Instituto Tecnológico y de Estudios Superiores de Monterrey
Campus Monterrey
School of Engineering and Sciences
Recovery and purification of exosomes using polymer-polymer aqueous two-phase systems (ATPS)
A thesis presented by
Abril Lorena Torres Bautista
Submitted to the
School of Engineering and Sciences
in partial fulfillment of the requirements for the degree of
Master of Science in Biotechnology
Monterrey Nuevo León, December 3rd, 2020
Dedication
To God, for all the love, blessings, and the wonderful view during my return to Monterrey, which I will treasure for the rest of my life.
To my parents, for encouraging my vocation to study, for being present and supporting me in every decision I make.
To my little sister Crhys and my brother Michael, who inspire me to be a better person and for watching my steps.
Acknowledgements
I would like to thank everyone who helped me in some way to consolidate this project.
To my advisor, Dr. José González-Valdez, for accepting me in his research group, for his unconditional support, confidence and patience.
To Dr. Alberto Mejía, M.Sc. Javier Donoso, M.Sc. Sergio Ayala and everyone in the Bioprocess Focus Group and members of the FEMSA Biotechnology Center for their help and support along my days in the laboratory.
To the members of my committee Dr. Alberto Mejía, Dr. Mario Torres and Dra. Ana Mayela Ramos, for accepting the invitation to review this work and for your time.
I would like to express my deepest gratitude to all those who have been side by side with me, each of you are a blessing in my life.
To my parents Rafael T.M. and Lorena B.V. for your unconditional love and support, for taking care of me, showing me how to be a good person and teaching me to give my best until I reach my goals.
To Bebs, my sister Crhys, for being my partner in crime, my inspiration, and the best friend that God has given me. You literally make me a better person and show me the light in the dark.
To Michael (Bitzito), my brother, who taught me to be strong and to keep my mind on the game despite adversity.
To my grandparents Don Jesús Mejía, Miguel Bautista, Amelia Valenzuela and María del Carmen Márquez for taking care of my parents and for their valuable advices.
To my aunts, Luz del Carmen and Gloria, and my uncle Memo who always remember me with love and welcome me happily.
To my godparents Gloria and Joaquín, for their love and for taking care of me.
To my unconditional new best friend Blanca (Boo) for being with me all this time, for your support and for making these years funnier.
To the Tecnológico de Monterrey and CONACyT for their support through scholarships.
This thesis was performed at Centro de Biotecnología FEMSA of Tecnológico de Monterrey, Campus Monterrey, with the financial support of the National Council on Science and Technology of Mexico (CONACyT).
Contents Page
List of Figures List of Tables
1. Introduction and Thesis Generalities Introduction
1.1 Biogenesis and characteristics of exosomes 1.2 Relevance and application of exosomes
1.3 Production, separation and purification of exosomes 1.4 Experimental models
1.4.1 CaCo2 cell line derives exosomes 1.4.2 Culture media contaminants 1.5 Hypothesis
1.6 General objective 1.7 Specific objectives 1.8 Thesis structure 1.9 References
2. Characterization and optimization of polymer-polymer Aqueous Two- Phase Systems for the isolation and purification of exosomes from CaCo2 cell-derived exosomes
Abstract 2.1Introduction
2.2 Materials and Methods 2.2.1 Cell culture
2.2.2 Exosome isolation
2.2.3 Exosome characterization
2.2.4 Construction of polymer-polymer ATPS 2.2.5 Protein quantification of isolated samples 2.3 Results and Discussions
viii xi
1 2 3 4 6 6 6 7 8 8 9 9
14 15
18 18 19 19 22 22
2.4 Conclusions
2.5 Acknowledgements 2.6 References
3. Conclusions and recommendations Vita
35 36 36 41 44
List of Figures
Page Figure 1.1.1 Biogenesis and release of extracellular vesicles. 3 Figure 2.3.1 Effect of TLL (%) w/w on ln KP of exosomes and
contaminants fractionation in aqueous two-phase systems.
30
Figure 2.3.2 Effect of NaCl upon top and bottom phases recovery yield of exosomes in the selected systems.
33
Figure 2.3.3 Simplified scheme for the optimized ATPS strategy to selectively fractionate a complex sample.
34
List of Tables
Page Table 2.2.1 Composition and parameters of selected polymer-
polymer ATPS for the fractionation of exosomes and their contaminants
21
Table 2.3.1 Effect of polymer type, molecular weight (MW) and tie-line length (TLL) upon the partition coefficient and recovery yield of exosomes and contaminants in each aqueous two-phase system.
24
Table 2.3.2 Effect of polymer type, molecular weight (MW) and tie-line length (TLL) upon the partition coefficient and recovery yield of exosomes and contaminants in each aqueous two-phase system.
31
Chapter 1
Introduction and Thesis Generalities
1. Introduction
In recent years, the biopharmaceutical industry has focused on the development of novel drugs to treat diseases in a consistent and predictable way, and in the development of selective drug delivery systems to reduce side effects and to improve efficacy and safety of the developed therapeutics [1]. A drug delivery system (DDS) is the term used to define a formulation or device with capacity cargo to transport therapeutic substances (i.e. drugs and other agents such as therapeutic genes) into the human body to reach its target cells, organs, or tissues with higher specificity and effectiveness, which becomes highly important for molecules with strong adverse pharmacological features such as chemotherapeutic agents [1,2].
Nanoscale drug delivery systems, including nanoparticles (i.e. polymers, synthetic vesicles, lipids and metals) and more recently, exosomes, have been studied for the past decades and used as drug delivery vehicles to improve the physicochemical and pharmacokinetic properties of the cargo substances, but mainly to avoid common limitations including adverse immune system responses, low solubility, ineffective cell penetration and invasive approach for normal tissues [3,4]. Exosomes are naturally occurring nanoparticles produced by almost all cell types that present attractive characteristics for their use as delivery systems as it will be later explained. Certainly, among the novel DDS, exosomes have presented interesting outcomes which can be attributed to their natural origin and their physicochemical characteristics that improve cell targeting. So far, the major challenges for therapeutic applications of exosomes are their low yield production strategies and heterogeneous composition; as well as the development of optimized methods for their isolation [5].
