Tổng hợp và đánh giá hiệu quả mang thuốc và tiêu diệt tế bào ung thư của một số hệ nanogel trên cơ sở polysaccharide sulfate (heparin, fucoidan) ghép các copolymer tương hợp sinh học TT TA

The research objectives of the thesis: Synthesize and evaluate properties of nanogel carrier based on polysaccharide sulfate heparin, fucoidan grafted with different pluronic to investi

GRADUATE UNIVERSITY OF SCIENCE AND

TECHNOLOGY -

NGUYEN NGOC THE

SYNTHESIS AND EVALUATION OF DRUG LOADING EFFICIENCY FOR NANOGELS BASED

CHEMISTRY PHD THESIS SUMMARY

HO CHI MINH CITY – 2021

Technology

1st Science instructor: Assoc Prof PhD Tran Ngoc Quyen

2nd Science instructor: PhD Nguyen Thi Thanh Thuy

The thesis can be found at:

- Library of Science and Technology Academy

- National Library of Vietnam

PREAMBLE

1 The urgency of the thesis:

Cisplatin (cis-diamminedichloroplatinum (II)) is one of the main anticancer drugs widely used and effective for the treatment

of many types of solid tumors However, the study of the clinical effect of cisplatin, later on, was limited by the poor selectivity of the drug between normal and tumor tissues At the same time, the toxicity of the drug has also caused many side effects on the kidneys, myelosuppression, chronic neurotoxicity, thereby leading to drug resistance and dosage restriction during treatment [2- 4] Therefore, to overcome the above limitations, nanoparticle drug carriers have been strongly developed by domestic and foreign scientists, based on increasing drug accumulation in

cancer cells and thereby reduce the adverse drug side effects

Nanogels are nanopolymer carriers that are attracting the attention of many scientists around the world Nanogel has many outstanding features compared to other nano-carrier systems such as flexible nano-size and size, capable of simultaneously responding to many stimuli from the external environment such

as temperature, pH, and intensity ionic strength, which

contributes to the effectiveness of controlled drug release [5-7]

Therefore, the direction of research and preparation of nanogel carriers to create high biocompatibility of the carrier, reduce the toxicity of the drug, contribute to improving the effectiveness of the drug and treatment will have many scientific implications and application The use of cisplatin complexed

hydrate with a carrier can also increase the ability to carry drugs and effectively destroy cancer cells

Because of the above reasons, we have carried out the project

"Synthesis and evaluation of drug loading efficiency for nanogels based on biocompatible grafted polysaccharide sulfate (heparin, fucoidan) copolymer against cancer cells growth”

2 The research objectives of the thesis:

Synthesize and evaluate properties of nanogel carrier based

on polysaccharide sulfate (heparin, fucoidan) grafted with different pluronic to investigate anticancer drug cisplatin and cisplatin hydrate effect Since then, the application of nanogel modulation in the transmission of two drugs combining cisplatin hydrate and nanocurcumin aims to reduce the volume of breast cancer tumors in mouse models (Mus musculus var Albino) carrying heterologous tumors from humans

3 The main research contents of the thesis:

1 Synthesize and evaluate the structure and morphology of the nanogels based on heparin grafted with pluronic P123, F127, F68, and F87 with different grafting ratios

2 To synthesize and investigate the effect of carrying the hydrate from cisplatin and cisplatin of the synthesized nanogels

3 Investigate the ability to release the drug as well as the effect of killing the breast cancer cell line MCF-7 of the nanogel Hep-P123 system carrying the drug

4 Synthesis and evaluation of nanogel structure and morphology-based on pluronic P123 conjugated fucoidan sulfate polysaccharide

5 Investigate the ability to release drugs as well as destroy the breast cancer cell line MCF-7 of the nanogel system Fud-P123 that carries the drug

6 Synthesize and investigate the ability to release drugs as well as the effect of destroying breast cancer cell line MCF-7 of nanogel Hep-F127 system with drug combining cisplatin hydrate and nanocurcumin

7 Evaluate the effect on volume reduction of breast cancer tumors on mouse models carrying heterozygous tumors from humans of the nanogel system with drug combined

The layout of the thesis: the thesis includes the Introduction, Conclusions, Recommendations, and 3 chapters (Chapter 1: Overview, Chapter 2: Research, Chapter 3: Results and discussion) The thesis has 25 tables, 71 figures, and 3 related works published The appendix includes 32 figures and 21 tables

of figures

CHAPTER 1: OVERVIEW 1.1 Nanogel

The term "nanogel" is defined as nanoscale particles formed

by the physical or chemical cross-links of polymer networks The size of the nanogels usually ranges from 20-200 nm, thereby reducing renal clearance and prolonging the plasma half-life [21-22]

These nanogels can trap hydrophobic molecules (anticancer drugs), proteins (enzymes, insulin, antigen proteins), and nucleic acids (DNA plasmids), so they are used as nano polymers in the

field of cancer treatment, protein distribution, and artificial vaccines [23]

