Toxicity evaluation of silkworm derived fibroin nanoparticles and prp based on iso 10993 5 standard and prp application in enhancing oocytes maturation in mice

VIET N AM N ATIO N AL U IVERSITY HO CHI MI H CITY N NFaculty of Biology and Biotechnology RESEARCH PROPOSAL TOPIC Toxicity evaluation of silkworm-derived fibroin-nanoparticles and PRP b

VIET N AM N ATIO N AL U IVERSITY HO CHI MI H CITY N N

Faculty of Biology and Biotechnology

RESEARCH PROPOSAL TOPIC Toxicity evaluation of silkworm-derived fibroin-nanoparticles and PRP based on ISO 10993-5 standard and PRP application in enhancing

oocytes maturation in mice

PRESENTER

Nguyen Khanh Nhu Quynh 21187261 VNUHCM University of Science Tran Thi My Phuc 21187257 VNUHCM University of Science Truong Phu Minh 21187236 VNUHCM University of Science

INSTRUCTOR

MSc Doan Nguyen Vu Department of Physiology and Animal Biotechnology MSc Nguyen Thuan Nho Department of Physiology and Animal Biotechnology

TABLE OF CO N TEN TS

I BACKGROUND 3

1.1 Platelet-rich plasma 3

1.1.1 What is Platelet-rich plasma? 3

1.1.2 How to obtain Platelet-rich plasma? 3

1.1.3 The application of PRP in research 4

1.2 Obtaining nanoparticles within silkworms 4

1.2.1 What is Nanoparticle? 4

1.2.2 What is silkworm? 5

1.2.3 Silkworm nanoparticle and its application 5

1.3 Cytotoxicity and ISO 10993-5 7

1.3.1 ISO and ISO 10993 5 - 2009 – 7

1.3.2 Cytotoxicity and ISO standards 8

1.4 Application of PRP for in vitro maturation 9

1.4.1 In vitro maturation method 9

1.4.2 2D and 3D cell culture 9

II Objective 10

III Research questions 10

IV Research questions 11

4.1 Experiments 1: Obtaining PRP from the blood of laboratory white mice 11

4.1.1 Principle 11

4.1.2 Materials 11

4.1.3 Method 11

4.2 Experiments 4.2: Creating nanoparticles from silkworm solution 12

4.2.1 Principles 12

4.2.2 Materials 13

4.2.3 Method 13

Creating nanoparticles from silkworms’ procedure 13

4.3 Experiment 3: PRP and silkworms’ nanoparticles cytotoxic evaluation based on ISO 10993-5 standard 15

4.3.1 Principle 15

4.3.2 Materials 16

4.3.3 Method 16

4.4 Experiment 4: Raising mice oocytes in 3D culture containing PRP 17

4.4.1 Principle 17

4.4.3 Method 19

REFERENCES 21

I BACKGROUND

1.1 Platelet- rich plasma

1.1.1 What is Platelet-rich plasma?

Blood composition contains plasma, Red Blood Cell (RBC), White Blood Cell (WBC), and platelets Platelets have an essential role in the blood clotting process and harbor potential for repairing tissue and regeneration The high natural concentration of platelets derived from whole blood is called Platelet-Rich Plasma (also known as PRP) PRP is widely used in many regenerative medicines, giving a new era of therapeutic approach This is due to many biological factors, such as Growth Factors (GFs), cytokines, lysozymes, and other adhesion proteins (Everts et al., 2020) The benefits of PRP include no negative effects, as well as simple preparation procedures, a broad application profile, and no restriction

of regulatory requirements (Andia & Maffulli, 2018) On the other hand, the compositions of PRP are

diverse platelet, WBC, and RBC content, which makes it difficult to determine concentrations

1.1.2 How to obtain Platelet-rich pla sm a?

There are various methods of obtaining PRP The open method involves a double-spin step of centrifugation to exclude unnecessary blood components (Dashore et al., 2021) The general step of PRP is illustrated in Figure 1

Figure 1: Method of collecting PRP by the double-spin open method (Dashore et al., 2021)

The first spin contains the heavy RBC, located at the bottom of the conical tube The lighter liquid at

the top is subsequently transferred to a new tube and centrifuged at a higher Round Per Minute (RPM)

spin than the first one The top two-thirds are then removed, leaving only the platelet pellet, which is gently resuspended in a small amount of plasma This approach is appropriate for dermatology due to its low cost, high platelet concentration, and variable volume of PRP synthesis, although this method has a significant risk of external contamination (Dashore et al., 2021) Another economical approach is

to use blood-filled syringe a for the first centrifugation After that, the supernatant is transferred to a different syringe by using a 3-way cannula to collect supernatant plasma prepared for the second centrifugation This is a closed approach since the blood remains within the medical disposable equipment, which reduces exposure to the external environment and the possibility of external contamination (Dashore et al., 2021)