It is well known that in pharmaceutical and therapeutic applications, bioprocessing schemes become relevant for the production, recovery and purification of the product of interest by ensuring quantity, quality, and lower production times and costs. So, some
of the presented engineering problems related to exosome procurement could be resolved with an optimized bioprocess to allow a large-scale production and to make the product accessible in an economical, effective, and safe way. In this context, Aqueous Two-Phase Systems (ATPS) have shown to be an alternative method to isolate biological molecules with promising expectations for exosomes [6]. In this chapter, some important characteristics regarding exosomes as natural nanocarriers and other applications, for example, in cancer research are discussed. Followed by an overview of the traditional and novel methods used to isolate these promising particles, and the description of this thesis generalities and characteristics.
1.1 Biogenesis and characteristics of exosomes
As it has been mentioned, exosomes are nano-sized extracellular vesicles (EV) produced by almost every normal and pathological cell [2,7]. Exosomes are composed by a lipid bilayer membrane which encapsulates cytosol and different molecular components such as associated proteins, genetic material (i.e. DNA, mRNA, miRNA), cellular receptors and their own unique biomarkers such as CD9 and CD81 [7-10].
These lipid vesicles are produced in late endosome formation by multiple invaginations of the cell membrane, acquiring a unique composition from the endosomal membrane of the producer cell. Multivesicular bodies (MVB) are formed after multiple budding into the endosomal lumen, then these MVB merge with the cytoplasmic membrane [4,11], releasing the newly-formed exosomes to the extracellular environment. Extracellular vesicle release and biogenesis is shown in Figure 1.1.1, as well as a schematic representation of exosome structure.
Once they are out of the cell, exosomes play an important role in micro and nano- molecule transportation, signal transmission and cell communication [8, 12, 13] by transferring and facilitating the exchange of their bioactive molecules contained into target cells [8, 14-16]. These processes are believed to be highly specific because of the same embedded biomolecules that are found in their membrane which target similar cells as the ones they are produced in. In this sense, exosomes are considered naturally
occurring nanocarriers and have shown promising functions as next generation drug delivery systems [4].
Figure 1.1.1 Biogenesis and release of extracellular vesicles. a) Exosomes are biological membrane vesicles secreted by almost normal and pathological cells. These nanovesicles consist in a lipid bilayer membrane that encapsulates cytosol and different molecular components including associated proteins, genetic material (i.e. DNA, mRNA, miRNA, siRNA, circRNA, lncRNA), enzymes, receptors and unique biomarkers from their parental cell. b) Extracellular vesicles can be classified into two distinct classes: exosomes and microvesicles. Microvesicles are formed by budding of the plasma membrane, while exosomes originate from late endosomes inside the multivesicular bodies. Exosomes are released out of the cell by fusion of MVBs and plasma membranes. EVs: extracellular vesicles;
MVBs: multivesicular bodies; circ: circular; lnc: long non-coding; si: small interfering; mi: micro; nm: nanometers.
1.2 Relevance and application of exosomes
Lately, exosomes have attracted research interest in novel engineered applications, such as nano-scale drug delivery systems, bioimaging, personalized therapies and their potential use as biomarkers in diagnosis, prognostication, and treatment monitoring [11, 17-19] Exosomes have shown multiple advantages over synthetic nanocarriers like liposomes [7, 10, 20], offering a high cargo capacity specificity towards target cells, improved body tolerance, better cellular internalization, longer circulating half-lives and non-cytotoxic effects [8, 10]. Hence, those characteristics make them useful as therapeutic targets for several pathological conditions such as cancer, diabetes, neurodegenerative diseases, among others [7, 13, 20, 21]. Recent studies have demonstrated their important physiological roles in the immune, cardiovascular, and nervous systems [11, 16, 21]. In the context of cancer, it has been demonstrated that
tumor cells have their “own language” and are giving out signals that cannot be intercepted by our immune system [14]. Exosomes are recognized as significant mediators of extracellular communication and it is now well-known, they can enable cancer progression by transferring cancer-promoting cellular contents to tumor surrounding cells or through blood circulation to distant sites in the body [15, 22, 23].
In this sense, exosomes have potential in cracking the communication code of cancer cells by giving information about cancer stages and progression when studying their natural-contained biomarkers such as tumor-specific proteins and nucleic acids [24, 25].
Furthermore, they have a significant impact in cancer therapy by targeting chemotherapeutic drugs and allowing their direct arrival to kill cancer cells without damaging the healthy ones [4, 20, 22]. With these advancements, exosomes are appearing as a very powerful and promising tool in the sight of understanding intercellular communication by studying the impact of transmission of their chemical and genetic content information in diseases.
1.3 Production, separation, and purification of exosomes
Exosomes can be obtained from body fluids including serum, saliva, cerebrospinal fluid, bile, milk, urine, plasma and produced by cell culture for research purposes [9, 12, 15, 17]. The production of heterogeneous and productive exosomes remains a challenge for therapeutic applications were large amount of these vesicles will be needed with commercial purposes. Even more, the following engineering steps towards isolating and purifying them present several setbacks that should also be addressed.
So far, one of the major challenges is the development of optimized methods for exosome isolation [5]. To date, there is no consensus on a standardized exosome purification process and there are still many opportunities in applying different isolation methods to obtain these nanoparticles limiting the proposal of further exosome applications [26]. There are, however, several traditional and novel methods reported for the extraction of exosomes from complex samples like human fluids [16]. Traditional exosome isolation methods include centrifugation, ultracentrifugation, filtration,
polymeric precipitation, size-exclusion, and liquid chromatography techniques [3, 5, 12, 17, 27]. However, these methods have demonstrated disadvantages such as equipment requirements, expensive reagents, complex and costly operating procedures with low productivities purification factors and yields [5, 16, 27].
