Prostate cancer (PC) is the most common type of cancer and the second cause of death in men in the UK. It has been primarily treated with radiotherapy and hormonal therapy but this approach is effective at the early stages of the treatment. Overtime, androgen independent PC cells emerge and novel approaches to manage this disease have emerged.

With the advances in drug delivery and nanotechnology, a wide range of nano-sized delivery systems has been developed to improve efficacy and reduce side effects of existing chemotherapeutics. Exosomes are nano-sized, cell-derived vesicles that carry proteins and RNAs for intercellular communication. They are increasingly seen as possible alternatives to liposomes as delivery carriers, since they could deliver their cargo across the plasma membrane and delay premature drug transformation and elimination. A new approach has been proposed to produce exosome-like vesicles by extrusion of monoblastic cell line through different pore size membranes in order to increase the yield and generate genetically modified exosome-like vesicles.

The aim of this project is to prepare PSMA (prostate-specific membrane antigen) targeted, exosome-like vesicles, encapsulating doxorubicin prodrug that will selectively target metastatic PC cells. To reduce the systemic toxicity of doxorubicin, PSA (prostate specific antigen) – activated doxorubicin prodrug will be used in this project, where drug activation takes place only in PSA-producing PC cells.

Table of contents

1. Introduction

1.1. Prostate gland and prostate cancer

1.2. Selective approach in prostate cancer treatment

1.2.1. Prostate – specific membrane antigen (PSMA)

1.2.2. Prostate-specific antigen (PSA)

1.3. Nanotherapeutic drug delivery systems

1.4. Extracellular vesicles

1.4.1. Extracellular vesicles as drug delivery systems

1.4.2. Methods of isolation

1.4.3. Drug loading into exosomes

1.4.4. Achieving targeted therapies

1.5. Exosome – like vesicles (ELVs)

2. Hypothesis and objectives

2.1. Hypothesis

2.2. Aim

2.3. Objectives

3. Materials and methods

3.1. Materials

3.2. Methods

3.2.1. Cell culture conditions

3.2.2. Preparation of exosome-like vesicles (ELVs)

3.2.3. Labelling ELVs with Dil dye

3.2.4. Spectrofluorometry

3.2.5. PD10 purification

3.2.6. qEV original purification

3.2.7. Ultracentrifugation

3.2.8. Dynamic light scattering (DLS)

3.2.9. Optimization of lysis buffer

3.2.10. ELVs lysis

3.2.11. Bicinchoninic acid (BCA) assay

3.2.12. Nanoparticle tracking analysis (NTA)

3.2.13. Transmission electron microscopy

4. Results

4.1. Preparation of ELVs

4.2. ELVs purification

4.2.1. Size exclusion chromatography using PD10 column

4.2.2. Ultracentrifuge

4.2.3. qEV original

4.3. Determination of ELVs protein content using BCA

4.4. ELVs stability overtime

4.4.1. Monitoring stability by DLS

4.4.2. Monitoring stability by protein content (BCA assay)

4.5. Nanoparticle tracking analysis (NTA) measurements

4.6. ELVs structure elucidation using transmission electron microscopy (TEM)

5. Discussion

6. Conclusion

7. Future research plan

8. Progress to date (PPD)

9. References

Abbreviations

 

ADT -Androgen deprivation therapy

BBB – Blood brain barrier

BCA – Bicinchoninic acid

BRCA 1 – Breast cancer type 1 susceptibility protein

BPH – Benign prostatic hyperplasia

CEL – Celastrol

CRPC – Castration-refractory prostate cancer

DLS – Dynamic light scattering

DOX – Doxorubicin

ELVs – Exosome – like vesicles

EPR – Enhanced permeability and retention

ESCRT – Endosomal sorting complex required for transport

GNP – Gold nanoparticle

ILVs – Intraluminal vesicles

LFA – Lymphocyte function – associated antigen

LHRHa – luteinizing hormone-realising hormone analogues

MAPK – Mitogen-activated protein kinase

MHC – Major histocompatibility complex

NP – Nanoparticle

NTA – Nanoparticle tracking analysis

MVBs – Multivesicular bodies

PC – Prostate cancer

PSA – Prostate – specific antigen

PSMA – Prostate-specific membrane antigen

PTX – Paclitaxel

RVG – Rabies viral glycoprotein

SNAREs – Soluble N-ethylmaleimide-sensitive factor attachment protein receptors

TEM – Transmission electron microscopy

TNF – Tumor necrosis factor

TRUS – Transrectal ultrasound

1.     Introduction

1.1. Prostate gland and prostate cancer

Prostate is a walnut size and shape gland, which develops after puberty in men’s lower abdomen. It is an accessory sex gland composed of ducts and alveoli that are lined by a tall columnar epithelium within a stroma of fibromuscular tissue (70%). The main functions of prostate gland are prostatic secretion and control of urine and semen flow. The prostatic fluid liquefies the semen. It is slightly acidic (pH 6.4) and forms about 20% of semen volume ¹.  Anatomically, prostate gland is divided into 4 zones (Figure 1): the transition zone, which consists of 5-10% of the glandular tissue and it is responsible for most of the benign prostatic hyperplasia that affects older men. Central zone (25% of prostate tissue) is placed around the ejaculatory ducts and accounts for 1-2% of prostate cancer. The peripheral zone makes up 70% of the prostatic volume and 70% of prostate cancers arise from this zone. The anterior fibromuscular stroma makes up approximately 5% of the prostate volume and extends to the transition zone ^2,3.

Figure 1. Zonal anatomy of the prostate. Adapted from reference ⁴

The most common prostate diseases, which affects man, are benign prostatic hyperplasia (BPH), prostatitis and prostate cancer. BPH affects mostly middle-aged men, and results in reduced urinary stream and frequent urination. Prostatitis is an inflammatory disease causing pain and discomfort, and affects younger and middle aged man. Prostate cancer (PC) is the most serious disease, and affects mostly elderly man ⁵. Prostate cancer is the most common type of cancer in man in the UK with around 46 700 cases diagnosed in 2014 ⁶. In addition, it is the second most common cause of cancer death in men in the UK, around 11 300 deaths in 2014 ⁶. Incidence rates for prostate cancer are expected to rise by 12% in the UK between 2014 and 2035 ⁶. In 2017, the most common cancers expected to occur in man are prostate, lung, bronchus and colorectal cancer, with prostate cancer accounting for almost 1 in 5 new diagnosis in the United States ⁷.

Leading factor for prostate cancer is age. Each year in the UK 54% of cases have been diagnosed in man aged 70 and over, and it is correlated with cell DNA damage which increases over time ⁶. Genetic factors including mutations in BRCA 1 and BRCA 2 genes contribute to the development of prostate cancer ⁸. Ethnicity, familial and other risk factors, including obesity and smoking cigarette appear to increase prostate cancer incidence ^6,9.

The majority of prostate cancers are adenocarcinomas (95%) while sarcomas and neuroendocrine tumours are less common. Adenocarcinoma cells have hyperchromatic, large nuclei with prominent nucleoli and abundant cytoplasm. Peripheral zone is the most likely cancer developing zone, as demonstrated in 70% of prostate cancer patients, while transition and central zone account for 20% and 10% of prostate cancer, respectively ⁹.

The main diagnostic tool for prostate cancer is the measurement of PSA (prostate specific antigen) in serum. PSA is a glycoprotein secreted only by the epithelial cells of the prostate gland. PSA level is increased in prostate cancer, BPH and prostatitis. This test is sensitive, but non-specific for cancer diagnosis as it can show false negative or false-positive results ⁹. That is why additional examinations are required, including digital rectal examination, transrectal ultrasound and biopsy (TRUS), as well as template and targeted biopsies ^9–11.

Based on the microscopic assessment of the glandular architecture of prostate, prostate cancer is graded using Gleason Scoring system and after additional examinations, extent of the disease is determined and patient is assigned to a risk group. The Gleason Grade indicates the degree of tissue differentiation: well-differentiated tumour indicates as grade 1 and poorly differentiated tumour indicates as grade 5. The two grades are counted together to give the Gleason score which ranges from 2 to 10 (for example 4+3=7) ⁹. Pathological staging relies on a TNM system (Table 1) which describes the extent of tumour (T), lymph node involvement (N), and presence of metastatic disease (M). Four main stages T1-T4 are indicating the prostate and surrounding tissues involvement ¹².

Table 1. Definitions of the American Joint Committee on Cancer TNM Criteria. ¹²

CATEGORY	CRITERIA
Clinical (cT)
TX	Primary tumor cannot be assessed
T0	No evidence of primary tumor
T1	Clinically inapparent tumor that is not palpable
T1a – T1c	Tumor incidental histologic finding in 5% or less of tissue resected, or more than 5%, Tumor identified by needle biopsy found in one or both sides, but not palpable
T2	Tumor is palpable and confined within prostate
T2a – T2c	Tumor involves one-half of one side or less, more than one half or both sides
T3	Extraprostatic tumor that is not fixed or does not invade adjacent structures
T3a –T3b	Extraprostatic extension (unilateral or bilateral), Tumor invades seminal vesicle(s)
T4	Tumor is fixed or invades adjacent structures other than seminal vesicles, such as external sphincter, rectum, bladder, levator muscles, and/or pelvic wall
Pathologic (Pt)
T2	Organ confined
T3	Extraprostatic extension
T3a –T3b	Extraprostatic extension (unilateral or bilateral) or microscopic invasion of bladder neck, Tumor invades seminal vesicle(s)
T4	Tumor is fixed or invades adjacent structures other than seminal vesicles, such as external sphincter, rectum, bladder, levator muscles, and/or pelvic wall
N category
NX	Regional lymph nodes were not assessed
N0	No positive regional lymph nodes
N1	Metastases in regional lymph node(s)
M category
M0	No distant metastasis
M1	Distant metastasis
M1a – M1c	Nonregional lymph node(s), Bone(s), Other site(s) with or without bone disease

The TNM staging used in combination with Gleason scoring and PSA level monitoring is the standard practice for prostate cancer diagnosis, and it is crucial in treatment decision-making ¹². Treatment options for prostate cancer depends on the stage and cancer type. For instance, localised cancer is treated by active surveillance, radical prostatectomy, external beam radiotherapy (EBRT) and brachytherapy ⁹ . If the cancer is locally advanced, treatment options are extended to therapy known as androgen deprivation (ADT) to lower the testosterone levels and disrupt androgen receptor signalling ⁹ .

The main challenges in treatment arise when metastatic prostate cancer occurs. Around 70% of patients with metastatic prostate cancer die within 5 years ⁶. Castration-sensitive cancer is treated with bilateral orchiectomy, luteinizing hormone-realising hormone analogues (LHRHa) and chemotherapy. When castration-refractory prostate cancer (CRPC) develops, resistance to hormone therapy occurs and PSA levels rise. There are some therapeutic options that showed small benefit to the advanced prostate cancer patients, but the overall efficiency is still very low. These treatment options include novel androgen receptor (AR) targeted therapies, such as abiraterone and enzalutamide; cytotoxic chemotherapies (docetaxel); palliative radiotherapy and bone-directed therapies such as bisphosphonates (binds to the mineralized bone matrix due to its structure); monoclonal antibodies (inhibits osteoclast maturation and bone turnover by mimicking native antibody interactions); and radioisotopes (similar to calcium and taken up to the osteoblastic activity site) ^9,13.

1.2. Selective approaches in prostate cancer treatment

Chemotherapy is most widely used to treat advance and metastatic prostate cancer, but its efficacy is usually limited because of its lack of specificity and systemic toxicity. The most advancing approach in overcoming those chemotherapeutical obstacles is active targeting. The main purpose of active targeting is delivery of chemotherapeutic drugs to the specific cancer cells. This approach includes specially designed nanocarriers which are modified with targeting ligands that can bind to prostate cancer specific antigens and they are loaded with specifically designed prodrugs. In the second approach to decrease side effect of anticancer drugs is by engineering prodrugs that will be selectively activated at tumour tissues, based on changes in pH, enzymes, etc. Several specific enzymes which are overexpressed in tumour microenvironment as well as prostate cancer relevant antigens are used for targeted drug delivery design and they showed promising results in therapeutic efficiency (Figure 2) ^14,15. PSMA and PSA are well studied markers that have been clinically used as therapeutic targets and they will be discussed in more details in the next section.

