence (IF) utilizes the affinity of antibodies to their corresponding antigens to
direct fluorescent dyes towards specific biomolecules within a cell. As a result, it enables the
examination of the spatial distribution of the targeted molecule across the sample using a
fluorescence microscope. The specific methods are as follows. Twelve coverslips were immersed in 100% ethanol and subjected to flame treatment to remove
any residual alcohol. Four wells were set up for transfection with pCMV6-lamp2b-Myc-DDK, another four wells were as control without primary antibody, and an additional four wells were
for without transfection. 1mL of HEK293T cells was aseptically seeded on coverslips in a
12-well plate at a density of 2.2*10^5 cells per well. The plate was incubated at 37℃ in the
incubator for 24 hours. To transfect cells, lipofectamine 3000 Transfection Kit and OPT-MEM were employed with the
following dosage (Table 8). Solution B was prepared first. The plasmid was added to OPT-MEM
initially, followed by P3000 to prepare solution A. Solutions A and B were incubated separately
for 5 minutes. Subsequently, solution B with solution A were combined and incubated for 30

34
minutes. 100μl of the mixture was symmetrically added into each well of the cell plate. For the
non-transfection group, 100μl of OPT-MEM was added instead. The cells were incubated at a
temperature of 37℃ overnight. Table 8. Dosage of the transfection solution
Original
0.916μg/μl
Solution A
P3000 20μl
Plasmid(μl) OPT-MEM(μl)
0.6μg/well 6.55 473.45
Slution B
Lipofectamine™3000 20μl
OPT-MEM 480μl
The media was removed and the cells were gently rinsed with 1ml/well of warm PBS twice for 5
minutes each. The plate was incubate overnight. Cells were fixed with a 1:1 mixture of methanol and acetone for intracellular protein analysis, at -20℃ for 15 minutes. The cells were washed three times with PBS for 5 minutes each. Non-specific binding sites were blocked by incubating the cells in 1% BSA in PBS on a shaker at
room temperature for 1 hour (prepared by dissolving 0.5g BSA in 50 mL PBS). The primary
antibody was diluted in 1% BSA according to the ratios specified in Table 9. 60 µL of diluted
antibodies was dispensed onto Parafilm for each coverslip, then carefully placed the coverslip
cell side down onto the Parafilm surface. The plate containing coverslips and Parafilm was
incubated overnight at 4℃, a small volume of water was added to the plate to ensure cellular
hydration was maintained.

35
Table 9. Dilution ratios of primary antibody in IF
Primary antibody Dilution Antibody
amount
Blocking Buff
amount
Anti-LAMP2B antibody
(abcam ab18529)
1:100 10 µl 990 µl
Anti-DDK (FLAG) monoclonal antibody
(ORIGENE TA50011-30)
1:1000 1 µl
After the primary incubation, the coverslips were carefully placed with the cell side facing up
back onto the plate. Subsequently, three washes were performed with PBS for 15 minutes each. A
solution of secondary antibodies was prepared by diluting them in 1% BSA, as the Table 10
showed, and 60 µL onto Parafilm was added for each coverslip. Then, the coverslips were gently
reverted so that their cell side faced down on top of the Parafilm. The plate was incubated at a
temperature of 37℃ for one hour. Table 10. Dilution ratios of secondary antibody in IF
Secondary antibody Dilution Antibody
amount
Blocking
Buff amount
Goat anti-Rabbit IgG (H+L) Cross-Adsorbed
Secondary Antibody, Alexa Fluor™ 488 (Invitrogen™
A -11008)
1:200 4 µl 786.4 µl
Rabbit anti-Mouse IgG (H+L) Cross-Adsorbed
Secondary Antibody, Alexa Fluor™ 568 (Invitrogen™
A-11061)
1:200 4 µl
Dapi 1:500 1.6 µl
Alexa Fluor™ Plus 647 Phalloidin(Invitrogen™
A30107)
1:200 4 µl
The coverslips were transferred back to the cell plates and proceeded with three washes using
PBS for 15 minutes each. Subsequently, the coverslips were delicately mounted onto slides with