In this context, alternative and less-used exosome isolation techniques have emerged to overcome some of these limitations. In general, these novel methods offer advantageous features such as scalability, lower processing times, effectiveness, lower economical burdens and in some cases, portability [18, 28]. Particularly, Aqueous Two- Phase Systems (ATPS) have proved to be an effective, fast, suitable, accurate and reliable method that has traditionally been used in the recovery of biological molecules and biological nanoparticles which could also be extrapolated for the recovery and purification of exosomes [16]. ATPS is a liquid-liquid extraction technique where two liquid solutions (typically a polymer and a salt or two polymers), are mixed above a critical concentration to form two immiscible phases that allow the separation of biological mixtures into their constituents. In these types of ATPS applications, it is desired for target solutes to partition towards a phase different from that of the contaminants to allow their recovery and further purification. In ATPS, the partitioning of solutes depends on the physicochemical properties of the molecules added (e.g.
molecular weight, surface charge, hydrophobicity, etc.), the characteristics of the phase- forming chemicals (e.g. polymer molecular mass, concentration and physicochemical properties) and system design parameters such as Tie-Line Length (TLL), volume ratio (VR) and pH which will be described in chapter 2 [17, 21, 29]. So far, only a few scientific works have reported exosome isolation using ATPS, but polymer-polymer systems have presented the best biological compatibility because of their natural characteristics [6, 26, 27]. In this context, exosomes have shown preference for the Dextran-phase over the Polyethylene glycol (PEG)-phase in PEG-Dextran ATPS after phase separation and equilibration using centrifugation (~1,000 x g, 10 minutes) in early experiments.
Additionally, ATPS are considered easy to operate, fast, economically feasible and non- destructive, maintaining the biological activity and specific functions of the sample components [30-32]. As it will be explained in Chapter 2, this method can allow the
primary purification of exosomes at ideal conditions. However, because there are many alternatives regarding polymer selection and system design parameter optimization more information in exosome recovery and purification using ATPS need to be obtained.
In this sense, this work focuses in studying the effect of polymer composition, characteristics and system design parameters in the primary recovery and purification of exosomes using ATPS. The following sections in this chapter will provide an overview of this work by describing the experimental models used, the hypothesis and objectives for the experimental procedures to do so.
1.4 Experimental models
A cancer cell line and the media used to culture these cells were used as experimental models in this thesis for exosome and contaminant obtention, specifically, human colon cancer (CaCo2) cells and contaminants from wasted cell culture media. These models are briefly described in the following subsections.
1.4.1 CaCo2 cell line derived exosomes
CaCo2 is an epidermal growth factor receptor colon cancer cell line isolated from epithelial cells of a 72-year-old Caucasian male with colorectal adenocarcinoma disease. CaCo2 cells have been used for exosome production and isolation because of their availability and scale-up easiness, and their ability to generate heterogeneous and productive exosomes with similar quality [3, 33, 34]. The CaCo2 cell line is well- described and has been used thoroughly used as an in vitro model in cancer research to simulate natural intestine conditions [33]. Our lab has found some of the best exosome production levels in this cell line when compared to other available mammalian cell lines [33]. Exosomes produced in this cell line present typically found particle diameters 30-200 nm [21, 35], z-potentials and protein and biomarker contents normally reported in exosome characterization.
1.4.2 Contaminants from wasted cell culture media
In vitro cell cultures require specific growth media to proliferate. Basal medium for mammalian cell lines consists in a mixture of nutrients, mostly growth medium.
Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM / F-12) is a widely used growth medium for mammalian cells, which is formed by a mixture of DMEM (glucose, amino acids and vitamins at high concentrations) and Ham’s F-12, and modified with L-glutamine and Phenol Red. DMEM / F-12 does not contain proteins, lipids, or growth factors, so it may require supplementation with Fetal Bovine Serum (FBS), as a source for them. The major component of FBS is a globular protein known as Bovine Serum Albumin (BSA), which in the research environment is commonly used as model protein and standard for protein quantification methods. Considering that cell- derived exosomes will be recovered from this media, the use of it as model contaminant source for bioengineering applications is consequential.
1.5 Hypothesis
Because of the multiple advantages that ATPS present in the isolation and primary recovery of a large number of biological molecules such as microorganisms, proteins and nucleic acids, this bioseparation method has been greatly used as an alternative strategy to recover and purify highly-valued commercial molecules. In this sense, it is believed, that ATPS would present multiple advantages in the bioengineered procurement of cell-derived exosomes. On their part, exosomes are mostly obtained from complex samples in which they are commonly present in low concentrations and with a considerable number of contaminants. It is therefore of enormous interest to finding ATPS conditions that allow a high recovery yield and purification factor for these promising nanovesicles. With this, the present work revolves around the following hypothesis:
The determination of optimal system design parameters in polymer- polymer aqueous two-phase systems, like polymer type, molecular weight, tie-line length and neutral salt concentration, will allow the selective
primary recovery and purification of CaCo2 cell-derived exosomes from common cell culture contaminants in attractive yields and purity levels above 50% for further applications.
1.6 General objective
The general objective stated to reach the hypothesis of this work is to study the partition behavior of CaCo2 cell-derived exosomes in a polymer-polymer ATPS, to find the optimal design parameters for their selective recovery and purification.
1.7 Specific objectives
The hypothesis and the general objective mentioned above can be addressed by achieving the following series of specific objectives:
1. To obtain purified exosomes from a mammalian cancer cell line (CaCo2) cultured in vitro.
2. To characterize the exosomes extracted from the CaCo2 cell line by size distribution, zeta potentials and CD63 biomarker presence.
3. To prepare samples (exosomes and media with contaminants) and polymer- polymer ATPS according to their respective composition established by the corresponding binodal curves of each system and validate the phase separation protocol.
4. To evaluate the discrete partition behavior of exosomes and protein contaminants by measuring protein concentration in the top and bottom phase by calculating recovery yield and partition coefficients of each system.
5. To analyze the obtained data to select the systems with better performance in discrete experiments for further optimization with neutral salt (NaCl) addition to enhance partition behaviors.
6. To select the system with better performance in discrete experiments and propose an optimized ATPS strategy for the purification of exosomes from complex samples.
By fulfilling these specific objectives, it is expected to establish the basis for this novel method to recover and purify this promising nanovesicles from complex samples.
1.8 Thesis structure
This work attempts to analyze different polymer-polymer ATPS composition and parameters for the recovery of exosomes and describe their effect over the partition behavior of these nanovesicles. To achieve this, the thesis is divided into 3 chapters.