Figure2: Prostate cancer specific antigens and enzymes in the tumour microenvironment ¹⁴

 

1.2.1. Prostate – specific membrane antigen (PSMA)

Prostate – specific membrane antigen (PSMA) is a 100kDa type II transmembrane glycosylated protein. It consists of a glycosylated extracellular domain of 707 amino acids and an intracellular domain of 19 amino acids. It has three different structural and functional domains: protease domain, apical domain and C-terminal domain ¹⁶. PSMA is expressed on the prostate epithelial cell membrane and is overexpressed in prostate cancer as it progresses ¹⁶. Because of this properties PSMA has been extensively used as a targeted antigen in targeted drug delivery systems ^17–22. Different PSMA targeting agents have been described, developed and utilized in nanocarriers to improve the targeting efficiency to the prostate cancer tissue. For example, aptamer conjugated micelles resulted in significantly high drug uptake in PSMA positive cancer cells ¹⁸. Also, antibodies which bind to PSMA have been conjugated with dendrimer nanoparticles increased their uptake in the PSMA expressing cells ¹⁹. Peptide sequences which can bind to PSMA and inhibit its enzymatic activity have also been identified ^17,20–22 . Due to aptamer and antibodies disadvantages as large size, possible immunogenicity and instability, peptides were more considered for targeted drug applications. They have small molecular weight, high permeability, good stability, they are less immunogenic, easy to synthesize and flexible in chemical conjugation ²⁰. Phage display technology has been used to screen and identify binding peptides such as KYLAYPDSVHIW and WQPDTAHHWATL that can bind to PSMA and inhibit its glutamate carboxypeptidase activity ^17,23. Those sequences can be transfected and expressed on the recipient cells improving their targeting to PSMA-expressing prostate cancer ²⁴.  Several PSMA based diagnostic and therapeutic agents are in clinical trials, implicating that this approach has great potential in the future prostate cancer targeted therapies ¹⁴.

1.2.2. Prostate-specific antigen (PSA)

The other commonly used approach to target prostate cancer is through PC specific enzymes that are overexpressed in tumour microenvironment. It is based on designing a specific protease activated prodrug that can release the active drug following enzymatic cleavage. Alternatively, can be encapsulated in the nanocarriers which can be degraded in the tumour microenvironment the presence of overexpressed enzymes, resulting in better penetration efficacy ¹⁴.

Prostate-specific antigen (PSA) is secreted by the normal human prostate epithelium and it is one of the most abundant proteins in semen. It is a serine protease that belongs to the glandular kallikrein family and cleaves seminal fluid protein semenogelin I and II. PSA enters the blood stream in its active or inactive form where active PSA is bound to protease inhibitors and inactive stays in unbound state ²⁵. Due to the disruption of prostate gland in prostate cancer patients, the total PSA level is increased and by measuring PSA levels in blood, PC can be diagnosed. The concentration of active PSA is much higher in tumour tissue compared to the circulation and that’s justify the development of PSA-substrate drug conjugate as a novel targeted therapy for prostate cancer (Table 2) ^15,26.

Peptide sequence (His-Ser- Ser-Lys-Leu-Gln-Leu) that can be cleaved specifically by PSA has been identified ²⁷ and prodrug was designed by conjugating this peptide to doxorubicin ²⁸. In another study different PSA-specific peptide Nglutaryl-(4-hydroxyprolyl)Ala-Ser-cyclohexaglycyl-Gln-Ser- Leu-CO2H have been described and by conjugation with doxorubicin reduced cytotoxicity in the cells that do not secrete PSA but increased antitumor toxicity compared with free doxorubicin ²⁹. Successful application of those prodrugs resulted in first clinical trial of the PSA prodrug (L-377202) and it was well tolerated in patients³⁰ . To enhance delivery and solubility of the prodrug, they can be encapsulated in nanocarriers and successfully delivered to the tumour site ^31,32.

Table2: Summary of PSA – activatable prodrugs. Adapted and modified from ²⁶

Construct	Drug	Reference
Mu-His-Ser-Ser-Lys-Leu-Gln-Leu-X	Doxorubicin,5 fluorodeoxyuridine, L12ADT	28,33–35
Mu-His-Ser-Ser-Lys-Leu-Gln-EDA-X	Paclitaxel	36
4-O-(Ac-Hyp-Ser-Ser-Chg-Gln-Ser-Ser-Pro)-X	Vinblastine	37,38
HO2C(Ch2 )3CO-Hyp-Ala-Ser-Chg-Gln-Ser-Leu-X	Doxorubicin	39
N-glutaryl-(4-hydroxyprolyl)-Ala-Ser-chGly-Gln-Ser-Leu-X	Doxorubicin	29,37,40

Prodrugs are playing significant role in improvement of pharmaceutical properties of drug and its efficient delivery to the specific prostate cancer tissue. To further improve the advance cancer therapies, proper delivery system needs to be designed. Today, nanomedicine is in great focus and by choosing the most suitable system which can be modified with targeting peptides and encapsulate specific prodrugs, advanced prostate cancer could be treated effectively with less side effect for the patient.

1.3. Nanotherapeutic drug delivery systems

They are called nanomedicines or nanotherapeutics because of their nanosized structure, ranging from 1nm to 1000nm ⁴¹.  Many nanosized delivery systems have been engineered such as liposomal, solid lipid, inorganic nanoparticles, nanodiamonds, carbon nanotubes, quantum dot nanocarriers, polymeric and dendrimeric nanoparticles and virus-mediated nanocarriers (Figure 3) ⁴². The initial goal of nanomedicine is to increase the drug concentration in the targeted tissues or cells and enhance therapeutic efficacy, while simultaneously decreasing the exposure of healthy tissues to the drug to reduce its toxicity. Through innovative nanomedicine design and creative management of tumour microenvironment it is possible to minimize toxicity and retain the drug efficacy ⁴³. Drug delivery systems are representing the new approach in cancer management because they can overcome the obstacles in cancer treatment, such as  challenges in targeting and delivery of therapeutic drugs and chemoresistance ⁴⁴. They can deliver various therapeutic as small-molecule drugs, peptides, proteins and nucleic acids alone or in different combinations⁴². Numerous pharmaceutical studies about applications of engineered nanomaterials in medicine resulted in 43 approved nanomedicine drug formulations and several undergoing clinical trials ^45,46. Apart from their therapeutic applications, they have been used in diagnostics and imaging as biomarkers and biosensors ⁴².

Figure3. Illustration of various nanoparticles (NPs) used in drug delivery: (A) Liposomal nanoparticle; (B) solid lipid nanoparticle; (C) gold nanoparticle; (D) nanodiamond; (E) magnetic nanovector;(F) carbon nanotube; (G) quantum dot nanocarrier; (H) polymeric nanoparticle;(I) dendrimer nanoparticle and (J) virus-mediated nanocarrier. Adapted from Jabir et al. ⁴²

Compared to the micrometer-sized delivery systems, nano-meter-sized systems can easily penetrate into tissues. They are also  taken up by cells 15-250 times higher than microparticles ⁴⁷. Studies have shown that nanometer-size delivery systems could target the tumor tissues through passive or active targeting (Figure 4) ⁴⁸. Passive targeting relies on the anatomical differences between normal and malignant tissues, which enables nanoparticles accumulation in the tumor tissue due to the leaky vasculature. This phenomenon is known as enhanced permeability and retention (EPR) effect. It is also observed at the site of inflammation ⁴⁹. The second approach of drug targeting is localized delivery of anticancer drugs using nanoparticles for treatment of local cancers. For example, nanoparticles containing therapeutics are directly injected in the local cancers such as prostate, head or neck cancers ⁴⁸. Alternatively, active targeting includes the conjugation of nanocarriers with receptor specific ligands, which can promote targeting to the specific tissue. Numerous targeting ligands including antibodies, antibody fragments, aptamers, peptides, whole proteins and different receptor ligands have shown great targeting capacity when conjugated to nanoparticles ⁵⁰.

Figure 4. Scheme of passive and active targeting: (A) Passive targeting: By EPR effect nanoparticels reach the tumour region; (B) Active targeting: enhance the therapeutic efficacy of drugs by the increased intracellular uptake via receptor-mediated endocytosis. Nanoparticles can be functionalized with ligands that bind to endothelial cell surface receptors. Adapted from Bamrungsap et al.⁵¹

Except for synthetic nanocarriers, naturally secreted extracellular vesicles have been widely described and proposed as drug delivery systems due their natural ability of cellular communication and macromolecules delivery.

1.4. Extracellular vesicles

Membrane vesicles secreted form cells were detected 50 years ago. They were considered as a cellular waste, but their first description as “exosomes” occurred in 1987 ⁵². They were initially discovered by studying the loss of transferrin during the maturation of reticulocytes into erythrocytes ⁵³. Their biogenesis was first described in the transferrin-gold conjugates biodistribution study where it was reported that intraluminal vesicles (ILVs) inside larger multivesicular bodies (MVBs) can be realised to the extracellular space following plasma membrane fusion ⁵⁴.

Extracellular vesicles have been described and divided into three main classes depending on their biological and physicochemical properties, biogenesis and surface markers (Table 3). They can be released form a wide range of cells into biological fluids, such as serum, plasma, urine, cerebrospinal fluid and breast milk ^55,56.

Table 3. Summary of properties and biogenesis of the different extracellular vesicles described in the literature.

Nomenclature	Properties	Biogenesis	Reference
Exosomes	Homogenous population with a size of 40 -100nm in diameter.  Exosomal markers include Tetraspanins (CD9, CD63), Alix, Tsg 101, ESCRT.	End of endocytic pathway, released from cells when multivesicular bodies fuse with plasma membrane.	57–61
Microvesicles	Heterogenous population with a size of 100-1000nm. Markers: Integrins, selectins, CD40 ligand.	Budding off/fission directly from the plasma membrane.	59–62
Apoptotic bodies	Variable size 500-2000nm in diameter. Consist of cytoplasm with tight packed organelles.  Marker: histones.	Released as part of apoptotic cell death. Extensive plasma membrane disintegration occurs followed by fragmentation of nucleus and separation of cell fragments during budding process.	59–61,63,64

Subpopulations of extracellular vesicles have been classified as ectosomes ⁶⁵, cardiosomes ⁶⁶, prostasomes ⁶⁷ and vexosomes ⁶⁸ based on their tissue/cell origin, size, composition and function. Numerous studies revealed that exosomes contain proteins derived from cell membrane, endosomes and cytosol which are not related to other cellular organelles and that makes them separate subcellular compartments⁶⁹. Exosome surface can be characterized by the presence of specific protein markers, which distinguish them from microvesicles and apoptotic bodies (Figure 5). Those proteins belong to different families, such as tetraspanins (CD63, CD81, CD9), lysosomal proteins (Lamp2b), heat shock proteins (Hsp 70) and fusion proteins (CD9, flotillin, Anexin). However, Kowal et al. recently described that smaller exosomes purified form several human cell lines can be divided into different subtypes based on the presence or absence of typical exosomal markers (CD63, CD81 and CD9) ⁷⁰. Specific databases have been created to summarize all proteomic studies of extracellular vesicles such as EVpedia ⁷¹, Vesiclepedia ^72,73 and ExoCarta ⁵⁷, but further research is necessary to distinguish different exosome subpopulations, and to understand their physiological and pathological functions in vivo.

Figure 5. Composition of exosomes. Exosomes are composed of various proteins:  major histocompatibility complex (MHC)-II, integrin, cluster of differentiation (CD), tetraspanins, heat shock protein (Hsp), Ras-related protein (Rab), etc. Exosomes also contain lipids, such as sphingomyelin and cholesterol. Exosomes are found to contain nucleic acid, including miRNA, mRNA and non-coding RNAs. Adapted from Ha et al.⁷⁴

Biogenesis pathway of exosomes from eukaryotic cells is schematically shown in Figure 6. While microvesicles bud directly from the plasma membrane, exosomes as intraluminal vesicles (ILVs) are formed via endosomal route by budding inwards into early endosomes or multivesicular bodies (MVBs). Molecules involved into biogenesis of ILVs are ESCRT machinery (Endosomal Sorting Complex Required for Transport), lipids (ceramide) and the tetraspanins. The late multivesicular bodies can be fused with the lysosomes or with the plasma membrane, which allows the release of exosomes into extracellular matrix. Proteins involved in the transport of MVBs to the membrane and exosome secretion include RAB proteins (RAB11, RAB27 and RAB35) which can act on different MVBs. Also, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are related in fusion process of MVBs with the cellular membrane ⁶⁹.