36
a drop of ProLong
TM Gold antifade reagent (Invitrogen ™ P36934) in the middle and allowed
them to air-dry overnight before sealing them meticulously with nail polish. Images were
captured using a confocal microscope. 2.12. iZON Size Exclusion Column
The qEVoriginal Columns (iZON), a size exclusion chromatography column designed for
efficient isolation of extracellular vesicles, were employed to isolate exosomes from cell media
in this study. To collect the cell media, HEK 293T cells were seeded in a 6-well plate and transfected one day
prior. Subsequently, the existing media was removed and replaced with fresh 2ml/well of DMEM
supplemented with 10% FBS, followed by overnight incubation. The cells was gently washed
twice using 2ml OPT-MEM/well and replenished with an additional 2ml OPT-MEM/well. After a
further 24 hours, the media was carefully collected, placed on ice and then centrifuged at 2000
rpm for 5 minutes at a temperature of 4℃ to separate any cellular debris. The resulting
supernatant was transferred to new tubes for subsequent processing. The collected media was
concentrated utilizing Amicon® Ultra Centrifugal Filters-100K (Millipore Sigma, UFC9100) at
room temperature by centrifuging at a speed of 4000 rpm for 40 minutes (modified from Table
11). Finally, the filter device was inverted and spun for two minutes at a force of
approximately1,000×g to obtain concentrated solute samples.

37
Table 11. Typical concentrate volume vs. Spin time (Swinging-bucket rotor)
Spin conditions: 4,000 × g, room temperature, 15 mL starting volume. Shaded volumes were
used for the calculation of protein recovery. (From SigmaAldrich.com)
The columns were pre-warmed at room temperature for 1.5 hours, and all liquid undergone
filtration using Millipore filters followed by centrifugation at 200rcf for 5 minutes to remove any
air bubbles. Each column was utilized up to five times with the same type of sample. The liquid
within the column was poured off, and then the column was washed with 17mL (2 times the
volume of the column) of PBS solution. Next, 500μl of the concentrated solute sample was
added and the effluent was begun to collect. When it was below the solid level in the column, PBS solution was topped up to maintain a volume of approximately 1-2mL above the solid level
to maintain flow rate consistency. After collecting an effluent volume of 2.9mL, exosome
effluent was collected specifically measuring around 1.2mL. To wash the column thoroughly, 8.5 mL of NaOH solution (0.1mol/L) was rinsed, followed by
17mL PBS solution until only about 1-2 mL was left. The remaining solution was poured out.

38
Finally, 2 mL 20% alcohol was added before closing the lid and the column was stored at 4℃. 2.13. ZetaView Nanoparticle Tracking Analysis
ZetaView Nanoparticle Tracking Analysis (ZVNTA) with corresponding software enables the
measurement of particle diameter, zeta potential, and particle concentration in the solution. Before starting, an automated machine wash or manual addition of 2mL bubble-free quality
water was performed for effectively removing any remaining particles in the machine until the
particle count in each channel remained below 10^5. The standard particle solution was gently
shaken and slowly added 2mL into the machine while carefully monitoring sensitivity and liquid
speed to ensure optimal conditions for measurement. After measuring, a machine wash was
performed. The sample was diluted using quality water based on its concentration and 1mL
diluted sample was introduced into the machine. Both sensitivity and liquid speed were verified
that were suitable before proceeding with sample measurement. Measurements were repeated
twice for each sample to enhance accuracy. Finally, a machine wash was performed and followed
by air pumping within the system. 2.14. Immunoprecipitation (IP)
Immunoprecipitation, a technique for selectively isolating a protein antigen from solution using
an antibody that specifically binds to the target protein, was employed to extract the tag protein
and examine its combination with LAMP-2B. This allows for the detection of transfection effects
on LAMP-2B expression. The methods used in this study is as follows. After collecting the WCE or lysates, 100 μL samples were heated at 95°C for 5 min to denature