The first chapter provides a general overview of the background and relevance of exosomes and, traditional and novel methods for the extraction of exosomes reported in the last years, as well as the experimental models, hypothesis and objectives. Chapter 2 consists in a manuscript for a research article entitled: “Characterization and optimization of polymer-polymer Aqueous Two-Phase Systems for the isolation and purification of exosomes from CaCo2 cell-derived exosomes” intended to be published in a scientific journal. Therefore, this chapter has its own introduction, methodology, results, conclusions, and references. Finally, chapter 3 is constituted by the general conclusions obtained from this work and recommendations for future research.
1.9 References
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Chapter 2
Characterization and optimization of polymer-polymer Aqueous Two- Phase Systems for the isolation and purification of CaCo2 cell-derived exosomes
Abstract
Exosomes are a type of extracellular vesicles that present natural and attractive characteristics such as nano size (30-200 nm) and unique structure for their use as drug delivery systems for drug therapy, biomarkers for prognostic, diagnostic and personalized treatments. So far, one of the major challenges for therapeutic applications of exosomes is the development of optimized methods for their isolation. In this context, Aqueous Two-Phase Systems (ATPS) have shown to be an alternative method to isolate biological molecules and particles with promising expectations for exosomes. In this work, fractionation of exosomes obtained from a CaCo2 cell line and the contaminants present in their culture media was performed in 20 different polymer- polymer aqueous two-phase systems (ATPSs) in a discrete way. The effect of system design parameters such as polymer composition, molecular weight, and TLL (15-30%) on PEG-Dextran, Dextran-Ficoll and PEG-Ficoll systems were studied keeping phase volume ratio (VR) and pH constant at 1.0 and 7.0, respectively. After partition analysis, 4 out of the 20 systems presented the best exosome fractionation from the contaminants under initial conditions and were selected for further partition optimization via salt addition (NaCl) to observe the behavior of both samples, individually, and improve the efficiency of collection. The PEG 10,000 gmol-¹ – Dextran 10,000 gmol-¹ system at TLL 25% w/w, with the addition NaCl to reach a final concentration of 25 mM, showed the best isolation efficiency resulting in exosome purification factor of 2 and furtherly demonstrated that ATPS has the potential for the selective recovery these promising nanovesicles.
Keywords: aqueous two-phase system, polymer-polymer, exosome, PEG-Dextran,
2.1 Introduction
Intercellular communication involves precise coordination of the emission and reception of biological signals and molecular information, presenting impact in normal and pathological conditions and in different physiological processes [1, 2]. Repair systems and healing mechanisms are influenced by cell interactions which become relevant for homeostasis maintenance in multicellular organisms, that is returning a damage to an anatomical and physiological normal state.
In this sense, the correct intercellular transfer of extracellular vesicles (EVs) plays a pivotal role [3-6] in the search for biological tools to achieve this goal, exosomes – as EVs –have appeared as a very powerful and promising alternative to harness the power of intercellular communication in multiple therapeutic applications. To date, exosomes have demonstrated to play a crucial role in maintaining cellular homeostasis and as mediators in anti-tumor responses [4, 6].
Exosomes are biological and endogenous nano-sized (30-150 nm) membrane vesicles [2, 7] produced by normal and pathological cells which can be obtained from virtually all body fluids [6]. Exosomes are originated from late endosomes and consist of a lipid bilayer membrane [2, 7] which encapsulates cytosol and different molecular components such as associated proteins, genetic materials (i.e. DNA, mRNA, miRNA), enzymes, receptors and their own unique biomarkers, like CD63, CD9 and CD81 [8-10] which are also present in their origin cell. These naturally occurring nanoparticles are released from the cell upon fusion of a multivesicular body (MVB), containing exosomes produced in late endosome formation, with the plasma membrane. Once they are secreted to the extracellular environment, exosomes play an important role in cell communication by transferring and facilitating the exchange of their bioactive molecules contained into a specific recipient cell based on their composition [5, 7].
Due to their natural characteristics such as size and unique structure, exosomes can overcome some molecule transport limitations and have also shown multiple advantages over synthetic nanoparticles in pharmaceutical applications [7] offering a high cargo capacity, longer circulating half-life, well body toleration, excellent cellular internalization, no cytotoxic effects and target specificity [8-10]. For those reasons, they
are being explored as nanodevices for the development of new therapeutic and diagnostic tools, such as biomarkers for prognostic and diagnostic of disease, nanocarriers for drug therapy and personalized treatments [11, 12].
So far, the major challenges for exosomal therapeutic applications are the production of heterogeneous and productive exosomes, and the development of optimized methods for their isolation and storage in specific formulations [13]. To date, there are several traditional and novel methodologies for exosome isolation from complex samples like human fluids, with their corresponding advantages and disadvantages for clinical trials, point-of-care settings, and scale-up scenarios [12]. Traditional methods include centrifugation/ultracentrifugation, filtration, microfluidic isolation, polymeric precipitation isolation, size-exclusion and liquid chromatography techniques [2, 11, 12, 14, 15].
However, these methods present several common limitations such as large equipment requirements, expensive reagents, complex and costly operating procedures, and low productivity, purity and yields [11, 12]. To date, ultracentrifugation is the most used methodology in exosome procurement, but novel strategies are emerging such as immunoaffinity, microdevices, liquid-liquid extraction and hydrophobic interaction chromatography with promising results for yield, purity and purification factor, while offering advantageous features such as scalability, lower processing times, effectiveness, lower economical burdens and in some cases, portability [4, 14-17]. In summary, there is a strong need to standardize effective, fast, suitable, accurate and reliable methods for exosome separation that achieve high product concentrations and purity levels, ensuring nanoparticles free of polluting materials [12]. This situation has raised the need to seek for other alternative methods for exosome purification and isolation. In this context, aqueous two-phase systems (ATPS) are believed to present advantages over other methodologies.
ATPS is a liquid-liquid extraction technique in which two liquid solutions (typically a polymer and a salt or two different polymer solutions) are mixed above a critical concentration to form two immiscible phases composed mainly of water [18, 19]. When a particular mixture is added to the systems, the physicochemical interactions between
design parameters offer an environment in which molecules partition to either the top or bottom phase [20]. If conditions are ideal, and the molecules of interest migrate to a phase different from that of the contaminants, a primary purification of said molecules can be achieved. Because ATPS are mainly composed of water, this purification strategy presents interesting advantages for the recovery and purification of exosomes.