Figure 6: Intracellular machineries of exosome biogenesis and secretion ⁶⁹.

 

Exosomes can be secreted from various cell types, which determines their role in human physiology. They can play a role in immune response against tumor cells due to their antigen presentation complexes and activation of B and T-cells ⁷⁵.

1.4.1. Extracellular vesicles as drug delivery systems

Natural drug delivery carriers including viruses, bacteria and cells have been studied in order to understand their mechanisms in communication or avoiding immunological recognition. These studies could be essential to improve the development of our synthetic nano-carriers ⁷⁶. However, over the past decade, the interest in exosomes has increased because of their natural biological properties as intercellular communication and transmission of macromolecules between cells, which can result in structural changes at the RNA, protein and phenotypic levels.  They also have a role in tumor growth and metastasis, making them therapeutic targets and diagnostic/prognostic biomarkers ⁷⁷. Because of their natural membrane composition, they have been proposed as vectors for drug delivery rather than synthetic drug delivery systems due to the better tolerability by the host body ⁷⁷.

Some of the earlies studies showed that secreted exosomes can carry major histocompatibility complexes (MHC II) that are recognized by T cells and participate in antigen presentation and suppression of tumour growth in vivo ^78,79.  Exosome therapies have been explored in anti-cancer studies and clinical trials and they can be used to treat cancers at lower doses than conventional treatments with reduced toxicity and without evoking an immune response ^74,80–86. In contrast, synthetic phospholipid-bilayer vesicles, or liposomes, have been used as drug carriers for decades. Many formulations are in clinical trials and several liposomal nanomedicines are clinically approved ⁸⁷. Liposomes and exosomes have many similarities but their differences need to be studied and assessed in order to establish improved drug carriers, which will advance the nanomedicine field and improve patient outcomes. Liposomes are spherical vesicles that spontaneously self-assemble when phospholipids are dispersed in aqueous environment.  By extrusion through polycarbonate membranes with pore sizes 800, 400 and 100nm their diameter can be downsized and adjusted to match their biological applications. Furthermore, they can incorporate both hydrophilic and hydrophobic drugs and could provide physical barrier from rapid elimination of drug. Long-time studies enabled them to achieve optimized properties and overcome rapid clearance, stability, targeting, drug loading and release issues. However, the main problem with using liposomes is their toxicity and immunogenicity due to the synthetic lipids used in formulations ^88,89.  On the other hand, exosomes as naturally occurring vesicles are well tolerated in the body.

The donor cell type gives the specific characteristics for the drug delivering exosomes and the optimal choice of that initial cell line is necessary to establish a good system. The cell should secrete non-immunogenic exosomes to prevent any systemic immunogenic responses. They should be stable to withstand the circulation and deliver the cargo. Immature dendritic cells are relevant exosome donor cells because they have shown significant anti-inflammatory properties due to their surface protein composition ⁹⁰. Mesenchymal stem cells have also been described as ideal source of exosomes for drug delivery due to their low immunogenicity, intrinsic therapeutic property in reducing tissue injury, and reproducible exosome production in higher yield compared to the other cell lines ⁹¹. Exosomes also have intrinsic homing abilities, which means that they can target and accumulate in the specific tissues from which they originate. For instance, exosomes derived from melanoma cells preferentially localize in the sentinel lymph nodes and promote tumour metastasis ⁹². In addition, tumour – derived exosomes have higher preferences of localising into cancer cells than immortalised non-tumour cell lines ⁸³. Moreover, glioblastoma-derived exosomes showed high incorporation ability towards their parental cells, breast cancer cells and fibro -sarcoma, independently of surface protein ligands on exosomes ⁹³. Therefore, further studies are still necessary to completely understand the mechanism of intracellular communication in exosomes; this understanding will help in selecting exosomes with most innate affinity to the tissue of interest.

As drug delivery vehicles, exosomes can incorporate various therapeutic cargos, including small interfering RNAs, micro RNAs and cytotoxic therapeutic agents. Due to a huge potential of RNA in gene-based therapy but its low stability and rapid degradation in the circulation, exosome incorporated RNA system have been intensively studied ^94–96.  Results showed that exosome-delivered siRNA against RAD51 protein, induced post transcriptional gene silencing and caused cell death of recipient fibro-sarcoma cells ⁹⁴. Also plasma and cell derived exosomes can deliver MAPK1-siRNA to monocytes and lymphocytes resulting in a specific gene knockdown ⁹⁵. MicroRNAs have been also incorporated in exosomes for therapeutic applications resulting in inhibition of tumour growth ⁹⁶. Encapsulating widely used cytotoxic drugs and phytochemical compounds in the exosomes has resulted in encouraging preclinical results showed in Table 4. One of the most common adverse effects of long-term doxorubicin administration is cardiomyopathy, but this problem can be effectively resolved by encapsulating the doxorubicin drug  in exosomes (ExoDOX) ^80,82,97,98. In addition, the biodistribution and safety profile of paclitaxel (PTX) and natural cytotoxic compounds can be improved by exosome encapsulation (ExoPTX) ^81,86,99.

Curcumin shows promising anticancer activities in vitro, but its poor solubility has been considered challenging. Encapsulating curcumin in exosomes resulted with Phase 1 study to test the pharmacokinetics and pharmacodynamics of curcumin-loaded exosomes and their efficacy in the treatment of colorectal cancer after oral administration (NCT01294072) ¹⁰⁰. Furthermore, there is phase II clinical trial that evaluates the effect of chemotherapeutic drugs (methotrexate, hydroxyl camptothecin and cisplatin) encapsulated in exosomes on malignant ascites and pleural effusion (NCT01854866) ¹⁰¹ and it was based on the study by Tang et al. ¹⁰² .

Table 4. Summary of studies describing exosomes and drug delivery systems.

Therapeutic agent	Exosomal origin	Loading method	Outcomes	Ref.
Doxorubicin  (Dox)	MDA-MB-231 (human breast cancer) and HCT-116 (human colon carcinoma) cell	Electroporation	Better safety profile of exoDox in vivo.  Cardiac tissue appeared normal in exoDox (15mg/kg) treated mice and the heart showed 40% reduction of exoDox, explaining the reduced toxicity. ExoDox had similar antitumor effect similar to free Dox, but with reduced toxic side effects.	82
	MDA-MB-231 (human breast cancer) and STOSE (mouse ovarian cancer)	Electroporation	Dox encapsulated in exosomes was less toxic, which allowed treatment of mice using higher doses.  The volume of breast and ovarian tumours was smaller when treated with exoDox compared to free Dox. Concentration of exoDox in tumor was higher than mice treated with free Dox.	97
	Immature  mouse dendritic cell line (imDC)	Electroporation	ImDCs were engendered to express Lamp2b membrane protein fused on to a αv integrin-specific iRGD peptide (CRGDKGPDC). iRGD exosomes showed high efficient targeting and Dox delivery to αv integrin-positive breast cancer cells in vitro. Dox was specifically delivered to tumor site.	80
	HEK-293 (human embryonic kidney cells) with or without expressing Cx43 (gap junction protein connexin43)	Electroporation	The presence of Cx43 in exosomes did not show a statistically significant difference in terms of therapeutic effect of Dox but the presence of Cx43 in exoDox reduced the cardiotoxicity in mice.	98
Gold nanoparticles conjugated (GNP)-Doxorubicin	H1299 and YRC9 cells (lung cancer cell line)	Passive incubation of GNP-Dox with exosomes	“Nanosomes” were created by incubating exosomes and gold nanoparticles-conjugated Dox (GNP-Dox). GNP were conjugated with Dox in order to enhance the loading of Dox due to the opposite charge (The surface charge of GNP-Dox is positive, while exosomes are negatively charged).  Delayed and sustained released of Dox up to 72 h producing cytotoxicity which was observed when nanosomes were added to tumor cells. They enable pH-controlled drug release and delay cytotoxicity.	103
Doxorubicin (Dox) and Paclitaxel (PTX)	Brain neuronal glioblastoma-astrocytoma (U-87 MG), endothelial  (bEND.3), neuroectodermal tumor (PFSK-1), and glioblastoma A-172 cell lines	Passive incubation with exosomes	Drugs delivered in bEND.3 exosomes penetrated BBB.  The exosomes delivered cytotoxic levels of doxorubicin and paclitaxel intracellularly to the brain cancer cells in zebrafish in vivo.	85
Paclitaxel (PTX)	RAW 264.7 macrophages	Passive Incubation with exosomes, electroporation and sonication	Sonication was the best loading method. Incorporation of PTX into exosomes increased cytotoxicity more than 50 times in drug resistant MDCKMDR1 (Pgp+) cells compared to Taxol.	86
	LNCaP and PC-3 PCa cell lines	Passive incubation with exosomes	Loading of PTX to autologous prostate cancer cell-derived exosomes increased its cytotoxic effect.  EVs delivered PTX into the cells via the endocytic pathway even after the removal of their surface proteins indicating that further studies are necessary to understand all aspects of exosomal targeting before clinical applications.	81
	SR4987 ( bone marrow mesenchymal stromal cell line)	Incubation of cells with PTX for 24h	PTX was loaded inside the Golgi-derived vesicles of SR4987 cells instead of accumulating in microtubules thus inhibiting the cytotoxicity.  PTX-loaded microvesicles (MVs) retained an anti-tumor effect, indicating that the pharmacological activity of PTX was not affected during the physiological biogenesis of MVs. Purified MVs were between 20 and 150 nm in diameter, confirming that MVs are actually exosomes.	99
Celastrol (CEL)	Milk from pasture-fed Holstein and Jersey cows	Incubation in the presence of ethanol (10%)	Exosomes loaded with CEL exhibited enhanced anti- tumor efficacy as compared to free CEL against lung cancer cell xenograft.  Celastrol did not exhibit any gross or systemic toxicity in wild-type C57BL6mice. Exo-CEL inhibited the TNFα-induced activation of NF-κB and reduced anti-proliferative effect of tumor cells.	104
Curcumin	EL-4 (mouse lymphoma cell line)	Passive incubation with exosomes	Encapsulation of curcumin into exosomes increase its solubility, stability, and bioavailability.  Curcumin was self-assembled into the lipid bilayer of exosomes through the hydrophobic interactions.	105

1.4.2. Methods of isolation

Exosomes are secreted in various bodily fluids and cell culture media as mentioned earlier. They can be isolated using different techniques, which have been developed over past 30 years. The advantages and disadvantages are summarised in Table 5. The main challenges encountered so far are the low yield, reproducibility, stability of exosomes and ease of application.

Table 5. Comparison between different exosome isolation methods. Summarized from references ^106,107.

Isolation methods	Mechanism	Advantages	Disadvantages
Differential centrifugation	Medium containing exosomes is exposed to the centrifugal force. Several centrifugation steps are applied to remove cells, large particles, cellular debris, organelles and precipitate exosomes.	This method is “gold standard” for isolating exosomes.  It is the common and described method used to isolate exosomes from biological fluids and media.	Density of the solution (plasma, serum) affects how exosome behave under differential centrifugation.  It is not completely able to separate exosomes by size.
Density gradient centrifugation	Combination of ultracentrifugation with sucrose density gradient	Separation of low-density exosomes from cellular debris and other particles.	Unable to separate exosomes from other extracellular vesicles with similar density.  Time consuming, low yield, possible ruptures of exosomes.
Size exclusion chromatography	Separation of macromolecules and particles based on their size. The method set up includes the column which is packed with porous polymeric beads.	Fast and specific separation of proteins, HDL, and exosomes.  The structure of exosomes is retained comparing with ultracentrifugation.	Small-scale purification due to the long running time if multiple samples are applied.
Immunoafinty chromatography	Immunological methods based on the recognition of specific markers on the exosome surface. Magnetic beads bounded to antibodies are used and ELISA-based separation.	Quick protocol and requires low plasma volume.  Allows the separation of specific exosome subtypes and it can be applied for characterization of exosomal proteins.	Absence of well-defined EV markers is a limitation factor.  Small-scale purification and isolated vesicles can lose the functional activity.
Ultrafiltration	Various pore size membranes are used to separate exosomes form macromolecules. Exosomes are concentrated on the membrane after filtration.	Isolation of more specific subset of exosomes and separation from soluble molecules, fast protocol and it concentrates exosomes on the membrane.	Lower recovery due to the exosome adherence to the membrane and exosome can be damaged if additional force for filtration is applied.
Polymer precipitation	The serum is mixed with solution containing polyethylene glycol (PEG)	Simple protocol, which does not affect the exosome stability.	It can isolate different contaminants as lipoproteins and polymer material from exosomes.
Microfluidic technologies	Includes three main techniques: immunoaffinity, sieving and trapping exosomes in the microfluidic channel.	Requires smaller volume of starting material, results in highly pure exosomes in a short time.	It is in an early stage of development so it needs to be validated.