39
the lysate. The suspension was diluted with 0.9 mL of non-denaturing lysis buffer (Cell Lysis
Buffer (10X), Cell signaling technology, CAT#9803) containing a protease/phosphatase inhibitor
at a ratio of 1:100. The solution was gently mixed. The DNA was fragmented by passing the
lysed suspension through a needle attached to a 1 mL syringe for approximately 5-10 times. This
mechanical disruption was repeated until the viscosity was reduced to manageable levels. Incomplete digestion and fragmentation of DNA can potentially interfere with subsequent
centrifugation steps for separating pellet and supernatant fractions. The mixture was incubated on
ice for 5 min. To initiate immunoprecipitation, 1000 μL of culture supernatant and 5 μg of LAMP-2B antibody
were introduced into a microcentrifuge tube placed on ice. The sample was incubated overnight
at 4°C with gentle agitation in the presence of the antibody. The duration of this incubation
period was adjusted based on the protein quantity and affinity characteristics of the antibody
employed. 50 µL of Dynabeads™ Protein G (ThermoFisher Scientific, CAT#10003D) was
prepared for each sample by resuspending Dynabeads™ magnetic beads in their vial through
vortexing for more than 30 seconds. 50 µL (1.5 mg) of these magnetic beads was transferred to a
separate tube and placed onto a magnet to facilitate separation from any remaining solution. Subsequently, the supernatant was removed without disturbing the beads. Finally, pipette tips
were handled by cutting off their ends to prevent potential damage to these valuable beads during
usage. The slurry was thoroughly mixed and 50 μL of the beads were added to each sample, ensuring samples were kept chilled on ice. To minimize bead adherence to the pipette tip,

40
movement was limited and a tip cut was performed 5 mm from the top. The lysate-bead mixture
was incubated at 4°C with rotary agitation for a duration of 4 hours. To remove non-target proteins, the tube was placed on a magnet and the supernatant was
discarded from the beads. The protein of interest was specifically bound to the antibody coating
on the beads. The beads were washed three times with 100 μL of PBS to eliminate any
non-specific binding. For each wash, the beads were gently mixed with PBS, centrifuged at 4°C
or placed the tube on a magnet, and the supernatant was discarded. The wash buffer was
carefully removed as much as possible from the beads. The complex was prepared for elution
from the beads. To elute the target protein, 50 µL of beads were heated in 50 µL of 2x SDS loading buffer
without DTT at 50°C for 10 minutes. The beads were pelleted, the supernatant was transferred to
a new tube, and 100 mM DTT was added in 2x SDS loading buffer (50 μL). The eluted samples
were boiled for 5 minutes. The sample content was analyzed using western blotting techniques.

41
Chapter Three
3. Results
3.1. The amplified plasmid is identical to the original plasmid. The pCMV6-lamp2b-Myc-DDK plasmid is transformed into DH5α chemically competent cells
for amplification, followed by selection of the cells using kanamycin. To validate the amplified
plasmid sequence, forward sequencing primer VP1.5 and reverse sequencing primer XL39 are
employed to prepare the sequencing solutions, which were subsequently sent to Azenta Life
Sciences' GENEWIZ, a reputable sequencing company. The report received from GENEWIZ
confirmed that the amplified plasmid is identical to its original counterpart. 3.2. Verification of the concentration of the vector to transfect HEK293T cells
The amplified plasmids are transfected into HEK293T cells and established different
concentrations of plasmid transfection groups (0.5, 1.5, 2.5, 3.5μg/well), with each well
containing approximately 6.5*10^5 cells. Whole cell extract (WCE) was collected after 48 hours
post-transfection for subsequent analysis by Western blotting to evaluate the efficiency of
plasmid transfection. As anticipated (Figure 7), a dose-dependent effect was observed, reaching
saturation at a concentration of 1.5μg/well. Based on these results, we will proceed with using
this amount as our standard transfection plasmid dosage.

42
Figure 7. Comparison of different plasmid transfection concentration The blot is incubated
with mouse anti-DDK (tag) antibody as primary antibody (Figure 11A) and the expected size is
100-110kDa (LAMP-2B and tag size). The following picture is the same blot incubated with
mouse anti-GAPDH antibody, of which the expected size is 38 kDa(Figure 2B). 3.3. LAMP-2B intracellular and extracellular amount change with plasmid concentrations
The amplified plasmids are transfected into HEK293T cells and established different
concentrations of plasmid transfection groups (0.5, 1.5, 2.5, 3.5, and 4.5 μg/well). After a
48-hour incubation period, whole cell extract (WCE) was collected for analysis. Western blotting was initially performed using β-mercaptoethanol (BME) to disrupt the disulfide
bonds within the protein. The transfer was carried out using iBlot 2 NC Regular Stacks
(Invitrogen by Thermo Fisher Scientific, REF IB23001). Subsequently, the blot was incubated
with Anti-LAMP2 antibody (abcam CAT#ab13524, diluted at 1:1000) and Goat Anti-Rat
secondary antibody (IRDye 680LT CAT#D10512-25, diluted at 1:5000), followed by imaging in