Among them we can mention biological compatibility, low interfacial tension, high load capacity and scale up easiness. Additionally, ATPS are considered easy to operate, fast, economically feasible and non-destructive, maintaining the biological activity and specific functions of the sample components [18, 21, 22].
Several publications involving protein fractionation in polymer–polymer ATPS have been published [11, 20]. Some works have aimed to evaluate the influence of intrinsic parameters on the partition behavior of two biomolecules. Among the most important design parameters, the partition coefficient (KP), volume ratio (VR) and tie-line length (TLL) has been reported [18]. KP is defined as the ratio of the particle concentration between the top and bottom phase [11, 23]. Volume ratio (VR) is established as the volume relation of the top and the bottom phases [20]. Tie-line length (TLL) on its part, is related to the relationship between phase-forming chemical mass composition in each of the system phases and is presented in %w/w units. The end points of a tie-line determine phase compositions at equilibrium and lie in the binodal curve, which indicates the separation between the two immiscible phases [18].
The objective of the present work was to understand the effect of system design parameters (i.e. polymer composition, molecular weight, and TLL) on the discrete partition behavior of exosomes obtained from a CaCo2 cell line and the contaminants present in their culture media on 20 different polymer-polymer ATPS. This was done to propose a primary recovery strategy for these nanoparticles applicable to traditional cell culture conditions. The systems that presented the best performance were selected for further optimization by the addition of sodium chloride (NaCl) to improve the efficiency of collection. A potential ATPS strategy for the isolation and purification of exosomes from culture media contaminants is proposed with the aim of implementing it in intensified strategies for exosome procurement.
2.2 Research methods
2.2.1 Cell culture
Human colon cancer cell line CaCo2 (ATCC®, HTB37™) were cultured with DMEM-F12 medium supplemented with 10% exosome-depleted Fetal Bovine Serum (FBS; Gibco, USA) and 1% Penicillin-Streptomycin solution (Gibco, USA) at 37ºC and 5% CO2
atmosphere. CaCo2 cells (~1x106 cells/mL) were placed in 7 mL of media in 100mm x 20mm tissue-culture treated petri dishes (Corning, NY) for 48 h until cultures reached
~80% confluence. Then, the exosome-enriched culture medium was recovered and reserved in -20 ºC until use. Another 7 mL of fresh culture media were added to the same dishes and cells were cultured for an additional 24 h at the same conditions. The maintenance medium was also recollected and reserved. Both reserved culture media were used to isolate exosomes secreted by the cells as it is described in the next section.
2.2.2 Exosome isolation
The reserved exosome-enriched culture medium was centrifuged at 300 x g for 10 minutes at 4ºC to remove cells and debris. The supernatant was carefully collected and transferred to a new tube and centrifuged at 2000 x g for 5 minutes at 4 ºC. The obtained supernatant was then filtered using a 20 µm surfactant-free cellulose acetate (SFCA) filter (Corning, USA) and later ultracentrifuged at 125,000 x g for 90 minutes and 4ºC using a SW 32 Ti rotor (Beckman Coulter, CA) to isolate exosomes. Finally, the supernatant was decanted without disturbing the pellet and preserved at -20ºC until use, while the exosome pellet was suspended in 200 μL of 10 mM pre-filtered phosphate buffered saline (PBS; Caisson Labs, USA) at pH 7.2. Exosomes were concentrated using Amicon® Ultra-4 Centrifugal Filters Ultracel® -10K NMWL (Merck Millipore, Ireland) and stored at -80ºC until use. Exosomal protein concentration was determined by measuring absorbance at 280 nm in a NanoDrop® 1000 Spectrophotometer (Thermo Scientific, USA), using Bovine Serum Albumin as standard for the corresponding calibration curve, as described in section 2.2.5.
2.2.3 Exosome characterization
Exosomes were characterized by size distribution, zeta potential and the presence of exosomal marker CD63. Size distribution and zeta potential were performed by dynamic light scattering (DLS) using a Zetasizer Nano ZSP equipment (Malvern Instruments, Worcestershire, UK) at room temperature (~25ºC), 175 degrees angle and 633-nm laser [4]. The sample was prepared in a standard Spectrophotometer cuvette (10mm path length) considering the addition of 10 μL of sample diluted in 900 μL of bi-distilled water to measure particle size. The same diluted sample was used to measure zeta potential using a folded capillary zeta cell. Exosomal marker CD63 was detected in the samples by ExoELISA-Ultra CD63 Kit (System Biosciences, USA) following manufacturer instructions. Briefly, the sample was incubated at 37 ºC for 1 h on the provided micro- titer plate. After incubation and wash step, CD63 primary antibody was added to each well and incubated for 1 hour. After washing, the same incubation conditions were used for secondary antibody. Then, super-sensitive TMB ELISA substrate was added and incubated at room temperature for 15 mins with shaking. After Stop buffer, the presence of CD63 was visually verified.
2.2.4 Construction of polymer-polymer ATPS
ATPS construction was followed as established by González-Valdez (2011) et al., selected PEG-Ficoll, PEG-Dextran and Ficoll-Dextran systems gave place to 20 systems identified in Table 1 and were chosen to understand the effect of system composition, polymer MW and TLL on the partition behavior of exosomes and contaminants (i.e. supernatant preserved after ultracentrifugation representing the cell culture media) in a discrete way. Polymer stock solutions were prepared with predetermined quantities of each polymer: PEG of nominal molecular weight of 3,350 (PEG 3,350; 50%w/w) and 10,000 (PEG 10,000; 40%w/w) gmol−¹; Dextran of 10,000 (Dextran 10; 40%w/w), 70,000 (Dextran 70; 40%w/w) and 110,000 (Dextran 110;
30%w/w) gmol−¹; and Ficoll of 400,000 (Ficoll 400; 40%w/w) gmol−¹. The composition of each system was designed using the binodal curves presented by Zaslavsky (1994) for systems 1 to 16 [24] and by Croll et al. (2003) for systems 17 to 20 [25]. Each system
was prepared in 1.7 mL centrifugation tubes with a final mass of 0.5 g considering the addition of 10% w/w of the sample solution, the estimated amount of each polymer stock solution according to their respective final polymer composition (%w/w), and bi-distilled water as needed. In each system 50 μg of concentrated exosomes diluted in 50 μg of 10 mM PBS or 50 μg of recovered supernatant media with around 350 μg of protein contaminants were added separately as samples. Then, the constructed systems were vortexed and dispersed by mixing for 15 min. To achieve complete phase separation and equilibrium, the ATPS tubes were centrifuged at 10,000 x g for 10 min at 4ºC using an Eppendorf centrifuge 5804 R (Eppendorf, Germany). For discrete experiments, VR
and pH were kept constant at 1.0 and 7.0, respectively, for all systems. All experiments were carried out at least three times.