Among different isolation methods, ultracentrifugation is the most commonly used for exosome isolation, but new techniques have been developed to improve the purification process. Depending on the exosomes application, certain method is chosen for isolation, but the main problem with all those methods is scalable production for use in clinical applications. Overcoming those obstacles, novel approach has been proposed to increase yield and improve the reproducibility of exosome isolation. This alternative method is called “exosome-mimetic vesicles” which eventually may allow controlled production, thorough characterization and application in the clinical settings ¹⁰⁸.

1.4.3. Drug loading into exosomes

The success of exosomes in the drug delivery field will be influenced by the capability of exosomes to stably encapsulate therapeutic agents and at high drug concentrations. The presence of the membrane and exosomal endogenous content makes the loading challenging. Nevertheless, two methods for loading the cargo exists: loading the drug into cells form vesicles are going to be purified using the endogenous loading cell machinery, while the second method includes loading of vesicles after purification ^109,110. Before extracellular vesicles isolation, cells can be transfected to achieve loading of siRNA used for therapeutic gene silencing. For instance, various cells were transfected by vectors inducing expression of small RNAs in cells and eventually they were packed inside exosomes during their biogenesis ^111,112. The second approach of loading the therapeutics before exosome purification is the incubation of cells with various therapeutic drugs ^99,102. An interesting study also described liposome treated cells which could load hydrophobic and hydrophilic compounds in the secreted vesicles ¹¹³ . Following exosome purification, therapeutics can be loaded in different ways depending on the drug properties. Hydrophobic drugs, for example curcumin, can be loaded passively by incubation with purified exosomes at room temperature  for several minutes ¹⁰⁵. Paclitaxel and doxorubicin were also incubated with exosomes at 37°C for one hour with shaking and compared with electroporation and sonication loading methods ^81,86. Three methods were compared and sonication was the most successful method for drug loading resulting in 28.29 ± 1.38 % loading capacity, while electroporation loading capacity was 5.3 ± 0.48% and incubation 1.44 ± 0.38%.  Also, DLS confirmed that the size increased similarly, where paclitaxel-loaded exosomes had the largest diameter. Reorganisation of phospholipid membrane under the sonication enabled the drug to enter inside the vesicles and it did not significantly affect the membrane protein content ⁸⁶. Electroporation is the most commonly used method for drug loading where exosomes are mixed with drug in electroporation buffer. After application of a certain voltage to disrupt exosome structure, drug is spontaneously incorporated inside exosomes. To allow the exosomes fully recovery, sample is incubated at 37°C for several minutes ¹¹⁰. Several studies have implemented electroporation for doxorubicin loading ^80,82,97,98. However, electroporation can cause aggregation of exosomes and change their morphological characteristics ¹¹⁴. Other loading methods include saponin-treatment, freeze-thaw cycles, cholesterol conjugation and extrusion. Extrusion is characteristic for novel exosome-like vesicles which will  be described later ^109,110. Today, various methods for drug loading are available, however, it is crucial to develop the most suitable method for clinical application.

1.4.4. Achieving targeted therapies

Talking about exosomes as specific drug vehicles, targeting peptide or protein on the surface of exosomal membrane is very desirable especially in cancer treatment, where drugs are highly cytotoxic. Bioengineering of exosomes regarding the expression of specific peptides resulted in several published studies (Table 6), although this approach is still at the beginning of the research.

Table 6: Use of targeting peptides on the exosomal surface. Adapted and modified from reference ¹¹⁵

Targeting peptide	Target	Disease model	Loaded therapeutics	Method of conjugation	Ref.
iRGD	Integrin αvβ3	Breast cancer	Doxorubicin	Immature DCs were transfected with the vector expressing iRGD-Lamp2b fusion proteins using Lipofectamine	80
LFA1	Endothelial cell adhesion molecules	Colon adenocarcinoma	Doxorubicin	*N/A	108
Folate receptor α	Brain parenchyma	N/A	N/A	N/A	116
Antibody light chain	Effector T cells	Allergic cutaneous contact sensitivity		Mice was sensitized with hapten conjugated to self-antigens and suppressive nanovesicles were purified	117
GE11 or EGF	EGFR	Breast cancer	N/A	HEK293 cells were transfected with pDisplay encoding GE11 or EGF using transfection reagent	118
MHC-II	T-cells	Melanoma	N/A	Murine melanoma cells transfected with the CIITA gene to induce overexpression of MHC-II	119
RVG	Acetylcholine receptor	N/A	exogenous siRNA	Dendritic cells were transfected with pLamp2b derivative plasmids using transfection reagent	120

*N/A: Not applicable

Some targeting peptides are naturally occurring at the exosomal membrane which is great exosomal advantage regarding targeted delivery. However, some targeting peptides can be inserted into the exosomal membrane by transfection of the donor cells. For example, Tian et al. managed to engineer the lysosomal membrane associated protein 2b (Lamp2b) to express targeting peptide iRGD. This approach ensured the correct insertion of the protein in the exosome membrane after the transfection of dendritic cells ⁸⁰.

By modification of the exosomal membrane, different cancer tissues can be targeted and delivery of chemotherapeutics can be efficient increased. In order to apply such systems in vitro and in vivo, exosomes have to be isolated and purified using previously described methods in section 1.4.2.

1.5. Exosome – like vesicles (ELVs)

Novel drug and antigen delivery system has been described few years ago by Jang et al ¹⁰⁸. Trying to overcome the naturally secreted exosome limitations in drug delivery applications, cell line was “forced” to  produce the exosomes artificially, known as exosome-like vesicles ¹⁰⁸. The cells were extruded through porous membrane filters to form ELVs, which could be purified using gradient ultracentrifugation. More interestingly, using passive incubation or electroporation as shown in Figure 7. They are promising drug delivery systems because of controllable and sterile production. However, by pushing the cells through membranes, ELVs can lose some structural components, which can reduce their natural function as extracellular vesicles. Despite this disadvantage, Jang et al have described ELVs and proved that they have a good potential in drug delivery ^108,121,122. Using them mostly as drug carriers, different chemotherapeutics have been successfully loaded into ELVs ^108,122,123 .

Figure 7: Production of chemotherapeutic-loaded exosome like vesicles (ELVs). Adapted from Kim et al. ¹²⁴

 

In contrast to the low yield obtained with exosomes, preparation of ELVs resulted in 120-fold higher production yield compared to the naturally secreted vesicles from the same cell concentration ¹⁰⁸. Characterization of those ELVs exhibited similar size, surface marker proteins, and membrane topology to naturally secreted exosomes. Chemotherapeutic drugs loaded in ELVs were doxorubicin, carboplatin, gemcitabine and 5-fluorouracil and were specifically delivered to the tumour endothelium after systemic administration in vivo ¹⁰⁸. This approach showed efficient anti-tumour effects as well as naturally secreted vesicles proving that ELVs can truly mimic the natural exosomes and their role in drug delivery.  There are not many studies using ELVs because of the novelty of this approach, but a few interesting articles showed their potential applications (Table 7).

Table 7: Summary of all ELVs published studies.

Cell lines	ELVs preparation	Loading	Reference
U937, Raw264.7 and CT26 cells	Serial extrusion and ultracentrifugation	Doxorubicin (passive incubation with cells)	108
Autologous melanoma tumour cells	Tissue homogenisation and density ultracentrifugation	*N/A	125
U937 monocyte cells	Serial extrusion and ultracentrifugation	SiRNA (exogenous – electroporation, endogenous-transfection)	122
Non-tumorigenic epithelial MCF-10A cells	Serial extrusion and ultracentrifugation	siRNA (exogenous electroporation)	126
Genetically engineered E. Coli – protoplast cells	Serial extrusion and ultracentrifugation	Doxorubicin and idarubicin (passive incubation with protoplasts)	123
Genetically engineered E. Coli – protoplast cells	Serial extrusion and ultracentrifugation	Antigen loading by genetically engineered E. Coli	121
Raw 264.7 macrophage cells	Serial extrusion and ultracentrifugation	Radiolabelling agent 99mTc-HMPAO (passive incubation with ELVs)	127
NIH3T3  and MIN6 cells	Serial extrusion and ultracentrifugation	N/A	128

*N/A – Not applicable

In conclusion, ELVs produced form cells^{108,122,126–129} and bacterial protoplasts ^121,123 show great potential in overcoming the naturally secreted exosome limitations, while simultaneously retained therapeutic and delivery potential. They can be easily produced, effectively loaded with chemotherapeutic drugs, and bioengineered to express certain targeting ligands. All those properties make them promising next-generation nanocarriers for applications in drug delivery, vaccination and theranostics. However, they are also produced from a wide range of cells therefore their content must be fully determined to secure the maximum efficiency and safety. In addition, donor cells need to be selected carefully and the precise mechanism of vesicle targeting needs to be studied. The novelty of this approach is promising, nevertheless, future studies are necessary to overcome all the obstacles, and to develop safe and easily produced ELVs-based nanomedicine.

3. Materials and methods

3.1. Materials

Heat inactivated newborn fetal bovine serum (FBS) was obtained from first link, UK. Advanced RPMI 1640 medium, GlutaMAX^TM supplement 200Mm, Penicilin/Streptomycin solution liquid (1000units/ml), Trypan Blue Stain (0.4%) and Dulbecco’s Phosphate buffered saline (1X) (D-PBS) were purchased from Invitrogen Gibco® Life technologies, UK. Polycarbonate membrane filters (Whatman) 10µm, 5 µm, 1 µm, 400nm, 200nm and 100nm and SephadexTM G-25 (PD10) desalting columns, were purchased from GE Healthcare life Sciences, UK. OptiPrepTM density gradient medium, sucrose, Triton X-100, deoxycholic acid (DOC), ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulphate (SDS), sodium chloride (NaCl), trizma® base, protease inhibitor (1ML) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil) were obtain from Sigma (UK). Thermo Scientific™ Pierce™ BCA™ Protein Assay and ethanol (absolute 99.8+ %) were obtained from Thermo Fisher Scientific (UK). qEV original column was purchased from Izon Science (UK).

3.2. Methods

3.2.1. Cell culture conditions

Monoblastic cell line U937 (ATCC® CRL-1593.2™) were grown in advanced RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin and 2mM L-glutamine. Cell suspension was cultured in T-75 tissue culture flasks (Thermo Fisher Scientific, UK) at 37°C incubator with a humidified atmosphere of 5% CO₂. Cells were splited every four days using fresh media to maintain cell concentration at 5×10⁵ cells/ml.

3.2.2. Preparation of exosome-like vesicles (ELVs)

Cells were washed three times using Dulbecco’s phosphate-buffered saline (D-PBS), centrifuged for 5min at 1500rpm (Heraeus Megafuge 8R, Thermo Scientific, UK), and resuspended in D-PBS at a concentration of 1, 2, 3, 4 and 5×10⁶ cells/ml. Cell suspensions were sequentially extruded through 10µm (15x); 5µm (20x); 1µm (20x); 400nm (30x); 200nm (30x)  and 100nm (30x) polycarbonate membrane filters using a mini-extruder (Avanti Polar Lipids, AL USA). Also, cell suspension was extruded three times through 10µm (10x), 5µm (15x), 1µm (20x) and 0.4µm (20x) polycarbonate membrane filters (Whatman) using 10ml sterile plastic syringe (Thermo Fisher Scientific, UK) attached to the syringe filter holder (13mm diameter).

