**3.8 Cellular barriers**

EVs that eventually reach the target cell has to overcome the plasma membrane, escape the endosome and evade lysosomal degradation to release their cargo into the cytosol (**Figure 3**).

The plasma membrane is an intricate structure consisting of various domains formed *via* different mechanisms. Some of these mechanisms include the formation of plasma membrane protein fences to reduce lateral diffusion in the plasma membrane, the arrangement of plasma membrane proteins into a scaffold that interacts with certain plasma membrane lipids, and protein-lipid interaction to form lipid rafts [174]. These domains represent the first barrier that a freshly-secreted EV needs to cross and determine the way the target cell internalizes EVs. Environmental factors also influence the interaction of EVs with the plasma membrane. For instance, the uptake of EVs *via* fusion with the cell membrane is observed to occur at a higher rate under acidic conditions [175], while endocytosis is shown to be hindered by neutral pH or high cholesterol levels [14, 176].

EVs can be internalised by target cells *via* endocytosis, be it caveolae-dependent endocytosis, flotillin-dependent endocytosis, ARF6-dependent endocytosis or other forms of endocytosis [105]. Clathrin-mediated endocytosis (CME), or "receptor-mediated endocytosis", plays an especially prominent role in the uptake of small EVs [105],

#### **Figure 3.**

*Internalisation, lysosomal degradation and lysosomal escape mechanisms of extracellular vesicles (EVs) in target cells. Upon reaching the target cell, EVs may be internalised by the cell via endocytosis or direct fusion with the plasma membrane. EVs that are internalised via endocytosis are packaged into endosomes, which may fuse with lysosomes to degrade the EVs. To escape lysosomal degradation, endocytosed EVs may undergo retrograde trafficking to the trans-Golgi network, endocytic recycling to be secreted out of the cell, or another mechanism altogether. Endocytosed EVs that do not undergo lysosomal degradation fuse with the endosomal membrane via the mediation of soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins to release their cargo into the cytosol. EVs that fuse directly with the plasma membrane to release their cargo into the cytosol evade endosomal and lysosomal activity completely (created with BioRender.com).*

as supported by recent studies on the uptake of human epidermoid carcinoma EVs [177] and rat pheochromocytoma EVs [178] by human cervical carcinoma (HeLa) cells and rat bone marrow-derived mesenchymal stromal cells respectively. During CME, a temporary membrane scaffold forms as a result of membrane binding of Bin/amphiphysin/Rvs (BAR) domain-containing proteins which recruit clathrin. Clathrin then binds to the cytoplasmic tails of membrane proteins with the help of adaptor proteins, resulting in a clathrin-coated pit that internalises the EV [105]. EVs internalised *via* endocytosis are packaged into endosomes. These EVs then proceed to release their cargo into the cytoplasm by fusion of their membranes with the endosomal membrane, a process mediated by soluble *N*-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins which join the cytosolic sides of the EV and endosomal membranes together [3].

In general, the CME of EVs is initiated through the mediation of lectins, tetraspanins, cell adhesion proteins and other receptor-ligand interactions [18, 179]. For instance, exosomes from macrophages possess C-type lectin, which interacts with the C-type lectin receptor found on dendritic and brain endothelial cells [179, 180]. Galectin-5 on red blood cell (RBC) EVs enables them to be internalised by macrophages [45, 179]. Integrins on tumour EVs have been associated with the uptake of these EVs by lung fibroblasts and liver macrophages [42, 179]. Exosomes and target cells can exploit the interaction between intercellular adhesion molecule 1 (ICAM-1) and lymphocyte function-associated antigen 1 (LFA-1) in exosomal uptake [179–181]. Heparan sulphate proteoglycans on target cells bind to EV fibronectin to facilitate uptake of EVs [54, 179, 182]. The high levels of outward-facing PS on the surface of exosomes also enable the recognition and

*Extracellular Vesicles and Their Interplay with Biological Membranes DOI: http://dx.doi.org/10.5772/intechopen.101297*

uptake of these exosomes by antigen-presenting cells *via* T-cell immunoglobulin and mucin domain (TIM) receptors located on the antigen-presenting cells' surface [63, 179, 183].