43
the 700nm channel. However, the signal obtained was insufficiently strong and no signal was
observed in immunofluorescence analysis. Consequently, alternative antibodies were employed
for incubation; namely Anti-LAMP2 antibody from Invitrogen (CAT#PA1-655 LAMP2, diluted
at 1:1000) along with Donkey Anti-Rabbit secondary antibody (IRDye 680LT CAT#D20802-05, diluted at 1:5000), as well as Anti-LAMP2B antibody from abcam (CAT#ab18529) also paired
with Donkey Anti-Rabbit secondary antibody (diluted at a ratio of 1:50000; IRDye 680LT
CAT#D20802-05). Unfortunately, these attempts also yielded weak signals. Finally, the utilization of polyvinylidene fluoride (PVDF) membrane and HRP-conjugated
secondary antibodies enables a clear and robust manifestation of the signal. The NC membrane
exhibits superior affinity towards low molecular weight proteins in comparison to the PVDF
membrane, while the latter demonstrates enhanced capability for binding high molecular weight
proteins and glycoproteins [58]. Moreover, horseradish peroxidase (HRP), commonly employed
as an enzyme marker alongside secondary antibodies, effectively amplifies the detected target's
signal by facilitating substrate oxidation in the presence of hydrogen peroxide, resulting in either
colored precipitate formation or light emission. The western blotting was performed using rabbit
anti-LAMP2B antibody as the primary antibody to quantify the total amount of LAMP-2B
protein present. Subsequently, the blot was stripped and probed with mouse anti-DDK antibody
to specifically detect the LAMP-2B produced by transfected plasmids carrying an epitope tag. The results showed an increase in LAMP-2B levels with the epitope tag (Figure 8B) while
endogenous LAMP-2B appeared to be decreased (Figure 8A).

44
Figure 8. Comparison of LAMP-2B intracellular and extracellular amount The blot is
incubated with rabbit anti-LAMP2B antibody as primary antibody (Figure 7A) and the expected
size is 100-110kDa. The second figure is the same site incubated with mouse anti-DDK antibody
after stripping (Figure 7B). The following picture is the same blot incubated with mouse
anti-GAPDH antibody, of which the expected size is 38 kDa (Figure 7C). 3.4. Determination of LAMP-2B extracellular amount
As previously mentioned, my objective was to utilize extracellular exosomes containing
LAMP-2B as carriers for targeting proteins. To investigate this further, I conducted experiments
to assess the impact of transfection on the production of extracellular LAMP-2B using Western
blot analysis.

45
Lysate and media of cells were collected 48 hours post-transfection. Media was harvested 8
hours after media change. The media was concentrated from 2ml to 70μl. Lysate, with or without
plasmid transfection, exhibited a robust LAMP-2B signal; however, the media contained a low
amount of protein as indicated by BCA analysis and showed significantly reduced LAMP-2B
signal intensity (Figure 9A). Both lysate and media samples subjected to transfection displayed a
strong DDK tag signal, indicating successful transfection. However, the lysate sample with
transfection exhibited an unexpected signal below 102kDa instead of above it as anticipated
(Figure 9B). The GAPDH signals depicted in Figure 9C confirmed sufficient protein levels in
both groups of lysates but revealed limited protein content in the two media groups. The three
figures depicted above are derived from the same blot, and the sequence of strip and incubation
follows a consecutive order. Western blot analysis failed to determine the extracellular
abundance of LAMP-2B.