3.2.3. Labelling ELVs with Dil dye

Lipophilic Dil (Sigma Aldrich, UK) dye was dissolved in ethanol at a concentration of 1mM. U937 Cells were resuspended at a concentration of 1×10⁶ cells/ml and incubated in serum free media with 40ul of 1mM DiI solution for 40 min at 37°C. To remove free DiI, cell suspension was washed three times in D-PBS and centrifuged for 5min at 1500 rpm. Dil-labeled cells were extruded, as described above.

3.2.4. Spectrofluorometry

Dil-labelled cells and extruded labelled ELVs (200µl) were transferred to 96- flat bottom black well plates (Sterilin Thermo Fisher Scientific, UK) Fluorescence intensity of DiI was measured using excitation and emission wavelength of 244nm and 590nm, respectively. Fluorescence spectral analysis was done using Omega microplate reader (BMG labTechnologies) and MARS data analysis software. The results were expressed as the fluorescence intensity (a.u.) ratio of purified sample to unpurified sample.

3.2.5. PD10 purification

PD10 is a desalting column with Sephadex G-25 resin. It is used for buffer exchange, desalting and removal of small contaminants. The chromatography technique is gel filtration and different molecules are separated on the basis of differences in size. In principle, molecules larger than the beads pore size in the sephadex matrix are eluted first, and smaller molecules trapped in beads will be eluted later. The PD-10 column was equilibrated with 20ml of D-PBS before purification and 1ml of sample was loaded to the column. ELVs sample was eluted using 1ml of fresh D-PBS. Twenty fractions of 1ml were collected and further studied for size measurements, and protein quantification using dynamic light scattering and BCA assay, respectively.

3.2.6. qEV original purification

qEV original column contains Sepharose resin with a pore size approximately 75 nm. Proteins and other contaminating molecules smaller than 75nm enter the pores of the resin and eluting in later fractions. To purify ELVs from soluble proteins, 500 µl of extruded ELVs sample was loaded on the column. As sample entered the column top filter, more D-PBS buffer was added and 500µl fractions were collected. The first six fractions (3 ml) are considered as the void volume and did not contain any ELVs.

3.2.7. Ultracentrifugation

Density gradient was prepared by placing 2ml of 50% OptiPrep^TM (iodixanol) at the bottom of an Ultra-Clear™ 13 x 51 mm ultracentrifuge tube (Beckman Coulter), overlaid with 2ml of 10% iodixanol and 1ml of extruded samples (5×10⁶ cell/ml extruded through 10, 5 and 1µm membrane and additional sample extruded through 0.4 and 0.2µm membrane). The tubes were placed in SW 55Ti rotor and ultracentrifuged at 100000g for 2h at 4°C using Beckman-Coulter Optima XL-100K ultracentrifuge.

3.2.8. Dynamic light scattering (DLS)

The mean hydrodynamic diameter of exosome like vesicles and polydispersity (PDI) were measured by dynamic light scattering using Zetasizer Nano ZS (Malvern Instruments). The ELVs resuspended in D-PBS, unpurified and purified samples were placed in disposable polystyrene cuvettes (Thermo Fisher Scientific, UK) and measured at room temperature. The Z-average diameter (nm) ± SD and PDI were expressed as average of three measurements.

3.2.9. Optimization of lysis buffer

 

Two RIPA buffer concentrations were prepared for cell lysis: 1x RIPA (10% Triton X-100, 5% DOC, 1% SDS, 0.15M NaCl, 0.05M Tris pH 8.0) and 2.7 times concentrated RIPA buffer (27% Triton X-100, 13.5% DOC, 2.7% SDS, 0.4M NaCl, 0.14M Tris pH 8.0). U937 monocytes were resuspended in D-PBS at the following concentrations: 1, 3 and 5 x 10⁶ cells/ml. Supernatant was removed after centrifugation at 1500rpm for 5 min and 200µl of 1x RIPA or 2.7x RIPA buffer was added to the cell pellet. Cells were sonicated and vortexed for 10 min to disrupt the cell membrane and extract the proteins from the cytosol. Between sonication and vortexing, samples were left on ice for 30 min in total to protect the proteins form protease degradation. After lysis, samples were centrifuged at 14000 rpm for 20min at 4°C. Supernatant was collected in clean tubes and protein concentration was determined using BSA assay.

3.2.10. ELVs lysis

Unpurified and purified ELVs (1, 2, 3, 4 and 5×10⁶ cell/ml initial concentration) suspended in D-PBS were mixed with RIPA buffer (50, 100, 150, 200 and 250µl) on ice. The samples were sonicated two times for to cycles for 5 min at room temperature starting with ELVs prepared form cells with the highest concentration. Between the two sonication cycles they were vortexed and incubated on ice for 30min in total.

3.2.11. Bicinchoninic acid (BCA) assay

Protein concentration was measured using BCA protein assay kit (Thermo Fisher Scientific, UK). After RIPA lysis, 20µl of ELVs suspension was placed in 96-well plate and 160µl of working reagent was added for each well (working reagent: 50:1 ratio of assay reagents A and B). The palate was incubated at 37°C for 30min before being analysed using Omega microplate reader and MARS data analysis software. Protein concentration in each well was calculated using BCA standard curve.

3.2.12. Nanoparticle tracking analysis (NTA)

Nanoparticle tracking analysis is an innovative system to determine particle size distribution and concentration in liquid suspension. This technique is based on combining laser light scattering microscopy with a charge-coupled device (CCD) camera, which enables the visualisation and recording of nanoparticles in solution. The software is able to identify and track individual nanoparticles under Brownian motion and relates the movement to a particle size according to the formula derived from the Stokes-Einstein equation¹³⁰. NTA has become more reliable technique than DLS in determining the ELVs size. Larger particles scatter more light than smaller ones, so their signals can be over-shadowed by bigger particles in the DLS case. In addition, exosome quantification has always been unreproducible and challenging using the protein quantification assays, but with NTA the ultimate quantification method has been established. Our NTA measurements were performed with a NanoSight LM20 (Malvern Instruments) in collaboration with King’s College London. Purified ELVs were diluted in PBS at an appropriate concentration and injected in the sample chamber using sterile syringes. All measurements were performed at room temperature and analysed using NTA 3.2 Dev Build 3.2.16 software. Three measurements of the same sample were performed and mean size and SD values correspond to the arithmetic values calculated with the sizes of all the particles was analysed using the software. The concentration of ELVs in each fraction was expressed as particles/ml.

3.2.13. Transmission electron microscopy

A few drops of the purified ELVs were placed on a copper grid coated with 300 mesh carbon film (Agar Scientific, UK), stained with 1% phosphotungstic acid (PTA) (Agar Scientific, UK) and dried. TEM experiments were performed using a JEOL JEM-2010 microscope operated at 200kV.

4. Results

4.1. Preparation of ELVs

Initially, cell suspension was extruded  through 10, 5, 1 and 0.4 µm polycarbonate membrane filters (Whatman) using 10ml sterile plastic syringe (Thermo Fisher Scientific, UK) attached to a syringe filter holder (13mm diameter). During the extrusion, the pressure was not sufficient to push all the cells through the membrane, resulting in membrane replacements and sample leakage from the filter holder. In addition, the preparation of ELVs was not reproducible as was evidenced by different sample volumes obtained after each extrusion. Due to these limitations, mini-extruder (Avanti Polar Lipids) was used to sequentially extrude cell suspensions (Figure 8).

       

Figure 8: Extruder units used for ELVs production: (A) 10ml syringe with a plastic filter holder unit (Thermo Fisher Scientifc, (B) Mini-extruder (Avanti Polar Lipids)

Different cell concentrations were extruded starting from 1×10⁶ cells/ml to 5×10⁶ cells/ml in order to assess the influence of the concentration on the size, polydispersity and particle recovery.  The hydrodynamic diameter and polydispersity index (PDI) were measured using Nano Zetasizer (Malvern) (Table 8). Our results showed that the size of vesicles was determined by the size of the pore sized used. For instance, extrusion through 0.4µm pore size membrane, ELVs size was approximately 230 – 260 nm and ELVs size did not significantly differ between the initial cell concentrations. Regarding in vivo applications, smaller particle size is preferable for tissue penetration and high retention in tumours (around 150nm). Reducing the membrane pore size to the 0.2µm and 0.1µm, smaller ELVs were produced and size measurement showed particle size around 150 nm. However, DLS measures the hydrodynamic size i.e. particle diameter in solution, so presented size is not the actual size of our ELVs.

Table 8.  Hydrodynamic diameter (Z-Ave) and polydispersity index (PDI) of unpurified ELVs after extrusion through 0.4µm, 0.2µm and 0.1µm membrane filters. The results were expressed as mean (nm) ± SD, for three measurements per sample, at 25°C.

Extrusion cycles	15×10µm, 20×5µm, 20×1µm, 30×0.4µm
Cell concentration	1×106 cells/ml	2×106 cells/ml	3×106 cells/ml	4×106 cells/ml	5×106 cells/ml
Z-Ave d.nm ± SD	233 ± 4.957	240 ± 5.289	255 ± 2.608	259 ± 7.219	239 ± 1.858
Pdl ± SD	0.276 ± 0.027	0.290 ± 0.018	0.328 ± 0.030	0.338 ± 0.053	0.262 ± 0.009
Extrusion cycles	15×10µm, 20×5µm, 20×1µm, 30×0.4µm, 30×0.2µm
Cell concentration	1×106 cells/ml	2×106 cells/ml	3×106 cells/ml	4×106 cells/ml	5×106 cells/ml
Z-Ave d.nm ± SD	184.5 ± 5.021	179 ± 2.501	182 ± 2.100	178 ± 3.774	186 ± 6.495
Pdl ± SD	0.210 ± 0.029	0.185 ± 0.013	0.157 ± 0.035	0.180 ± 0.031	0.178 ± 0.006
Extrusion cycles	15×10µm, 20×5µm, 20×1µm, 30×0.4µm, 30×0.2µm, 30×0.1µm
Cell concentration	1×106 cells/ml	2×106 cells/ml	3×106 cells/ml	4×106 cells/ml	5×106 cells/ml
Z-Ave d.nm ± SD	154 ± 8.721	155 ± 4.149	162 ± 2.100	159 ± 2.050	150 ± 1.002
Pdl ± SD	0.260 ± 0.060	0.183 ± 0.017	0.215 ± 0.008	0.175 ± 0.023	0.181 ± 0.012

4.2. ELVs purification

In order to separate soluble proteins and cellular debris from ELVs after extrusion, all extruded samples have to be purified. Among all previously described methods, size exclusion chromatography and density gradient ultracentrifugation are the most commonly used for the exosome and ELVs purification ¹³¹. These methods were used to purify our ELVs, as will be described below.

4.2.1. Size exclusion chromatography using PD10 column

First purification method used was PD10 column.  The chromatography technique is a gel filtration method where molecules could be separated based on the differences in their size. Molecules larger than the beads pores in the sephadex matrix are eluted first (i.e. void volume), while smaller molecules penetrate the pores and exit the column later. To detect ELVs fractions during purification, cells (5×10⁶ cells/ml) were labelled with DiI lipophilic dye (Sigma Aldrich) before extrusion and afterwards ELVs were purified through PD10 column. DiI (Ex/Em = 550/563nm) is a fluorescent dye which is incorporated in cell membranes and  ELVs enabling their detection using fluorescence platereader. 1ml of ELVs was loaded to the PD10 column and the first fraction containing a void volume was discarded. Then, the sample was eluted by adding 1ml of fresh D-PBS and 20 fractions of 1ml were collected. Following purification, fluorescence was measured in each fraction in order to identify ELVs fractions. Fluorescence percentage was calculated by dividing the fluorescence of each fraction (1-20) by the initial fluorescence of the unpurified sample (which was expressed as 100% fluorescence) (Figure 9). Our results showed that fractions 3 and 4 contained ELVs with the high fluorescence percentage of 51% and 26%, respectively.