The systems that presented a better performance in the separation of exosomes from the contaminants were selected for further optimization. These systems were supplemented with neutral salt (NaCl) to reach a total final concentration of 25 mM and then analyzed under the same procedure as described in the next section.
Table 2.2.1. Composition and parameters of selected polymer-polymer ATPS for the fractionation of exosomes and their contaminants.
System Polymer 1 MW (g mol-1)
Polymer 2
MW (g mol-1) TLL Polymer 1 (%w/w)
Polymer 2 (%w/w)
1 PEG 3,350
Dextran 10
25 7.3 16.0
2 30 8.2 17.0
3 PEG 10,000 25 5.6 15.0
4 30 6.8 15.9
5
PEG 3,350
Dextran 100
15 3.5 8.2
6 20 4.0 11.1
7 25 4.9 13.5
8 30 6.8 15.0
9
PEG 10,000
15 5.4 10.0
10 20 5.4 12.1
11 25 6.1 14.1
12 30 7.0 15.5
13
Dextran 70
Ficoll 400
15 12.8 11.0
14 20 13.5 11.6
15 25 15.2 13.1
16 30 16.3 14.0
17
PEG 10,000
15 5.5 12.7
18 20 6.0 15.0
19 25 7.0 16.0
20 30 7.5 18.0
Systems were designed as described in materials and methods section. For all systems, VR and pH was kept constant at 1.0 and 7.0, respectively. TLL, VR and the composition of each system were estimated using the binodal curves presented by Zaslavsky and Croll et al. Systems 1, 3, 7 and 14 were selected for parameter optimization with the addition of NaCl.
2.2.5 Protein quantification of isolated samples
After stabilization, 50 μL were carefully taken from top and bottom phases with a micropipette in each system for total protein quantification. Protein content in each phase was determined by measuring absorbance at 280 nm in a NanoDrop® 1000 Spectrophotometer (Thermo Scientific, USA), using a previously prepared calibration curve (e = 0.0005, R2 = 0.9983) with Bovine Serum Albumin as standards at concentrations of 25, 50, 100, 250, 500, 750, 1000 and 1500 μg/mL. It is important to remember that because of their characteristics, exosomes present embedded proteins in their surface. After verification of exosome isolation according to the procedures previously presented and according to the exoELISA, 2 mg/mL of protein concentration was equivalent to 1x1010 particles/mL number of exosomes isolated from CaCo2 [4, 26].
Protein concentration was used to analyze results reported in this work. The volumes of the top and bottom phases in each replicate were estimated visually using graduated tubes to verify the VR in each system. The protein recovery yield for the exosome and contaminant samples was estimated for both phases, considering the interphase as part of the bottom phase. Partition coefficient (KP) was calculated as the protein concentration ratio between the phases [11, 23]. Data reported in this work is expressed as the average of the results with its corresponding standard error of at least three replicates for each system.
2.3 Results and Discussion
To determine the optimal design ATPS parameters for exosome isolation, twenty different polymer-polymer compositions along with at least two different TLL were studied as depicted in Table 2.2.1. As mentioned, the influence of system design parameters upon the partition behavior of exosomes and contaminants was evaluated in a discrete way in which exosomes and contaminant partition was evaluated separately.
Exosome size ranged between 30 and 150 nm according to the methodology used for isolation and analysis. In this work, exosomes were isolated from exosome-enriched
characterization experiments, particle size distribution and zeta potential results were consistent with exosome characteristics in previous published works [4, 26-28]. The diameter size of the obtained exosomes ranged from 40 nm to 235 nm with an average size of 152.00 ± 55.13 nm, well within the reported values [4, 28]. The average zeta potential of the particles was -29.00 ± 4.10 mV, which is expected because of the negatively charged phospholipid membrane of the particles [24]. Most importantly, this obtained zeta potential value acts as an indicator of particles stability in colloidal dispersions [29], which is influenced by the net surface charge. In this sense higher negative Z-potential values indicate a larger stability for hydrophobic particles [30].
An absorption-based quantitation method was chosen in this study to track the partitioning preference of the exosomes by calculating embedded protein concentration in the nanoparticle surface and, on the other hand, the contaminant proteins from the culture media. Since proteins are among the most abundant exosome components, it has been reported that embedded protein concentration in exosomes is directly related to the number of vesicles in the sample as demonstrated by simultaneous exosome quantification by Dinamic Light Scattering (DLS) and protein concentration using the Bradford method [31]. The partition behavior of exosomes and contaminants on each ATPS is shown in Table 2.3.1.
In general, this research shows that exosomes have a significantly higher preference to the bottom phase than to the top phase since this was observed in 18 out of the 20 systems under study, while recovery yields ranged from 55 ± 4.64 % to 99 ± 0.58% in this same phase. However, particular changes in system design parameters must be studied to find alternatives to manage exosome partition behavior and find the best conditions for its separation from culture-medium contaminants.
The first 4 systems (see Table 2.3.1) were evaluated at TLL values of 25% and 30%. It is important to mention that for these polymer combinations, lower TTL values, as presented for the rest of the systems, cannot be generated because of the high polymer concentrations needed to obtain the biphasic system. Nonetheless, in the PEG 3,350 - Dextran 10 systems, the increase of TLL by 5%, represented a decrease in the bottom
Table 2.3.1. Effect of polymer composition, molecular weight (MW) and tie-line length (TLL) upon the partition coefficient, recovery yield and selectivity of exosomes and contaminants in each aqueous two-phase system. Selected systems for further optimization are highlighted in gray.