46
Figure 9. The different signal of lysate and media with and without plasmid transfection
The blot is probed with rabbit anti-LAMP2B antibody as the primary antibody (Figure 8A), targeting a predicted size of 100-110kDa. Subsequently, the same site is probed with mouse
anti-DDK antibody after stripping (Figure 8B), also targeting a predicted size of 100-110kDa. Finally, the same blot is probed with mouse anti-GAPDH antibody, targeting an expected size of
38 kDa (Figure 8C). 3.5. Determination of the association between LAMP-2B and DDK
Immunoprecipitation (IP) was employed to assess the level of LAMP-2B associated with the
DDK tag. A volume of 100μl concentrated media from transfected cells, was incubated with
either 2μg or 5μg anti-LAMP2B antibody for IP initiation. It is essential to employ antibodies
derived from different species for both IP and western blotting procedures in order to prevent
any potential interference caused by heavy or light chain IgG. The anti-DDK antibody (1:2000)
was applied in Western blotting, however, no detectable signal was observed. Similarly, the
anti-GAPDH antibody did not yield any signal either. Nevertheless, a signal was detected in the
wash solution, indicating successful removal of other proteins. In reverse, 100μl of concentrated media was incubated with 5μg of anti-DDK antibody. The

47
Western blot analysis was performed using anti-LAMP-2B (1:1000), but no signal was detected. It is suspected that the strong detergent used may have disrupted the interaction between
LAMP-2B and DDK. Therefore, whole cell extract (WCE) was harvested under non-denaturing
conditions. However, despite these efforts, no signal could be observed. IP failed to determine
the extracellular amount of LAMP-2B. 3.6. Immunofluorescence analysis of transfected cells
The HEK293T cells were seeded onto cover glass one day prior to transfection. Following a
48-hour incubation period, the cells were fixed with a 1:1 mixture of methanol and acetone and
subsequently blocked in 1% BSA in PBS. Primary and secondary antibodies were utilized for
cell incubation, as depicted in Figure 10. Notably, the LAMP-2B signal was clearly evident both
with and without transfection, while no signal was observed for the secondary control. The DDK
signal was distinctly visible only within transfected cells; conversely, there was no DDK signal
detected among non-transfected groups or the secondary control. 10%-20% cells are transfected
in HEK293T cell culture. In terms of co-localization analysis, clear signals for both LAMP-2B
and DDK were observed solely within transfected cells; however, no such signals were present
among non-transfected groups or the secondary control.

48
Figure 10. Image of HEK293T cells with and without transfection and secondary control
Immunofluorescence staining was performed for LAMP-2B, DDK, and their co-localization. The
green fluorescence indicates the presence of LAMP-2B, while the pink fluorescence represents
DDK. Additionally, actin was visualized in red and the nucleus in blue. 3.7. Determination of exosomes
The media of HEK 293T cells was collected 48 hours after transfection and 8 hours after
replacing the media without fetal bovine serum (FBS). The media was concentrated from 19.2 ml
to 90 μl before being filtered using an iZON column to obtain exosomes. ZetaView
determination was repeated three times for each sample. The median particle sizes of exosomes
derived from both transfected and non-transfected HEK294T cells showed no significant

49
difference (p=0.185), while there was a statistically significant difference in the particle numbers
between the two groups (p=0.03), as depicted in Figures 11 and 12. The size distributions of
exosomes present in the cell culture media (CCM) obtained from transfected and non-transfected
cells followed a normal distribution pattern. (Figure 13)
Figure 11. The median particle size of exosome samples from HEK293T cells with and
without transfection Each data point represents one sample test. An independent sample t-test
was performed, p=0.185 (p>0.05, n=3).
Figure 12. The particle number of exosome samples from HEK293T cells with and without
transfection Each data point represents one sample test. An independent sample t-test was
performed, p=0.003 (p<0.05, n=3)