Figure 9. Elution profile of Dil-labeled ELVs in PD-column.Percentage of fluorescence was calculated by dividing the fluorescence intensity of each fraction (1-20) by the initial intensity of unpurified sample (S) x 100.

DLS measurements showed that only fractions 3 and 4 contained particles, approximately the same size and polydispersity as unpurified sample. Larger particles were eluted first, in fraction 3, followed by smaller vesicles, which were collected in fraction 4 (Table 9). To confirm purity, protein concentration was determined in all fractions using BCA assay. If purification was successful, fractions 3 and 4 should not contain free proteins. However, our results demonstrated that ELVs purification using PD-10 column was not efficient due to the high protein concentrations in those fractions, 600 µg/ml and 400 µg/ml, respectively (Figure 10). Therefore, other purification methods such as ultracentrifugation and qEV were studied.

Table 9. The hydrodynamic diameter (Z-Ave) and polydispersity index (PDI) of purified ELVs using PD10 column. The results were expressed as mean (nm) ± SD, for three measurements per sample, at 25°C.

Extrusion cycles	10×10µm, 15×5µm, 20×1µm, 20×0.4µm
Sample (1×106 cells/ml)	Unpurified sample	Fraction 3	Fraction 4
Z-Ave d.nm ± SD	246 ± 2.946	268 ± 3.612	202 ± 6.612
Pdl ± SD	0.440 ± 0.006	0,419 ± 0.032	0,438 ± 0.049

Figure 10. Total protein concentration in purified ELVs (fractions 3 and 4) after PD10 purification.

 

4.2.2. Ultracentrifuge

 

The next purification method used for removing proteins aggregation and cell debris from ELVs was OptiPrep^TM (iodixanol 60% w/v) density gradient ultracentrifugation. Following the previously reported use of ultracentrifuge as exosome and exosome – like vesicles purification method ^{108,122,123,129} , the density gradient was prepared by placing 2ml of 50% iodixanol at the bottom of an ultracentrifuge tube (Beckman Coulter), then overlaid with 2ml of 10% iodixanol and 1ml of extruded samples. The tubes were ultracentrifuged at 100000g for 2h at 4°C (Figure 11).

Figure 11. Sample preparation for ultracentrifuge

ELVs were collected from the interface of the 50% and 10% iodixanol layers, however, our size measurements did not show any particles. In fact, DLS was not able to measure the sample. Because of small sample volume and difficulties in sample extraction from the ultracentrifuge tube (Figure 12) different purification method was used.

Figure 12. ELVs position between 10% and 50% OptiPrep solution after ultracentrifugation.

4.2.3. qEV original

After unsuccessful PD-10 and ultracentrifugation purification, size exclusion chromatography using qEVoriginal column (Izon Science) was performed, as described previously ^132–134. The column contains a Sepharose resin with a pore size of 75 nm which efficiently separate exosomes and ELVs from soluble proteins. As 500µl of extruded sample was loaded to the column, then eluted using 500µl and 25 fractions (500µl each) were collected. The first six fractions were considered as void volume (3 ml) which did not contain vesicles, where fractions 7, 8, 9 and 10 contained the purified vesicles. Fractions beyond 10 usually contained higher protein and lower vesicle levels ¹³⁵.  Our DLS measurement confirmed the presence of ELVs in the fractions 7-10 (Table 10) and BCA protein quantification assay confirmed their purity. The presence of ELVs was further confirmed by the high protein content in fraction 8 and 9 following ELVs lysis using RIPA buffer (Figure 13) and concentration of ELVs expressed as particle/ml  was determined using nanoparticle tracking  analysis (NTA).

Table 10. Hydrodynamic diameter (Z-Ave) and polydispersity index (PDI) of purified ELVs using qEV column. The results were expressed as mean (nm) ± SD, for three measurements per sample, at 25°C.

Extrusion cycles		15×10µm, 20×5µm, 20×1µm, 30×0.4µm, 30×0.2µm
qEV Fractions	7			8		9		10
Samples		Z-Ave d.nm ± SD	Pdl ± SD	Z-Ave d.nm ± SD	Pdl ± SD	Z-Ave d.nm ± SD	Pdl ± SD	Z-Ave d.nm ± SD	Pdl ± SD
1×106 cells/ml		218 ± 13.65	0.248 ± 0.087	205 ± 5.672	0.129 ± 0.030	197 ± 4.100	0.107 ± 0.015	180 ± 1.557	0.148 ± 0.065
2×106 cells/ml		213 ± 4.176	0.128 ± 0.060	202 ± 6.207	0.095 ± 0.023	194 ± 5.029	0.084 ± 0.031	172 ± 3.258	0.173 ± 0.025
3×106 cells/ml		211 ± 11.52	0.251 ± 0.066	201 ± 5.079	0.079 ± 0.031	195 ± 3.026	0.095 ± 0.031	179 ± 3.748	0.124 ± 0.004
4×106 cells/ml		205 ± 5.272	0.254 ± 0.087	192 ± 4.310	0.113 ± 0.028	177 ± 0.7095	0.114 ± 0.038	168 ± 4.347	0.128 ± 0.010
5×106 cells/ml		230 ± 7.347	0.391 ± 0.026	206 ± 4.508	0.141 ± 0.026	190 ± 2.548	0.129 ± 0.013	172 ± 2.608	0.157 ± 0.013

Figure 13. Protein concentration in fractions 7-25 after ELVs purification (1×10⁶ cells/ml) before and after lysis, with RIPA buffer.

4.3. Determination of ELVs protein content using BCA

 

BCA Protein assay (Thermo Scientific) was used for protein quantification in order to confirm the purity of the ELVs and to determine total protein concentration from pure ELVs (Figure 13). To accurately measure ELVs protein content, ELVs should be lysed to extract membrane and cytoplasmic proteins. To determine the sufficient RIPA buffer concentration to completely extract proteins from ELVs, RIPA buffer was prepared at two concentrations and used for cell lysis: 1x RIPA and 2.7x RIPA buffer, respectively. In this experiments, cells were resuspended in PBS at the following concentrations 1, 3 and 5 x 10⁶ cells/ml. Supernatant was removed after centrifuge at 1500rpm for 5 min and the cell pellets were lysed using 200µl of 1x or 2.7x RIPA. The lysed pellet was sonicated and vortexed for 10 min and left on ice for 30 min to ensure complete lysis. Then, samples were centrifuged at 14000 rpm for 20min and the supernatant was collected in the clean tubes, and protein concentration was determined using BCA and BSA calibration curve (Figure 14).

Figure 14. Calibration curve of 1x RIPA with known BSA concentrations.

Figure 15 depicts the total protein concentration of different concentration of cells lysed with RIPA buffers (1x and 2.7x). Lysis was more efficient with 2.7x RIPA in 1×10⁶ and 5×10⁶ cells/ml but it was not statistically significant.  By comparing the two RIPA concentrations, we decided to continue using 1x RIPA for lysis of all ELVs before and after purification.

Figure 15. Total protein concentrations from lysed cell pellets.

After selecting the optimal concentration of RIPA buffer to lyse cells, we moved next to determine the protein content in purified ELVs. Fractions containing exosomes (7-10) were mixed with 1x RIPA buffer in increasing volumes to ensure complete lysis in all samples, despite their concentration (Table 11). 20µl of each lysed fraction containing ELVs was placed in 96 well plate and protein content was determined using BCA. After incubation, the colour of lysed and unlysed samples changed from green to purple, indicating the presence of proteins (Figure 16).

Table 11. The volumes of 1x RIPA buffer used to lyse ELVs samples of different concentrations.

 

Samples (cell/ml)	1×106	2×106	3×106	4×106	5×106
Sample : 1xRIPA  (V/V)	1:1	1:2	1:3	1:4	1:5
Sample : 1xRIPA (µl/µl)	100 µl:100 µl	100 µl: 200 µl	100 µl: 300 µl	100µl: 400 µl	100 µl: 500 µl
Final dilution	1:2	1:3	1:4	1:5	1:6

           

Figure 16. Colour change between lysed and unlysed samples: a) 96-well plate with unpurified (purple) and purified (green) samples without lysis, b) 96-well plate with unpurified and purified samples with lysis.

Figure 17. a) depicts the protein concentration in purified ELVs prepared using different cell concentrations (1-5×10⁶ cells/ml). These concentrations were determined following lysing ELV fractions (7-10) using 1x RIPA buffer and BSA standard curve. A linear correlation was observed from lower to higher cell concentration samples, where fractions 8 and 9 contained the highest protein concentration. Sample with 5×10⁶ cells/ml showed the highest total protein concentration (1832 µg/ml), while samples with 1,2,3 and 4×10⁶ cells/ml showed lower protein concentration (25 µg/ml, 137 µg/ml, 833 µg/ml and 1410 µg/ml). Protein concentration in sample with the lowest cell concentration (1×10⁶ cells/ml) was only measured in fractions 8 and 9, while fraction 7 and 10 did not contain any proteins. In addition, other samples showed higher protein concentration in fractions 8 and 9 suggesting they were the most abundant ELVs fractions.

Figure 17. Total protein concentration and percentage of protein recovery in each fraction after ELVs lysis: a) protein concentration in unpurified and purified ELVs (fractions 7-10) prepared using different cell concentrations (1-5×10⁶ cells/ml), b) Protein recovery from fractions 7-10 immediately after purification expressed as a percentage by dividing the protein concentration of each fraction by the total protein concentration from the unpurified samples

 

Protein recovery of each ELVs fraction was also expressed as a percentage by dividing the protein concentration of each fraction by the total protein concentration from the unpurified samples (Figure 17.b). Protein recovery from 1×10⁶ cells/ml sample was only 7%, following 20% recovery from 2×10⁶ cells/ml sample. Sample with initial cell concentration of 3×10⁶ cells/ml had a uniform recovery in all fractions (15%) with the total recovery of 60%, while recovery percentage further increased in 4×10⁶ cells/ml sample (68%). ELVs prepared form the highest cell concentration (5×10⁶ cells/ml) showed the highest recovery (79%), which implicates that optimal cell concentration for highest ELVs yield was 5×10⁶cells/ml.

4.4. ELVs stability overtime

4.4.1. Monitoring stability by DLS

To determine the stability of ELVs after extrusion, the size of all the samples, purified and unpurified was measured every day after extrusion. Samples are always stored at 4°C in D-PBS. Tables 12-16 summarise the hydrodynamic diameter (Z-Ave) and polydispersity index (PDI) of unpurified and purified samples prepared using different cell concentrations. Monitoring size, polydispersity and count rate it was possible to demonstrate ELVs stability overtime. When the size and polydispersity of the particles increased, the count rate decreased which meant that sample was no longer stable and DLS measurement did not show good quality report. It is shown that unpurified sample prepared using initial concentration of 1×10⁶ cells/ml maintained the stability up to five days, while samples with higher ELVs concentration maintained their stability up to two weeks. On the other hand, purified ELVs from fractions 7 and 10 showed a lower stability over time because of their aggregation. ELVs in fractions 8 and 9, which were prepared from high cell concentrations (3,4 and 5×10⁶ cells/ml), maintained their stability for a longer time (approximately one to two weeks).

Table 12.  Size stability of purified and unpurified ELVs over two weeks (1×10⁶ cells/ml) as determined by DLS.