1.1.3 The application of PRP in research

The application of PRP in both in vitro and in vivo is diverse in many types of research Polycystic Ovary Syndrome (PCOS) affects 15% of women of reproductive age PRP application can enhance

follicular growth and induce antiandrogenic and antioxidant effects to improve PCOS-induced pathogenesis (Merhi et al., 2023) However, the overuse of PRP to boost ovulation can cause the pathomechanism of Ovarian Hyperstimulation Syndrome (OHSS), which is potentially life-threatening due to the vasoactive substances released from overstimulated ovaries (Nagy et al., 2023)

1.2 Obtaining nanoparticles within silkworms

1.2.1 What is Nanoparticle?

Nanoparticles possess special properties that contribute to improving the development of many fields, such as medicine, food, agriculture, pharmaceuticals, and cosmetics Although nanoparticles possess a relatively small size, in the range of 1-100 nm, they are still considered an important milestone in the new technological revolution in science Using nanoparticles as a covering material helps protect the substances inside, limiting pollution and their impact on each other Some nanoparticles, such as MgO NPs, TiO NPs, and CuO NPs, are used in the antibacterial treatment of some types of bacteria plants 2 in

In addition, silica is also considered a nanoparticle, helping to control and combat the incidence of disease-causing pests on farms Nanoparticles are in the commercialization stage, so they need to be

clinically tested to reduce negative effects as well as increase the benefits of the material before being put into use (Fometu et al., 2021)

1.2.2 What is silkworm?

Silkworm is an organism that is used widely, especially in the experimental research, because it possesses many beneficial characteristics, such as being an invertebrate animal, small, having a short generation time, good fertility, easy to grow under different conditions, having a wide variety of species, possessing a complete genome, and being considered an available protein database Using silkworms is also easier compared to fruit flies The reason is that organs and genes from silkworms can be obtained with the naked eye without using a microscope, like fruit flies Silkworms are currently considered one

of the outstanding model organisms because they play many important roles in many different scientific fields, as well as do not causing any ethical issues in the research process However, when using silkworms, it is necessary to check toxicity very carefully before use so as not to bring any risks to the tested animals (Fometu et al., 2021)

1.2.3 Silkworm nanoparticle and its application

The use of nanoparticles from silkworm into materials technology not only helps develop the tosurvival rate of silk and promote growth and development, but also helps improve the quality of silk fibers (Fometu et al., 2021) Fibroin is a dominant protein that is well-suited as a biomaterial for drug delivery Fibroin is extracted mainly from silkworm cocoons A silk strand of fibroin consists of about 75% of the core and about 25% of sericin (a protein that coats the outside of fibroin) Fibroin consists

of 2 subunits: a heavy chain and a light chain linked together through a sulfide bond at the C-terminus

of the heavy chain and Cys at the light chain Due to the non-repetitive amino acid sequences in the structure, the light chain is more hydrophilic and has low water resistance, so fibroin has good elasticity

as well as a semi-crystalline structure with hardness and high durability Fibroin has crystallinity that can change into a variety of different polymorphic structures, called silk I, silk II, and silk III, respectively With silk I, this silk fiber is found mainly in silk glands, with the lowest crystallinity among all three types of silk Possessing a rather zigzag structure, silk fiber easily dissolves in water and is thermodynamically unstable Compared to silk I, silk II appears mainly in silk fibers and has high

crystallinity Thanks to the hydrophobic interactions between hydrogen atoms and methyl groups, silk

II is thermodynamically stable Therefore, silk II is insoluble in water, has good heat resistance, and is stable Silk III is found in naturally formed membranes on surfaces and is a special crystalline form of fibroin Due to the crystallinity formed from silk I and silk II, it has high mobility The silk production process uses Na2CO3 boiled at 100 degrees Celsius for 1 hour, then washed three times with water and dried naturally at room temperature, converting from raw silk to silk In case we want to switch from IIsilk II back to silk I, we use Ca(NO with a salt ratio of 1:4, heat from 60 to 90 degrees Celsius, then 3)2dialyze to remove salt (Pham et al, 2020)

Figure 2: Process of collecting silk fibroin from silkworm (Pham et al, 2020)