50
0 200 400 600 800 1000
0.00
2.50×10
6
5.00×10
6
7.50×10
6
1.00×10
7
1.25×10
7
1.50×10
7
1.75×10
7
2.00×10
7
Size /nm
P
a
rticle
a
m
o
u
n
t
/
m
L
C
C
M
CCMsize distribution
Transfected
W/O transfection
Figure 13. The size distribution of exosomes in the cell culture media (CCM) from
transfected and non-transfected cells
To confirm the presence of exosomal proteins in the cell media, WB was employed. Initially, an
attempt was made to collect 36ml of media, both with and without transfection, followed by
collection of the media after 8 hours. However, Western blot analysis did not yield any signal due
to insufficient protein content in the collected media. Therefore, it was decided to increase the
volume of cell culture and extend the incubation time in order to address this issue. After
collecting 48 ml of cell media, both with and without transfection, the media was collected 24
hours after replacing it without FBS. Subsequently, concentration was achieved using a 100K
filter followed by isolation of exosome particles using iZON. The total protein per lane on the gel
amounted to 4 μg/lane.The primary antibodies used were anti-DDK (1:1000), GAPDH (1:5000), and anti-LAMP2B (1:1000). Due to low protein amounts, all signals observed were weak. The

51
DDK signal was detected in both cell media with plasmid transfection and lysate with
transfection groups. The GAPDH signal was only observed in the lysate with transfection group. A very weak signal of LAMP-2B was detected in the lysate with transfection group. (Figure 14)
To obtain a stronger signal, it may be necessary to collect more concentrated media samples.
Figure 14. The different signal of exosomes in media with and without plasmid transfection
The blot is probed with mouse anti-DDK antibody as the primary antibody (Figure 13A), targeting a predicted size of 100-110kDa. Subsequently, the same site is probed with mouse
anti-GAPDH antibody after stripping (Figure 13B), also targeting a predicted size of 38kDa. Finally, the same blot is probed with rabbit anti-LAMP2B antibody, targeting an expected size of
100-110kDa (Figure 13C).

52
Chapter Four
4. Discussion
The results of this study demonstrate successful transfection of HEK 293T cells with the plasmid, pCMV6-lamp2b-Myc-DDK, facilitating the incorporation of DDK tags into LAMP-2B. This has
been validated through Western blot analysis in cell lysates and Immunofluorescence analysis in
cells; however, attempts to further confirm the interaction between LAMP-2B and DDK using
immunoprecipitation was not successful. Additionally, the findings revealed an elevation in LAMP-2B levels with the epitope tag while
endogenous LAMP-2B appeared to be reduced in WB. Nevertheless, protein levels in the media
were insufficient for detection even after increasing cell culture volume and extending incubation
time. Therefore, Western blotting cannot be employed for testing the target protein in cell culture
media. It is imperative to collect more media samples to compare the abundance of target
proteins between transfected and non-transfected cell media in further study. The unexpected signal below 102kDa of DDK molecular weight was observed in the lysate
sample following transfection, as opposed to above it as seen in the media sample (Figure 8B). While cleavage may have occurred, it is uncertain in which fraction. The cationic liposome-mediated transfection efficiency in this study was determined to be
10%-20% based on immunofluorescence analysis. Strategies for enhancing transfection
efficiency included optimizing cell density by transfecting cells when they covered 70% of the

53
plate area, as excessively high cell density could compromise transfection effectiveness. Additionally, only cells in optimal condition were selected for transfection and transfected for a
minimum of 36 hours. Following the addition of transfection reagents, close monitoring of cell
morphology was conducted to assess any changes. Upon observing signs that the toxicity of the
transfection reagent had impacted cell morphology to some extent, the medium was replaced
accordingly. A significant difference in exosome numbers was observed between cell culture media with and
without transfection during NTA analysis, which has not been previously reported. It is plausible
that plasmid transfection may stimulate the release of exosomes, offering a potential rationale for
this observation. Another conjecture is that the upregulation of LAMP-2B gene expression could
be accountable for this augmentation. Another issue that needs to be addressed is the determination of DDK positivity in the additional
exosomes. Due to insufficient levels of exosomes in the media for accurate detection, this aspect
cannot be resolved within the scope of this study. Further analysis using additional media
samples or alternative techniques is warranted. The NanoFCM is considered to determine the
proportion of DDK positive exosomes and can also enhance the detection of low-abundance
exosome signals. This observation could potentially support the hypothesis that there might be shared molecular
components between cellular trafficking pathways and exosome biogenesis [4]. For example, when an engineered protein is introduced, it may lead to functional changes like those observed