	Unpurified		Fraction 7		Fraction 8		Fraction 9		Fraction 10
1×106 cells/ml	Z-Ave d.nm ± SD	Pdl ± SD	Z-Ave d.nm ± SD	Pdl ± SD	Z-Ave d.nm ± SD	Pdl ± SD	Z-Ave d.nm ± SD	Pdl ± SD	Z-Ave d.nm ± SD	Pdl ± SD
Day 1	184.5± 5.021	0.210 ± 0.029	218 ± 13.65	0.248 ± 0.087	205 ± 5.672	0.129 ± 0.030	197 ± 4.100	0.107 ± 0.015	180 ± 1.557	0.148 ± 0.065
Day 3	196 ± 22.49	0.176 ± 0.012	NP		224 ± 8.358	0.118 ± 0.011	218 ± 4.670	0.063 ± 0.034	191 ± 6.621	0.047  6.621
Day 5	180 ± 3.958	0.189 ± 0.012	NP		217 ± 7.139	0.094 ± 0.015	203 ± 3.132	0.078 ± 0.020	176 ± 85.91	0.189 ± 0.012
Day 7	520 ± 83.33	0.920± 0.105	NP		228 ± 15.65	0.075 ± 0.035	212 ± 4.660	0.071 ± 0.035	NP
Day 10	*NP		NP		223 ± 12.26	0.077 ± 0.041	216 ± 15.81	0.060 ± 0.036	NP
Day 14	NP		NP		NP		218 ± 14.25	0.070± 0.019	NP

*NP: Not possible to measure due to poor quality of the sample

Table 13.  Size stability of purified and unpurified ELVs over two weeks (2×10⁶ cells/ml) as determined by DLS

	Unpurified		Fraction 7		Fraction 8		Fraction 9		Fraction 10
2×106 cells/ml	Z-Ave d.nm ± SD	Pdl ± SD	Z-Ave d.nm ± SD	Pdl ± SD	Z-Ave d.nm ± SD	Pdl ± SD	Z-Ave d.nm ± SD	Pdl ± SD	Z-Ave d.nm ± SD	Pdl ± SD
Day 1	179 ± 2.501	0.185 ± 0.013	202 ± 6.207	0.128 ± 0.060	202 ± 6.207	0.095 ± 0.023	194 ± 5.029	0.084 ± 0.031	172 ± 3.258	0.173 ± 0.025
Day 3	184 ± 4.124	0.189 ± 0.011	226 ± 5.408	0.174 ± 0.036	204 ± 7.074	0.105 ± 0.018	198 ± 6.161	0.100 ± 0.025	192 ± 0.9899	0.111 ± 0.059
Day 5	183 ± 2.364	0.210 ± 0.015	220± 18.06	0.160 ± 0.103	202 ± 4.850	0.084 ± 0.042	195 ± 1.510	0.104 ± 0.027	188 ± 5.096	0.098 ± 0.026
Day 7	184 ± 5.254	0.223 ± 0.009	218 ± 6.045	0.189 ± 0.021	204 ± 6.090	0.088 ± 0.009	194 ± 5.108	0.103 ± 0.029	191 ± 2.052	0.144 ± 0.036
Day 10	183 ± 4.303	0.195 ± 0.012	*NP		207 ± 8.808	0.070 ± 0.017	194 ± 1.012	0.105 ± 0.022	176 ± 2.173	0.207 ± 0.010
Day 14	174 ± 3.866	0.206 ± 0.016	NP		206 ± 8.503	0.098 ± 0.010	192 ± 2.779	0.088 ± 0.015	182 ± 2.442	0.150 ± 0.040

*NP: Not possible to measure due to poor quality of the sample

Table 14.  Size stability of purified and unpurified ELVs over two weeks (3×10⁶ cells/ml) as determined by DLS.

	Unpurified		Fraction 7		Fraction 8		Fraction 9		Fraction 10
3×106 cells/ml	Z-Ave d.nm ± SD	Pdl ± SD	Z-Ave d.nm ± SD	Pdl ± SD	Z-Ave d.nm ± SD	Pdl ± SD	Z-Ave d.nm ± SD	Pdl ± SD	Z-Ave d.nm ± SD	Pdl ± SD
Day 1	182 ± 2.100	0.157 ± 0.035	211 ± 11.52	0.251± 0.066	201 ± 5.079	0.079 ± 0.031	195 ± 3.026	0.095 ± 0.031	179 ± 3.748	0.124 ± 0.004
Day 3	187 ± 4.188	0.173 ± 0.015	*NP		207 ± 2.263	0.103 ± 0.018	199 ± 5.862	0.082 ± 0.0027	184 ± 5.020	0.120 ± 0.045
Day 5	182 ± 3.950	0.199 ±0.029	NP		201 ± 2.829	0.102 ±0.15	196 ± 0.3055	0.104 ± 0.013	181 ± 0.8083	0.128 ± 0.016
Day 7	179 ± 1.872	0.181 ± 0.015	NP		202 ± 1.858	0.077 ± 0.005	196 ± 2.021	0.108 ±0.009	184 ± 1.436	0.080 ± 0.024
Day 10	174 ± 2.060	0.202 ± 0.016	NP		199 ± 2.413	0.117 ± 0.006	195 ± 2.722	0.084 ± 0.022	178 ± 1.716	0.109 ± 0.013
Day 14	171 ± 1.845	0.155 ± 0.0006	NP		194 ± 2.381	0.055 ± 0.016	196 ± 2.868	0.110 ± 0.031	183 ± 5.248	0.099 ± 0.028

*NP: Not possible to measure due to poor quality of the sample

Table 15.  Size stability of purified and unpurified ELVs over two weeks (4×10⁶ cells/ml) as determined by DLS.

	Unpurified		Fraction 7		Fraction 8		Fraction 9		Fraction 10
4×106 cells/ml	Z-Ave d.nm ± SD	Pdl ± SD	Z-Ave d.nm ± SD	Pdl ± SD	Z-Ave d.nm ± SD	Pdl ± SD	Z-Ave d.nm ± SD	Pdl ± SD	Z-Ave d.nm ± SD	Pdl ± SD
Day 1	178 ± 3.774	0.180 ± 0.031	205 ± 5.272	0.254 ± 0.087	192 ± 4.310	0.113 ± 0.028	177 ± 0.7095	0.114 ± 0.038	168 ± 4.347	0.128 ± 0.010
Day 3	186 ± 16.44	0.185 ± 0.001	*NP		217 ± 26.27	0.146 ± 0.021	196 ± 9.234	0.094 ± 0.015	177 ± 9.091	0.141 ± 0.012
Day 5	189 ± 19.81	0.196 ± 0.012	342 ± 57.19	0.515 ± 0.084	207 ± 13.17	0.127 ±0.034	196 ±13.60	0.090 ±0.008	174 ± 5.636	0.132 ± 0.005
Day 7	164 ± 1.836	0.178 ± 0.035	NP		190 ± 4.428	0.126 ± 0.031	181 ± 2.730	0.090 ± 0.027	170 ± 2.629	0.117 ± 0.010
Day 10	165 ± 1.457	0.185 ± 0.007	NP		194 ± 2.063	0.113 ± 0.016	178 ± 2.079	0.105 ± 0.017	169 ± 1.002	0.110 ± 0.017
Day 14	165	0.179	NP		195 ± 4.729	0.105 ± 0.025	182 ± 3.630	0.107 ± 0.019	171 ± 1.804	0.122 ± 0.025

*NP: Not possible to measure due to poor quality of the sample

 

Table 16.  Size stability of purified and unpurified ELVs over two weeks (5×10⁶ cells/ml) as determined by DLS.

	Unpurified		Fraction 7			Fraction 8		Fraction 9		Fraction 10
5×106 cells/ml	Z-Ave d.nm ± SD	Pdl ± SD	Z-Ave d.nm ± SD		Pdl ± SD	Z-Ave d.nm ± SD	Pdl ± SD	Z-Ave d.nm ± SD	Pdl ± SD	Z-Ave d.nm ± SD	Pdl ± SD
Day 1	186 ± 6.495	0.178 ± 0.006	230 ± 7.347		0.391 ± 0.026	206 ± 4.508	0.141 ± 0.026	190 ± 2.548	0.129 ± 0.013	172 ± 2.608	0.157 ± 0.013
Day 3	185 ± 5.773	0.170 ± 0.011	247 ± 12.94		0.308 ± 0.083	215 ± 8.011	0.155 ±0.040	193 ± 3.612	0.145 ± 0.017	178 ± 1.607	0.132 ± 0.028
Day 5	186 ± 6.252	0.174 ± 0.024	264.9 ±44.35	0.311 ± 0.161		214 ± 8.907	0.137 ± 0.019	194 ± 4.100	0.116 ± 0.029	178 ± 4.061	0.111 ± 0.010
Day 7	175 ± 1.716	0.192 ± 0.032	223 ± 3.612	0.195 ± 0.071		209 ± 3.816	0.146 ± 0.015	192 ± 1.069	0.131 ± 0.035	173 ± 3.855	0.106 ± 0.014
Day 10	174 ± 0.8145	0.188 ± 0.026	215 ± 8.300	0.221 ± 0.063		208 ± 0.9452	0.144 ± 0.021	189 ± 1.212	0.133 ± 0.025	173 ± 0.4163	0.127 ± 0.015
Day 14	178 ± 7.001	0.159 ± 0.013	220 ± 4.701	0.191± 0.006		208 ± 5.313	0.116 ± 0.030	192 ± 6.000	0.091 ± 0.019	173 ± 1.539	0.132 ± 0.006

*NP: Not possible to measure due to poor quality of the sample

4.4.2. Monitoring stability by protein content (BCA assay)

 

Next, we assessed the stability of the encapsulated proteins in ELVs overtime. Figure 18 summarise the protein concentrations of unpurified and purified samples prepared using different cell concentrations (1-5×10⁶ cell/ml). After one week, both purified and unpurified samples with lower initial cell concentration showed decreased protein concentrations, while samples with 4 and 5 x 10⁶ cells/ml, maintained the initial protein content (Figure 18.b). After two weeks, all purified samples had significantly lower protein concentrations indicating that ELVs were no longer stable and their proteins degraded (Figure 18.c). On the other hand, unpurified sample with the highest initial cell concentration (5×10⁶ cells/ml) maintained the same protein concentration over two weeks.

Although concentrated fractions 8 and 9 from high initial cell concentrations (3,4 and 5×10⁶ cells/ml) maintained their size stability for two weeks (Table 14, 15, 16) , their protein concentration significantly decreased after one week. Regarding the size stability and protein concentration over time, purified samples can be stored at 4°C within one week. In terms of stability and protein recovery, it can be concluded that our optimal cell concentration for ELVs production was 5×10⁶cells/ml.

Figure 18. Protein stability of purified and unpurified ELVs overtime as determined by BCA. a) Protein concentration in unpurified and purified ELVs (fractions 7-10) prepared using different cell concentrations (1-5×10⁶ cells/ml) immediately after purification: b) protein concentration from the same samples measured after one week from purification; c) protein concentration from the same samples measured after two weeks from purification.

4.5. Nanoparticle tracking analysis (NTA) measurements

NTA measurements were performed in collaboration with King’s College London to quantify the ELVs yield and determine the ELVs size distribution. Two cell concentrations (1 and 5×10⁶ cell/ml) were freshly extruded through 200nm pore size membrane and purified using qEV column a day before NTA measurement. Additionally, one-week-old sample (initial cell concentration 5×10⁶ cells/ml) was also measured using NTA. Prior to NTA, size was determined by DLS measurements in order to compare the two methods (Table 17). NTA measures the random movement of single particles in a sample while DLS measures intensity of scattered light caused by particles Brownian motion. Due to the stronger signal from bigger particles, DLS always shows slightly bigger size than NTA.

Table 17. Hydrodynamic diameter (Z-Ave) and polydispersity index (PDI) measured using DLS compared with mean size distribution measured using NTA.

	DLS						NTA
Initial cell concentration (cells/ml)	1×106		5×106		5×106  (one week old sample)		1×106	5×106	5×106  (one week old sample)
	Z-Ave ± SD	PDI ± SD	Z-Ave ± SD	PDI ± SD	Z-Ave ± SD	PDI ± SD	Mean (nm) ± SD
Fraction 7	204 ± 3.313	0.066 ± 0.030	182 ± 4.120	0.098 ± 0.038	184 ± 1.375	0.093 ± 0.034	164 ± 14	136 ± 29	212 ± 17
Fraction 8	198 ± 2.401	0.093 ± 0.023	174 ± 0.4583	0.101 ± 0.015	179 ± 0.8083	0.064 ±0.013	99 ± 5.4	102 ± 5.3	149 ± 11
Fraction 9	177 ± 5.408	0.158 ± 0.027	158 ± 2.173	0.135 ± 0.019	169 ± 0.5292	0.126 ± 0.023		189 ± 12.2	159 ± 8
Fraction 10	*NP	*NP	129 ± 3.225	0.222 ± 0.040	150 ± 1.873	0.137 ± 0.027			134 ± 10

*NP: Not possible to measure due to poor quality of the sample

All samples measured using NTA showed smaller mean size distribution except for fraction 9 (5×10⁶ cell/ml sample) and fraction 7 (old 5×10⁶ cell/ml sample). NTA showed a size distribution of the purified ELVs graphically, with a peak diameter of approximately 130 – 200 nm (Figure 19). Fraction 7 ( 5×10⁶ cells/ml, one week old sample) contains mostly larger particles with peak diameter of 146 and 200nm because the largest particles are eluted first during purification process (Figure 19.a). Other fractions (8-10) showed smaller peak diameters of 135, 153 and 143nm (Figure 19. b, c, d). To obtain monodispersed sample, next step is to extrude the cells through 100nm, so larger particles will be removed.