EVs internalised *via* endocytosis might risk being degraded by lysosomes in the cytosol [3]. The fusion of the membrane of endosomes containing the endocytosed EV with the lysosomal membrane is mediated by SNARE proteins and involves the active transport of vesicles along the cytoskeleton [18]. EV size may play a role in determining the fate of EVs upon uptake *via* endocytosis, as EVs larger than 100 nm may require macropinocytosis for their uptake, which tends towards lysosomal degradation more than other internalisation mechanisms accessible to smaller EVs [177, 184, 185]. Endocytosed EVs might escape lysosomes *via* pathways similar to those of viruses, like the CD81 positive lysosome-associated membrane protein 1 (Lamp-1) negative route in dendritic cells which resembles that of HIV-1 uptake [186]. A study showed that HEK293 exosomes internalised by human fibroblastic, hepatic and renal cells were transported to the endoplasmic reticulum where they released their cargo, a pathway that might be a potential escape route from lysosomal degradation [187]. EVs may also evade lysosomal degradation *via* endocytic recycling out of the cell [188] or retrograde trafficking from the endosomal pathway to the trans-Golgi network [189].

EVs have also been reported to deliver their cargo into target cells *via* direct membrane fusion with the cell membrane, with EV surface proteins syncytin-1 and syncytin-2 seemingly playing a significant role in this [190–192]. Originally found on the plasma membranes of placental trophoblast cells [190, 191], gamete cells [190, 193] and various cancerous and non-cancerous cells known to fuse directly with other cells [190, 194–197], these proteins have also been detected on EVs secreted from these cells [190, 192]. In light of this, incorporating these surface proteins into EVs to increase their uptake *via* direct membrane fusion might be a possible way to evade endocytosis and lysosomal degradation completely.


Preface

Chapter 7 **Aging Auditory System and Amplification 111**

**Learning Simulations 131**

**Section 6 New aspects of Auditory periphery 165**

**World Science 153**

**Loud Music 167**

Yoshiharu Soeta

**Sense Organ 207** Raphael R. Ciuman

Chapter 13 **Ototoxicity: Old and New Foes 233** Agnieszka J. Szczepek

**Section 7 Ototoxicity 231**

**Section 5 Cochlear Implants 129**

**VI** Contents

Payton Lin

Christiane Marques do Couto and Izabella dos Santos Brites

Chapter 8 **Temporal Filterbanks in Cochlear Implant Hearing and Deep**

Chapter 9 **Treatment of Partial Deafness: The Polish School in**

Chapter 11 **Psychophysiological Evidence of an Autocorrelation**

Henryk Skarżyński and Piotr Henryk Skarżyński

Chapter 10 **Various Aspects of Auditory Fatigue Caused by Listening to**

Andrzej Dobrucki, Maurycy J. Kin and Bartłomiej Kruk

**Mechanism in the Human Auditory System 187**

Chapter 12 **Cochlea – A Physiological Description of a Finely Structured**

The objective of the present book is to diffuse information related to a number of technologi‐ cal and procedural advances in the area of clinical audiology. This book was made possible through the generous contribution of 20 authors.

The format for this book is purely educational, targeting graduate courses in audiology, speech pathology and hearing science, and neurosciences and basic graduate courses in ENT. The authors have been asked to present a generous background of their topic, in the "Introduction" section of their chapter, and an extensive list of technical terms used through‐ out the chapter.

The book is divided into six sections as follows:

It was my intention to use multimedia material wherever possible, but for the majority of contributors, this was not feasible, during the publication time. We were only able to locate a few interesting videos on wideband tympanometry, and the web references are included in the Appendix section of the relative chapter.