54
in other cellular vesicular pathways such as Golgi, lysosomes, and autophagy. These alterations
could subsequently have a secondary impact on the production or secretion of exosomes [59]. The presence of various factors like differences in parent cell types, variations in culture
conditions, and the lack of standardized methods for generating and characterizing exosomes can
all contribute to experimental variability. To improve classification accuracy, it is crucial to
employ multiple complementary characterization techniques while also monitoring for any
co-isolated non-exosome components [60]. In addition, it is important to consider the unintended consequences of contamination caused by
mycoplasma and other microorganisms. These contaminants can modify the cellular physiology
of donor cells, leading to the release of their own exosomes. Furthermore, exosomes derived
from culture media should also be taken into consideration. Other factors such as processing and
storage methods can also impact the physiological properties of exosomes and consequently
influence exosome research outcomes [60]. Therefore, it is crucial to identify and address these
experimental artifacts in order to ensure reliable progress in exosome research.

55
Chapter Five
5. Conclusion
Numerous studies have emphasized the significant involvement of exosomes in intercellular
communication, shedding light on their physiological and pathological roles. Exosomes have
been identified as highly suitable vehicles for drug delivery due to their excellent
histocompatibility and ability to shield therapeutic agents from degradation. The cationic
liposome-mediated transfection utilized in this investigation exhibited an efficiency ranging from
10% to 20%. The engineering of exosomes enabled the manipulation of surface proteins for the
overexpression of target proteins. Size-based separation was employed for effective extraction of
exosomes and proved to be successful. In this investigation, the aim was to assess epitope-tagged LAMP-2B intracellular and
extracellular abundance. This study found that the amount of LAMP-2B with epitope tag
increased in comparison to the non-transfected HEK293T cells, while the endogenous LAMP-2B
appeared to decrease. Moreover, a statistically significant increase was observed in the exosomes
present in the media of transfected cells compared to those found in non-transfected cell media
(p=0.03). This thesis has provided a deeper insight into engineering the exosomes into a target drug carrier. An implication of this is the possibility of exosome extraction and separation from the cell
culture media. The subsequent procedure involves the integration of the target protein into

56
LAMP-2B, as outlined in Kyle L. Mentkowski and Jennifer K. Lang's thesis [29]. This would be
a fruitful area for further work.

57
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Abstract (if available)

Abstract Exosomes are small vesicles secreted by cells that aid in cell-cell communication and have received much attention as new carriers for drug delivery. It has been found that exosomes are ideal drug delivery carriers since they have good histocompatibility and can protect the encapsulated therapeutic agents from degradation. LAMP-2B, a member of the lysosome associated membrane protein families, is an exosome surface protein, whose N-terminal extracellular domain can be genetically fused with targeting peptides to achieve a targeting effect for these modified exosomes. The plasmid, pCMV6-lamp2b-Myc-DDK, contains the gene sequence of LAMP-2B fused to a FLAG epitope tag. The goal of this project was to isolate exosomes enriched in epitope-tagged LAMP-2B through transfection of this plasmid into HEK293T cells. In this project, the author developed a robust set of protocols capable of transfecting cells, tested transfection efficiency and determined epitope-tagged LAMP-2B intracellular and extracellular abundance. Western blotting and immunoprecipitation techniques were optimized to quantify protein levels, while nanoparticle tracking analysis was employed for determining the number of exosomes in the cell culture media. These protocols were adapted from the existing literature and modified to work with our samples. Compared with the LAMP-2B amount of HEK293T cells without transfection, the LAMP-2B amount with epitope tag increased, but the endogenous LAMP-2B appeared decreased. The exosomes present in the transfected cell media exhibit a statistically significant increase compared to those found in non-transfected cell media (p=0.03).

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Creator Bian, Shiyu (author)

Core Title Determination of LAMP-2B from HEK293T cells transfected by pCMV6-lamp2b-Myc-DDK

School School of Pharmacy

Degree Master of Science

Degree Program Pharmacy / Pharmaceutical Sciences

Degree Conferral Date 2024-05

Publication Date 05/21/2024

Defense Date 05/06/2024

Publisher Los Angeles, California (original), University of Southern California (original), University of Southern California. Libraries (digital)

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Advisor Hamm-Alvarez, Sarah (committee chair), Mackay, Andrew (committee member), Zaro, Jennica (committee member)

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