 

 

Figure 19: Size distribution of purified ELVs (initial cell concentration 5×10⁶ cells/ml) from different fractions measured by NTA; a) Fraction 7 was diluted 1:100 in PBS and size distribution of ELVs was measured with two peak diameters of 146 and 200nm. b) Fraction 8 was diluted 1:1000 in PBS and size distribution was measured with three peak diamters of 96, 135 and 195nm. c) Fraction 9 was diluted 1:100 in PBS and size distribution was measurd with peak diamter of 153nm. d) Fraction 10 was diluted 1:50 in PBS and size dstribution was measured with peak diamter of 143nm.

In order to quantify the ELVs, NTA was used to measure the particle count in the sample, which was expressed as particle/ml. We also measured protein concentration from the three samples before NTA analysis in order to compare the results (Table 18).  ELVs prepared using 1×10⁶ cells/ml, contained 145.89 µg/ml of total protein and 4.63 x 10¹⁰ particles/ml (sum of fractions 7 and 8). Increasing cell concentration to 5×10⁶ cells/ml, resulted in 735.1 µg/ml of total protein and 936 x 10⁹ particles/ml (sum of fractions 7, 8 and 9). One-week-old sample with 5×10⁶ cells/ml contains 888.48 µg/ml of total protein and 617.6 particles/ml sum of all fractions 7-10) indicating that particle and protein concentration decreased over time. By increasing the initial cell concentration, we increased the yield of total protein and particle number.

Table 18. Determination of ELVs total protein content and particle concentrations. 1 x10⁶ and 5 x10⁶ cells were extruded through 200nm membrane and purified using qEV. ELVs containing fractions were analysed to determine protein concentration and particle/ml concentrations using BCA and NTA respectively.

	BCA			NTA
	Protein concentration (µg/ml)			Particle concentration (particles/ml)
Initial cell concentration (cells/ml)	1×106	5×106	5×106  (one-week-old sample)	1×106	5×106	5×106  (one week old sample)
Fraction 7	74.86	607.43	184.57	1.34×1010	6.03×1011	5.36×1010
Fraction 8	71.05	327.43	207.43	3.29×1010	2.88×1011	4.45×1011
Fraction 9	63.43	340.29	269		4.50×1010	1.03×1011
Fraction 10	75.81	321.71	227.48			1.60×1010

 

4.6. ELVs structure elucidation using transmission electron microscopy (TEM)

TEM was used to study the size and morphology of ELVs. Initial cell concentration of 5×10⁶ cell/ml was extruded through 100nm membrane and purified using qEV. A preliminary examination of the purified ELVs only from fraction 7 revealed ELVs as aggregated spherical dark structures (Figure 20.a). The size of ELVs under TEM (50-70 nm) was smaller compared to DLS (182 ± 4.120nm) (Table 17). However, DLS probably measured the whole aggregated structure as one particle, resulting in bigger size presentation.  On the other hand, Figure 20.b) shows more dispersed ELVs with 100 – 130nm in diameter, which is consistent with the NTA measured size (Table 17). Further studies are still required to examine ELVs structures isolated in different fractions.

       

      

Figure 20: Morphological examination of ELVs using transmission electron microscopy. 5×10⁶ cells/ml were extruded through 100nm membrane and purified through qEV column. Fraction 7 was used for this TEM analysis. Sample was negatively stained with 1% PTA prior to imaging. a) Aggregated ELVs and b) Dispersed ELVs.

5. Discussion

Exosome like- vesicles (ELVs) derived from cells have been introduced by Jang et al. ¹⁰⁸  as a solution for overcoming naturally secreted vesicles limitations, especially regarding their low yield purification. Furthermore, ELVs retain the natural ability of cellular communication and are successfully applied in drug delivery. ELVs are mostly prepared from different cell lines ^{108,122,126–129}, but two studies also report bacterial protoplasts as source of ELVs ^121,123. Cells used for production of ELVs were monoblastic U937 cells that contain the counter-receptors for endothelial cell adhesion molecules (CAMs), for example lymphocyte function-associated antigen 1 (LFA-1). Abnormal angiogenesis in tumours enhance the rapid proliferation of  endothelial cells that express CAMs and support tumour growth 136. Therefore, chemotherapeutic-loaded nanovesicles generated from macrophages and monocytes have been reported to effectively bind to endothelial cells. To obtain a higher yield of cell-derived ELVs, in this study, human U937 monocytic cells were harvested and serially extruded using mini -extruder (Avanti Polar Lipids) as previously described 108,121,122. Different cell concentrations have been used for extrusion to assess the optimal cell concentration for ELVs production. Lower cell concentrations (1 and 2×106 cells/ml) were easy to extrude because they did not cause membrane saturation, therefore pressure was lower comparing to the higher initial cell concentrations (3, 4 and 5×106 cells/ml). Interestingly, ELVs size was not dependent on the initial cell concentration as confirmed by DLS (Table 8). ELVs size was dependant on the membrane pore size used for extrusion. Optimum size for drug delivery (<200nm) was achieved using 100nm membrane. These findings were supported using DLS (Table 8), NTA (Table 17) and TEM (Figure 20). NTA has become more reliable technique in determining the size because DLS measurements show larger size due to the intensity of scattered light caused by larger particles or aggregates. DLS measured the size of 182 ± 4.120nm, which corresponds to the TEM figure 20.a) showing the aggregated structure. Individual ELVs size from the aggregated structure was quite small, around 50-70nm, which could be explained by dehydration of the sample when observed under TEM.  NTA measures the random movement of single particles rather than a global analysis. Our results obtained by NTA showed the smaller mean ELVs size, with different peak diameters depending on fraction number (Figure 19) which is correlated with TEM image showing dispersed ELVs (Figure 20.b). Morphology of our ELVs can be described as round-shaped structures, dispersed or aggregated in clusters (Figure 20.a),b). Usually, cryotransmission electron microscopy (Cryo-TEM) is used to image those particles ^108,121,123 because it does not require chemical staining and enables observation in a near native state. In our case, TEM was the only available option for ELVs imaging but cryo-TEM imaging could be done in the future.

The major challenge in exosomes and ELVs research is the purification from proteins and cellular debris. Currently used isolation methods for exosome purification from cell culture media are time consuming, unreproducible, and resulting in low exosome yield. After cell extrusion in D-PBS, the samples contained free proteins, cellular fragments and other cellular debris, which had to be removed to obtain a pure sample. In this study, three different methods were used for sample purification. First method was size exclusion chromatography (SEC) using PD10 column. Purification was unsuccessful because of unsuitable column matrix with too small separation size. The next purification method used was density gradient ultracentrifugation. Ultracentrifugation has been widely used for purification of ELVs after extrusion, using OptiPrep (iodixanol 60% w/v) ^108,121,122. In our case, this method did not show efficient purification due to the small sample volume (Figure 12). The last purification method also included SEC but using commercially available qEV original column containing sepharose. There have been reports showing that exosomes could be isolated form plasma by SEC using self-packed sepharose 2B, with success in separating proteins from exosomes ¹³³. The qEV original  was also described as a promising commercial product for exosome purification with minimal protein contamination ^131,132,134. Although, use of qEV has never been reported before for ELVs purification, it was used in this study.  Indeed, ELVs were able to be separated from proteins effectively using this column, which was confirmed by DLS (Table 10) and BCA (Figure 13). Separation size of qEV is 70 nm, allowing a faster passage of all larger particles through the column and separation from small proteins and cellular debris. qEV showed promising results and it was decided to use this method in future experiments. Also, another application would be a separation of unencapsulated drugs form drug-containing ELVs for future in vitro and in vivo studies.

Regarding ELVs yield, results from DLS and BCA need to be supported by particle count measurements using nanoparticle tracking analysis (NTA) to have a more quantitative measurement. NTA measured the particle concentration from different fractions expressed as particles/ml.  In the literature, the yield of ELVs is always expressed as number of particles obtained from total protein mass ^108,128. BCA results showed that 73 µg/ml of total protein was obtained using initial cell concentration of 1×10⁶ cells/ml and 2.32 x 10¹⁰ ELVs/ml (sum of fractions 7 and 8) (Table 18). Using 5×10⁶ cells/ml, we obtained 637 µg/ml of total protein and 9.36 x 10¹¹ ELVs/ml (sum of fractions 7, 8 and 9) (Table 18). Jang et al. reported the yield of 2.10 x 10¹¹ particles and 203µg of total protein from 1×10⁷ U937 cells. On the other hand, naturally secreted exosomes were also quantified and the same number of cultured cells produced only 1.74µg of total protein and 2.00 x 10⁹ particles of exosomes confirming that extrusion is better method for higher yield purification ¹⁰⁸.

The difference between our and reported yield is probably because of the different production and purification methods. We were using qEV original column based on size exclusion chromatography, while in the paper density gradient ultracentrifugation was used for purification of ELVs.  It can be concluded that SEC successfully purified the ELVs form free proteins and increased the yield of ELVs comparing to the density gradient ultracentrifugation. However, our extrusion technique was slightly different because of additional membranes used for extrusion (400, 200 and 100nm). Furthermore, number of cycles was not reported in the literature, which could also affect the differences in obtained yield.

Stability of purified and unpurified ELVs was monitored overtime for two weeks. After extrusion and purification, samples are always stored at 4°C in D-PBS, and knowing their stability is important since not all published studies reported ELVs stability. Based on DLS, BCA and NTA measurements stability of different samples was determined regarding their initial cell concentration. Unpurified sample prepared using initial concentration of 1×10⁶ cells/ml maintained the stability up to five days (Table 12), while unpurified samples with higher ELVs concentration maintained their stability up to two weeks (Table 13-16). On the other hand, purified ELVs from fractions 7 and 10 showed a lower stability over time because of their aggregation. Concentrated fractions 8 and 9 from high initial cell concentrations (3, 4 and 5×10⁶ cells/ml) maintained their stability for a longer time (approximately one to two weeks). Higher initial cell concentration maintains the longer stability of the sample in D-PBS. Because of that reason, it was continued using 5×10⁶ cells/ml as initial concentration for extrusion. Higher concentrations (7×10⁶ cells/ml) were also tested, but extrusion process using more than 5×10⁶ cells/ml was difficult because of membrane blocking and disabling the vesicle formation. Membrane replacements would only cause the protein content loss, therefore using the higher cell concentration is unpractical. Monitoring stability by protein concentration, it was seen that longer storage of the sample (more than one week) causes protein degradation (Figure 18), although DLS shoved good quality results (except for 1×10⁶ cells/ml sample). Overall, 5×10⁶ cells/ml should be used for extrusion to maintain stability of ELVs for one week.

6. Conclusion

In this study, exosome-like vesicles (ELVs) were successfully prepared by extrusion of monoblastic U937 cell line using mini-extruder. Size of ELVs was manipulated based on the pore size of membrane filters used. Our results also showed that ELVs size was independent on the initial cell concentration, however, the particle size stability and protein content were superior in ELVs prepared using higher cell concentration (5×10⁶ cells/ml). Furthermore, our findings demonstrated the high purity of ELVs samples following qEV column size exclusion chromatography, and high ELVs yield, as demonstrated by BCA assay and NTA measurements.

Overall, ELVs showed huge potential in future drug delivery applications due to their easy and controllable production, purification and characterization. To improve their antitumor activity against prostate cancer, future steps of this project will combine the innate affinity of monocytes to tumour tissues with PSMA active targeting. This should be done by the bioengineering of ELVs following U937 cells transfection with pDNA to express  PSMA peptide on their membranes. Then PSMA-targeted ELVs will be loaded with Dox-PSA prodrug to enhance the therapeutic efficacy of this prodrug in prostate cancer cells.

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