#### **Acknowledgments**

I would personally like to thank all the contributing authors, the InTech personnel, and spe‐ cifically Ms. Ana Simcic for her continuous and generous assistance during the preparation of the volume.

> **Stavros Hatzopoulos, PhD** Clinic of Audiology and ENT, University of Ferrara, Italy

**Section 1**


4.2.1 Numbers

Numbers in cereal\_fwd are serialized and deserialized similar to cereal, but to ensure forward compatibility, all integer type fields are saved with size information attached. This makes it possible to load integers which have different sizes on writing and reading side. The attempt to load integer that cannot fit into type used for loading will result in thrown exception. For space-saving purpose, the size of the saved integer is determined as the minimal number of bytes needed to represent the number being stored.

```
Figure 9.
Basic cereal serialization procedure.
```
Figure 10. Serializing structure in cereal.

#### 4.2.2 Arrays and strings

The size of array or string is saved using variable-length integer encoding—the most significant bit is used to indicate if the next byte is part of stored number. This way for small arrays size is stored using only 1 byte. To speed up serialization and save archive space, all items in the array are assumed to have the same size—size information is stored only once per array.

## 4.2.3 Polymorphic types

To properly read object stored using polymorphic pointer, identifier of the object's most derived type is needed. In cereal\_fwd library, as well as in Boost. Serialization, for polymorphic pointers a unique type of identifier is saved to stream. Identifier can be any string; by default fully qualified name of the type is used. During reading process identifier is a key in factory which helps in creating correct object and selecting proper deserialization function.

Identifier for specific class is saved only once in stream, for the first occurrence of given type, accompanied with corresponding ordinal number. For every next instance, just the ordinal number is saved. While reading data saved by newer application, it may happen that identifier of polymorphic type will be connected to field which is unknown and will not be normally read. For that reason logic to process every occurrence of type identifier was added.

During code evolution new derived types for existing base classes may be added and saved as polymorphic pointers in archive. In the original version of cereal library, the attempt to read unknown polymorphic type results in exception being thrown, consequently stopping reading process. In cereal\_fwd library an option was added which changes that behaviour. For pointers of unknown type, nullptr value is set, and reading process is continued without interruption.

#### 4.2.4 Shared pointers

Existing cereal library supports saving several pointers pointing to the same object. In this situation only one copy of the data is saved into the stream. For every other occurrence of the same object, only numerical identifier of previously saved data is stored. For saving process a dictionary of stored pointers and their identifiers is maintained. If address is found in the dictionary, only identifier is saved. For loading process similar mapping between identifiers and shared pointers is

maintained. If during loading pointer identifier is already in the map, data is not loaded, and pointer is returned directly from the map.


*2.2.1 Advantages of vacuum drying method*

#### **Figure 4.** *Vacuum dryer.*

	- 1.*Cost*: Vacuum drying equipment is expensive and may not be feasible for smallscale production.
	- 2.*Maintenance*: Vacuum drying equipment requires regular maintenance to ensure proper operation and prevent contamination.
	- 3.*Complex process*: Vacuum drying is a complex process that requires expertise and specialized equipment.
	- 4.*Limited capacity*: Vacuum drying may not be suitable for large-scale production due to its limited capacity.
	- 1.*Drying of APIs (active pharmaceutical ingredients)*: Vacuum drying is commonly used for drying APIs, especially those that are sensitive to high temperatures [18, 19].
	- 2.*Drying of excipients*: Excipients are the inactive ingredients used in pharmaceutical formulations. Vacuum drying is used to remove moisture from excipients to prevent degradation or clumping.
	- 3.*Drying of finished dosage forms*: Vacuum drying is used for drying finished dosage forms, such as tablets or capsules, after they are coated with a protective layer.
	- 4.*Drying of raw materials*: Vacuum drying is used to dry raw materials, such as herbs or plant extracts, before they are used in the manufacturing process.