Nanoparticles in silkworms possess very high versatility, therefore widely used in therapeutic compounds Fibroin contains many diverse amino acid molecules, so it is also used to attach different molecules or antibodies to the target Some applications include infusion, medicine, transcutaneous administration, etc Fibroin nanoparticles have been used as capsules in pharmaceuticals, encapsulating drugs inside Using fibroin as a capsule will help control the direction of the drug, keeping stability inside the digestive tract (pH, acidity, enzymes) Due to its ability to adhere to mucous membranes, fibroin can adhere tightly to the intestinal mucosa, ensuring the endocytosis process between the drug and the blood and ensuring that the drug will enter the blood intact and effectively and the safest (Pham

et al., 2020) One of the typical medicinal applications for the presence of nanomaterials in silkworms

is as an anti-cancer agent Because fibroin in silkworms has hydrophobic properties, this will help cancer

drugs be less soluble in water, maintaining the stability and biological activity of the drug after release (Nguyen et al., 2019) For infusion, nanoparticles can be used to overcome the characteristics of conventional pharmaceuticals, such as low permeability, short half-life and especially drug resistance after treatment Because of their high versatility, fibroins are often used to deliver small molecules, focusing largely on cancer treatment and anti-inflammation In addition, fibroin is also used to deliver large molecules during protein treatment, increasing effectiveness quite clearly For skin administration, nanoparticles are quite small (average 4nm) and can go through the skin intact Because it has both ≤hydrophobic and hydrophilic regions, fibroin can easily and conveniently control particles through the skin (Pham et al., 2020)

1.3 Cytotoxicity and ISO 10993-5

1.3.1 ISO and ISO 10993 5 - 2009 –

ISO (International Organization for Standardization) is an international standardization organization founded on February 23, 1947 This is the body that sets international standards, providing commercial and industrial standards that are applied worldwide Currently, more than 160 countries are ISO members ISO's headquarters are currently located in Geneva, Switzerland Vietnam is the 77th country

to participate in the ISO standards system ISO standards are translated into Vietnamese and issued under the name Vietnamese Standards (abbreviated as TCVN) ISO standards are internationally standardized rules to help organizations to develop sustainably and create capabilities that enhance the value of businesses and organizations in all fields of production, trade, and service Product quality would meet the consumers’ needs when applying ISO standards Currently, ISO has so many different sets of standards; depending on the field, there are specific sets of standards As in these experiments,

we will use ISO 10993 in 2009, which is the latest set of standards for Biological Evaluation of Medical Devices ISO 10993 - 5 and ISO 10993 - 12 are being used in this experiment; ISO 10993 - 5 would help test the cytotoxicity of PRP and silk nanoparticles collected in the progress while ISO 10993 - 12 would involve sample preparation and reference materials These two standards would be operated throughout the experiments (ISO 10993 5: 2009 and TCVN 7391 5 : 2005) – –

1.3.2 Cytotoxicity and ISO standards

Cytotoxicity is the ability of a chemical or mediator to destroy living cells This could happen because

of some factors like chemical stimuli, exposure to other cells or the conditions of the experiment Cytotoxicity can let cells overgo one of these three pathways: necrosis (accidental cell death), apoptosis (programmed cell death) or cytostasis (a decrease in cell viability) (Çelik et al., 2018) Therefore, measuring cytotoxicity is especially important in evaluating biomaterials because in this experiment, we would want to exclude cytotoxicity in PRP and silk nanoparticles Samples and controls test should be repeatedly performed 3 times There are 3 types of cytotoxicity tests in general: direct test, indirect test,and test on extract (ISO 10993-5 : 2009 and TCVN 7391-5 : 2005)

a Test on extracts shows assessments of cytotoxicity in both quality and quantity

b Test by direct contact shows assessments of cytotoxicity in both quality and quantity

c Test by indirect contact separately determine cytotoxicity in quality with filter diffusion test and in quantity with agar diffusion test

Determine the effects of cytotoxicity in quantity or quality:

A Quality assessment: using cell dyes or assessing changes in general morphology The material cytotoxicity could be sorted into the following order:

1.4 Application of PRP for in vitro maturation

1.4.1 In vitro maturation method

In vitro maturation (IVM) is a process in which a woman’s eggs (also called oocytes) are collected

and raised outside the body This is also another part of an in vitro fertilization (IVF) procedure IVM

is considered for women with PCOS, OHSS and polycystic ovaries to prevent ovarian hyperstimulation

(ReproductiveFacts.org, 2015)

Platelets, the main component of PRP, contain more than 1100 different proteins and result in over