Exosomes Contaminants
Recovery Yield (% w/w) Recovery Yield (% w/w)
System Polymer 1 Polymer 2 TLL Top Phase Bottom Phase ln (KP) Top Phase Bottom Phase ln (KP) 1 PEG 3,350
Dextran 10
25 39.35 ± 09.62 60.65 ± 00.81 -0.43 ± 0.02 53.28 ± 07.56 46.72 ± 06.83 0.13 ± 0.00
2 30 44.97 ± 00.28 55.03 ± 04.64 -0.20 ± 0.03 44.37 ± 04.96 55.63 ± 13.84 -0.23 ± 0.01
3 PEG 10,000 25 42.68 ± 01.58 57.32 ± 03.41 -0.29 ± 0.01 15.65 ± 11.76 84.35 ± 04.05 -1.68 ± 0.01
4 30 03.50 ± 00.00 96.50 ± 29.55 -3.32 ± 0.00 20.06 ± 05.13 79.94 ± 02.58 -1.38 ± 0.01
5
PEG 3,350
Dextran 100
15 03.50 ± 16.13 96.50 ± 08.83 -3.32 ± 0.01 18.08 ± 01.10 81.92 ± 00.00 -1.51 ± 0.00
6 20 34.94 ± 00.00 65.06 ± 02.29 -0.62 ± 0.00 20.46 ± 00.06 79.54 ± 00.59 -1.36 ± 0.02
7 25 02.87 ± 00.00 97.13 ± 16.46 -3.52 ± 0.00 22.92 ± 00.54 77.08 ± 07.36 -1.21 ± 0.03
8 30 08.22 ± 00.00 91.78 ± 09.36 -2.41 ± 0.00 11.36 ± 00.88 88.64 ± 00.57 -2.05 ± 0.00
9
PEG 10,000
15 16.13 ± 00.00 83.87 ± 00.00 -1.24 ± 0.00 15.87 ± 00.60 84.13 ± 01.18 -1.67 ± 0.01
10 20 01.56 ± 01.30 98.44 ± 07.00 -3.74 ± 0.02 08.55 ± 01.07 91.45 ± 02.26 -2.37 ± 0.01
11 25 01.42 ± 00.00 98.58 ± 24.14 -4.05 ± 0.00 01.17 ± 01.17 98.83 ± 05.26 -4.43 ± 0.01
12 30 01.34 ± 04.55 98.66 ± 02.53 -4.30 ± 0.01 00.76 ± 00.76 99.24 ± 08.67 -4.87 ± 0.02
13
Dextran 70
Ficoll 400
15 01.76 ± 00.00 98.24 ± 28.21 -4.43 ± 0.00 38.28 ± 00.85 61.72 ± 02.67 -0.48 ± 0.01
14 20 15.60 ± 03.90 84.40 ± 00.00 -1.83 ± 0.00 62.73 ± 02.36 37.27 ± 03.42 0.52 ± 0.00
15 25 62.49 ± 04.19 37.51 ± 05.59 0.51 ± 0.00 73.29 ± 01.58 26.60 ± 01.27 1.01 ± 0.00
16 30 65.35 ± 00.00 34.65 ± 03.69 0.63 ± 0.00 65.76± 00.09 34.24 ± 00.19 0.65 ± 0.01
17
PEG 10,000
15 00.79 ± 00.00 99.21 ± 00.58 -5.10 ± 0.00 01.87 ± 01.87 98.13 ± 29.89 -3.96 ± 0.03
18 20 03.50 ± 00.00 96.50 ±13.93 -3.32 ± 0.00 01.82 ± 01.82 98.18 ± 00.00 -3.99 ± 0.00
19 25 02.69 ± 00.00 97.31 ± 09.48 -3.59 ± 0.00 02.79 ± 01.94 97.21 ± 00.00 -3.55 ± 0.00
20 30 06.92 ± 06.92 93.08 ± 06.16 -2.60 ± 0.00 08.38 ± 00.00 91.62 ± 01.94 -2.39 ± 0.00
For all systems, VR and pH were kept constant at 1.0 and 7.0, respectively.
Dextran-rich phase partition of exosomes from 60.65 ± 0.81 % to 55.03 ± 4.64 %;
whereas in the PEG 10,000 - Dextran 10 systems this same TLL increment enriched exosomes in the bottom Dextran-rich phase and increase recovery yield from 57.32 ± 3.41 % to 96.5 ± 29.55 %. Considering that the main difference between these two sets of ATPS (i.e. systems 1-2 and 3-4) is the increment in polymer molecular weight (MW), this parameter has a particular effect in this behavior since the higher polymer concentrations at the PEG-enriched phase decrease the available free volume between polymer molecules caused by the increment of PEG chain lengths rendering less volume for exosomes to partition towards this phase [20, 32]. Other works where similar polymers have been used, suggest that exosomes present a higher preference towards the Dextran-rich phase [11, 33, 38] which according to these results is true for systems where large PEG MW are used and free volume becomes a factor affecting partition.
The rest of the systems (5 to 20; see Table 2.3.1) were evaluated at 4 different TLL values; 15, 20, 25 and 30% w/w. PEG 3,350 - Dextran 100 and PEG 10,000 – Dextran 100 systems presented higher exosome partition towards the bottom Dextran-rich phase. In the case of the PEG 3,350 – Dextran 100 systems, the highest protein recovery yields (~97%) were obtained at TLL 15% and 25% with a negligible difference between them (0.63%; see systems 5 and 7 in Table 2.3.1). For the PEG 10,000 – Dextran 100 systems, no significant difference between the highest recovery yields (<98%) was observed between the TLL 20, 25 and 30% w/w systems. Considering these ATPS sets (i.e systems 5-8 and 9-12), the best exosome recovery yield increased only in around 1% when PEG MW increased. Therefore, results indicate that the partition of exosomes in these sets is mostly independent from TLL and PEG MW and is rather affected by polymer hydrophobicity. In this sense, PEG polymers present a relatively high hydrophilicity due to the availability of polar -OH moieties in its chemical structure but at the same time are less hydrophilic than Dextran. Furthermore, the hydrophobic character of PEG and the hydrophilic character of Dextran increase in proportion to their MWs. Therefore, in these systems, exosomes present a higher partition preference towards the Dextran-rich phase because of the high hydrophilicity this polymer presents, and the increments of hydrophobicity observed in PEG as the selected MWs increase
rather than by increments in their relative concentrations. Besides, this separation behavior is also expected because exosomes present extremely hydrophilic surfaces [20, 38]. It is important to mention that from this set of systems, the one evaluated at TLL 20% (i.e. system 6) presented a different behavior with a recovery yield of 65.06 ± 2.29% in the bottom phase, which is around 29% lower than the rest (<91%). This particular polymer composition showed that the separation could be affected by other variables in the process such as temperature or separation time that might have an effect in the load capacity of Dextran-rich phase. Nonetheless, this system was considered as an outlier which did not behave under the same tendencies as the rest of the PEG 3,350 – Dextran 110 systems.