1.2 Cancer and anticancer drug cisplatin

Cancer occurs due to a mutation in ADN, leading to an infinite proliferation of cells, disorganized, not obeying growth control mechanisms The most important issue in cancer is the distinction between benign and malignant tumors Through recent studies, cancer is genetic, that is, due to alterations in genes that lead to changes in cell function and division

Cisplatin has been used clinically to treat cancers To date, cisplatin is proven to be one of the effective anti-cancer chemotherapy drugs but needs to be combined with other drugs during treatment and more research is needed to improve treatments to reduce the Side effects of medications In which, the research direction of nanoscale slow-release carrier is considered as one of the most effective solutions to address limitations due to side effects and drug resistance of cisplatin

1.3 Previous studies

In the country: there are several research groups on nanoparticles to transport cisplatin or platinum complexes (II), typically: the research group of Nguyen Cuu Khoa and Tran Ngoc Quyen synthesizes dendrimer nanoparticles that carry anticancer drugs 5-fluorouracil and cisplatin; Tran Ngoc Quyen, Nguyen Dai Hai, and the research team in Korea have successfully synthesized the electronegative - heat-sensitive nanogel carrier based on heparin

Overseas: authors Xiang-Hong Peng (2011), Liu (2012), Yoon Ki Joung (2013), Lili Liu (2014), Yi Zhang (2018), Seyed Ebrahim Alavi (2019), Pai-An Hwang (2017), Merve Tutuncu (2018) studied a combination of anti-cancer drugs cisplatin and nano-carriers The study combining curcumin with the anti-cancer drug cisplatin has authors Mendonça (2013), Baharuddin (2016), Parveen Kumar (2017)

There are no domestic and foreign scientific works on the preparation and application of nanogel from fucoidan, as well as using the nanogel carrier system based on heparin-pluronic to create complexes directly with hydrate cisplatin in combination with nanocurcumin

CHAPTER 2: RESEARCH 2.1 Research content

- Synthesis of nanogels based on heparin, fucoidan grafted with pluronic P123, F127, F68, and F87 with different grafting ratios

- Determine the structure and morphology of products

- Investigate the CMC concentration of the synthesized nanogels

- Investigate the effect of carrying drugs cisplatin (Cis) and cisplatin hydrate (CisOH) of the synthesized nanogels

- Investigate the ability to destroy the breast cancer cell line MCF-7 of nanogel Hep-P123 (1:3) system carrying Cis and CisOH; Fud-P123 (1:3) system carries Cis and CisOH

- Investigate the release ability of nanogel Hep-P123-CisOH (1:3) and Fud-P123-CisOH (1:3)

- Synthesize and investigate the ability to release drugs as well

as the effect of killing breast cancer cell line MCF-7 of nanogel Hep-F127 system with drug combining cisplatin hydrate and nanocurcumin (Hep-F127-CisOH-Cur)

- Create a mouse model with a tumor

- Evaluate treatment effects of Hep-F127-CisOH-Cur nanogel system in mice carrying heterozygous tumors from humans

2.2 Research Methods

The process of synthesizing the grafted copolymer Hep-P123, Hep-F127, Hep-F68, Hep-F87, Fud-P123 is done through 4 stages, the method is referenced as previously published by the research team [50]

- Product structure is determined by 1H-NMR spectroscopy and FT-IR spectroscopy

- Using the TGA method to determine the percentage of pluronic content grafted to polysaccharide sulfate

- The morphology and size of products are determined by electron microscopy transmitted through TEM

- Using iodine and UV-Vis spectroscopy method to determine CMC value of nanogels

- Investigate the ability to destroy the breast cancer cell line MCF-7 of the drug nanogel system by SRB staining

- Investigate the ability of nanogels to carry and release drugs through plasma emission spectroscopy (ICP-OES)

- Model of a mouse carrying tumor by Xenograft method

- Evaluate tumor size in mice by measuring the tumor size and volume Evaluation of treatment efficacy using SOD2

immunohistochemical and H&E staining

CHAPTER 3: RESULTS AND DISCUSSION

3.1 Results of synthesis and survey of nanogel Hep-P123

Hep-P123 products with 4 different grafting ratios (1:3; 1:7; 1:10; 1:14 mmol/mmol) are synthesized from intermediate products NPC-P123-NPC, NPC-P123-Ami and Hep-DAB, white, colloidal The products were structured by FT-IR spectrum, 1H-NMR spectrum, TGA, TEM, and CMC