1500 protein-based bioactive factors after translational modifications These factors involve many pathways/positions in the body such as immune system messengers, growth factors, enzymes and their prohibitors and other factors which can join in the recovery process One more important thing is that PRP is an autologous product, collected from self-blood; this leads to no risks cross-contamination of

or disease affection or any immune reactions Besides damaged tissue regeneration, PRP supplies many nutrients depending on the components of platelets; PRP also stimulates the requirement, proliferation, and differentiation of the cells Growth factors in PRP might be the first source of nutrients for oocytes

in in vitro maturation Platelet-derived growth factor can enhance collagen synthesis and proliferate bone cells Vascular endothelial growth factor in platelets could stimulate angiogenesis and increase the permeability of the vessels Insulin-like growth factors would promote cell growth, differentiation and recruitment in bone, blood vessels, skin, and other tissues Fibroblast growth factor might promote the proliferation of mesenchymal cells, chondrocytes and osteoblasts and stimulate the growth and differentiation of chondrocytes and osteoblasts These components of our PRP would supply great crucial nutrients for oocytes to mature in IVM (Pavlovic et al., 2016)

1.4.2 2D and 3D cell culture

Mammalian cells growing in an environment that closely resembles the inside of the body help scientists get closer to the accuracy of their research Traditionally, we often grow cells on 2D surfaces

in vitro; However, 2D cell culture has many limitations, such as changes in cell morphology, interactions between the environment inside and outside the cells, or effects on cell polarity Therefore, the 3D environment was born, to improve the limitations of the 2D environment The 3D cultures provide cells

grown in vitro with an environment more like the in vivo environment 3D environments are divided into 3 types: i) suspension cultures on non-adherent plates; ii) cultures in concentrated medium or in gel-like substance and iii) cultures on a scaffold These cultures also help cells get equal nutrients from the environment; cells in these cultures also show as grown in the body about cellular topology, gene expression, signaling and metabolism These also help scientists to have more accurate observations and calculations (Kapałczyńska et al., 2018)

Figure 3: Comparison of 2D and 3D cell culture systems (A) 2D cell culture, (B) 3D cell

culture (Samimi et al., 2021)

II Objective

In this module, our objective is to obtain PRP in blood by centrifugation method, collecting fibroin nanoparticles within silkworms and successfully mature the mice oocytes in vitro by fibroin nanoparticles containing PRP without toxicity, death cell-induced and grow normally

III Research questions

Our experiment aims to answer the questions in terms of applying the nanoparticle containing PRP

to nourish mice oocytes These questions are stated below

1 How to collect PRP in mice blood efficiently?

2 How to collect fibroin nanoparticles remaining its native size and quality from silkworms?

3 Based on ISO 10993-5 standard, how are the PRP and fibroin nanoparticles toxicity evaluated to prepare for oocyte maturation application?

4 How does the supplementation of PRP culture affect the growth and quality of mice oocyte?

IV Research questions

4.1 Experiments 1: Obtaining PRP from the blood of laboratory white mice

4.1.1 Principle

Our group will use an open approach (using a blood-filled conical bottom tube to collect and generate PRP from laboratory mice This method not only creates good platelet concentration and flexible volume of PRP production, but also lower the contamination level when evaluating toxicity based on ISO 10993-5 standard An open approach is also an economical method because it requires only centrifugation machine and some tools that is available in the laboratory

4.1.2 Materials

• White mice (provided by biomaterials labora ory)

Tools & equipment

• 2 sterile conical plastic tubes

• Pipette

• Centrifugation machine

4.1.3 Method

Procedure

1 Collect a volume of blood mice using syringe and gauge needles

2 Transfer blood volume to conical tube and centrifuge at low RPM

3 Transfer plasma supernatants at the top layers to another conical tube using a sterile pipette

4 Centrifuge again at higher RPM than the first centrifugation

5 After second centrifugation, removing at approximate two-thirds of supernatants containing poor plasma

platelet-6 The platelet pellet is gently resuspended in the lower 1/3 of plasma, which contains PRP

Na2CO3 and Ajisawa are the two reagents we will use Na2CO3 is used to cause destruction and breakdown of a large amount of sericin inside silk, and completely break the side chain in the peptide, gradually reducing its mechanical properties (Zhang Y Q , 2018) Ajisawa is used to remove the glue layer of silk, preserving the silk fiber structure (Rizzo et al., 2021)

Scanning electron microscope (also known as SEM) is used to observe images of nanoparticles, thereby understanding the distribution of nanoparticles as well as the size, composition and shape of the particles The reactivity of particles, existing toxicity, and surface area of nanoparticles can be known, contributing to the application of nanoparticles in the fields of medicine, technology, electronics, etc SEM uses a focused electron beam, which in turn creates secondary electron signals and backscattered electrons Receiving these signals creates detailed images of the sample's surface Microsoft Visio allows to create many types of charts, images, and diagrams with ease (Guide, S (n.d.)).