Further on, it is evident that in the Dextran 70 - Ficoll 400 system, exosome partition behavior is dependent on TLL. As TLL increases, the partition behavior shifts towards the top Ficoll-rich phase. At TLL 15 and 20% w/w exosome recovery yields are higher at the bottom Dextran-rich phase, 98.24 ± 28.21% and 84.40 ± 0.00%, respectively, while at 25% and 30% the molecule has a higher preference to the top Ficoll-rich phase with recovery yields of 62.49 ± 4.19% and 65.35 ± 0.00% respectively (see systems 13 to 16 in Table 2.3.1). This behavior has been previously explained by Nisslein et al.
(2020) as an effect originated from the chemical similarity between polymers. Both Dextran and Ficoll are sugar-based polymers with a similar distribution of polar and non- polar groups. Moreover, since an increment in TLL causes an increase in polymer concentration in each phase (see Table 2.2.1), which in turn, decreases the free volume available for protein partition and modifies the density of both phases and surface tension at the interface [23], changes in the partition behavior of nanoparticles are expected. As a result, at lower polymer concentrations, the systems present lower surface tension allowing particles to move through the bottom Dextran-rich phase or to be trapped at the interphase [11]. Also, since the amount of Dextran 70 is higher than Ficoll 400 in this set of systems (i.e. 13 to 16), when the weight composition of polymers increases, the available free volume in the bottom phase is not enough to accommodate the exosomes [23].
The PEG 10,000 - Ficoll 400 systems presented a decrease in exosome partition towards the bottom phase while TLL values increased (see systems 17 to 20 in Table 2.3.1). Their recovery yield decreased from 99.21 ± 0.58 % to 93.08 ± 6.16%, and the highest yield was obtained at the lowest TLL value. This polymer-polymer ATPS present a high-density environment, in which PEG is predominantly found in the top PEG-rich phase with a larger hydrophobic character than Ficoll [20]. This behavior is consistent with other studies, in which hydrophilic proteins and particles have been found to prefer the bottom Ficoll-rich phase in PEG-Ficoll systems [34, 35]. As expected, the exosomes tended to move towards the less hydrophobic phase, while increments in TLL affected free volume in a similar manner as in the previously described cases.
If the purpose of this work is to propose an ATPS-based strategy for the recovery of exosomes from the contaminants found in cell-culture media, the study of the partition behavior of said contaminants should also be performed. On its part, protein contaminants in the culture media from which exosomes are isolated were also evaluated in the same ATPS to determine their partition behavior.
Contaminants exhibited a similar partition behavior to exosomes with a marked preference for the bottom phase in 15 out of the 20 systems studied. In the case of PEG 3,350 - Dextran 10 systems, the increase of TLL by 5% w/w, increased the bottom phase preference of the contaminants from 46.72 ± 6.83 % to 55.63 ± 13.84 %. These results suggest that their separation is dependent from TLL. However, protein contaminants are much smaller than exosomes, so they occupy lower volumes in the system making them able to remain at the top PEG-rich phase with similar recovery efficiency while increasing polymer concentration. PEG 10,000 - Dextran 10 systems also enriched contaminants in the bottom Dextran-rich phase with a higher recovery yield at the lowest TLL. At a lower polymer concentration, the interfacial tension is also lower, which allows the migration of biomolecules through the interphase easily [11]. From these systems, it could be assumed that the recovery yield is affected by TLL and polymer MW.
On their part, PEG 3,350 – Dextran 100 and PEG 10,000 – Dextran 100 systems (see systems 5-8 and 9-12 in Table 2.3.1) showed an increase in the recovery yield from 81.92 ± 0.00% to 88.64 ± 0.57 % and 84.13 ± 1.18% to 99.24 ± 8.67 %, respectively,
while TLL increased. Protein contaminants are hydrophilic and are expected to present a higher preference towards the most hydrophilic phase, that in this case is the Dextran- rich phase [20]. Increases in TLL which, as it has been said, represent increments in the polymer concentration at any given phase increase as well as the hydrophilic or hydrophobic character of the phases promoting these types of interactions between the phase-forming chemicals and the solutes.
Contaminants tested in the PEG 10,000 - Ficoll 400systems showed a significant high preference towards the bottom Ficoll-rich phase (see systems 17-20 in Table 2.3.1).
The increment in TLL from 15% to 30% caused a decrease in the recovery yield from 98.13 ± 29.89 % to 91.62 ± 1.94 %. As known, Ficoll is a high MW polymer and an increase in TLL might cause possible saturation of the Ficoll-rich phase, while it also affects density and surface tension at the interphase which probably causes entrapment of molecules in the interphase [34].
From the previous analysis, it is evident that the highest exosome recovery yields were achieved at the bottom phase in all systems, but this was also true for contaminants.
Considering the need to find a system in which both exosomes and contaminants were partitioned mainly towards different phases further analysis were performed to find and later optimize those alternatives. In doing so, the natural logarithm of the partition coefficients (ln(KP)) of both exosome and contaminants were analyzed with respect to TLL, because even when all systems are different in polymer composition they were evaluated considering the same TLL values. The effect of TLL on ln(KP) of exosomes and contaminants in each ATPS is depicted in Figure 2.3.1. This arrangement allows us to identify the optimal parameter selection for those systems which present a potential exosome isolation from their contaminants. A more negative value for ln(KP) indicates that the studied particle prefers the bottom phase, while a more positive value portrays a preference towards the top phase. As it is seen in Figure 2.3.1.a, for the PEG 3,350 - Dextran 10 systems the largest negative ln(KP) value (-0.43 ± 0.02) for exosomes was obtained at TLL 25% w/w while this system also presented the highest positive ln(KP)value for contaminants (0.13 ± 0.00) making it a good candidate for further