3.1.1 Results of determining the composition and structure of copolymer Hep-P123

3.1.1.1 Analysis results of FT-IR spectrum

The FT-IR spectral data in figure 3.1 are listed in table 3.1

Table 3.1 FT-IR results of P123, NPC-P123-NPC, NPC-P123-Ami

Spectral data in figure 3.6 are listed in table 3.3

Table 3.3 FT-IR results of NPC-P123-Ami, Hep-DAB, Hep-P123

groups

Wavelength (cm -1 ) NPC-

f -COO-NPC 1769

Through the summary of spectral data of substances in Table 3.3, there is the repetition of characteristic oscillating signals on the heparin molecule at position a, b and on the P123 molecule

at position c, d always appears in Hep-P123 molecule after synthesis The oscillation of the oscillating signal in the f position

to the g position indicates the formation of a urethane bond from the reaction between Hep-DAB and NPC-P123-Ami, followed

by the disappearance of the e-signal

3.1.1.2 Result of spectrum 1 H-NMR analysis

Spectral data in Figures 3.2 and 3.3 are listed in Table 3.2

Table 3.2 Results 1 H-NMR of NPC-P123-NPC and NPC-P123-Ami

Spectral data in Figures 3.5 and 3.7 are listed in table 3.4

Table 3.4 Results 1 H-NMR of NPC-P123-Ami, Hep-DAB; Hep-P123

3.1.1.3 TGA analysis results of Hep-P123

The thermal stability of the Hep-P123 grafted copolymer with heparin and P123 was analyzed by TGA measurements From the experimental results on the TGA scheme, we calculate the percentage of pluronic P123 mass grafted into heparin consistent with the transplant rate, as reported by Hong Liang Kang [121]

3.1.1.4 Results of analysis of CMC value of Hep-P123

Experimental results show that CMC of grafted copolymer measured from 0.028 - 0.041 (% wt) is larger than CMC value of pure P123, pluronic P123 after grafting with heparin will increase

the hydrophilic sheath of the conductive micelle to increase the CMC value of the sample solution

3.1.1.5 Analysis results TEM and DLS of Hep-P123

TEM results in Figure 3.11 show that the nanoparticles are spherical, with diameters arranged in the range from 45 nm to

153 nm The size distribution ranges from 94.4 nm to 182.4 nm

by DLS, at 25°C and depends on the amount of pluronic P123 is conjugated

Figure 3.10 TEM results of P123 pluronic

Figure 3.11 TEM and DLS results of (a): P123; (b): Hep-P123 (1:3);

(c): Hep-P123 (1:7); (d): Hep-P123 (1:10); (e): Hep-P123 (1:14)

3.1.2 Results of nanogel Hep-P123 synthesis carrying drugs cisplatin and cisplatin hydrate

3.1.2.1 Results of analysis of FT-IR spectrum of Hep-P123 carrying Cis and CisOH drugs

Figure 3.13 FT-IR spectrum of Hep-P123-Cis; Hep-P123-CisOH

The FT-IR spectral data in figure 3.13 are listed in Table 3.6

Table 3.6 FT-IR results of heparin, P123, P123-Cis,

Hep-P123-CisOH

groups

Wavelength (cm -1 ) Hep-

P123

P123-Cis

Hep- CisOH

D -C-O-S-

(complex)

Through table 3.6 summarizes the spectral data with repetition of characteristic oscillating signals on heparin molecule at position a, b and on molecule P123 at position c, d in molecule Hep-P123-Cis and Hep -P123-CisOH after synthesis

In the spectrum of Hep-P123-CisOH we see new absorption oscillations at positions A, B, C, D, showing the complex formation of CisOH and carboxylate, sulfate group on heparin While the spectrum of Hep-P123-Cis only appears oscillating signal at position A This proves that Hep-P123-Cis and Hep-P123-CisOH compounds have been synthesized successfully

3.1.2.2 Analysis results in ICP-OES of P123-Cis and P123-CisOH

Hep-Figure 3.14 Drug-bearing efficacy of the Hep-P123 carrier system

The results of figure 3.14 testing the drug-carrying capacity

of 4 types of Hep-P123 nanocarriers show that the more pluronic P123 is grafted to heparin, the more hydrophobic interaction on Hep-P123 molecule will be made, making the results carry higher Cis and lower CisOH complex formation Conversely, on the

structural circuit of heparin containing anionic groups (carboxylate/sulfate), these groups will form a chemical bond with the platinum hydroxide portion of CisOH, when more P123

is bound to the heparin, corresponding to the number of anionic groups decreases, resulting in lower complex formation as the grafting rate increases

3.1.3 Results of assessing the ability to release drugs of P123

Hep-The results of the ability of the Hep-P123-CisOH nanosystem

to release drugs showed that the CisOH release at pH 5.5 (nearly 75%) was higher than at pH 7.4 (65%) after 24 hours The release

of CisOH indicates that the Hep-P123-CisOH complex is rapidly hydrolyzed under acid conditions This result is useful for the carrier system because the pH is lower in some tumor sites than

at the normal tissue site

3.1.4 Cell toxicity results (on MCF-7 cell lines and fibroblasts)

Experimental results after 48 hours showed that nanogel platinum exhibited high proliferative activity against human breast cancer cell line MCF-7 Hep-P123 complexing with hydrate cisplatin also significantly increases cytotoxicity compared to Hep-P123-Cis nanosystem

Hep-P123 nanocarrier system (1:3) is also evaluated to test for fibroblast toxicity by the SRB staining method The results showed that the Hep-P123 nanocarrier system at the concentration of 100 µg/mL, after 48 hours, the cell survival rate was about 90%