Procedure for Toxicological Activity Survey Using Srb Method

2.2.4.1. Cell culture method

Breast cancer (MCF-7), lung cancer (NCI H460), cervical cancer (HeLa), liver cancer (Hep G2) and leukemia (Jurkat) cell lines provided by ATCC (USA) were cultured in E'MEM medium (MCF-7, NCI H460, HeLa and Hep G2) or RPMI (Jurkat) supplemented with L-glutamine (2 mM), HEPES (20 mM), amphotericin B (0.025 μg/mL), penicillin G (100 IU/mL), streptomycin (100 μg/mL), 10% (v/v) fetal bovine serum FBS and incubated at 37 o C, 5% CO 2 .

2.2.4.2. Procedure for surveying toxic activity using the SRB method

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Single cells were seeded on 96-well culture plates at densities of 104 cells/well (for HeLa, Hep G2 and MCF-7 cell lines); 7.5.103 cells/well (for NCIH460 line) and 5.104 cells/well for Jurkat cell line. After 24 hours of culture, the cell population was incubated with the assay material at various concentrations for 48 hours. Then, total protein from the test cells was fixed with cold 50% Trichloroacetic acid (Sigma) solution (70% for Jurkat) and stained with 0.2% Sulforhodamine B solution (Sigma). The results were read using an ELISA reader at two wavelengths of 492 nm and 620 nm. The experiments were repeated three times and the results are presented as mean values ± standard deviation. Processing of results:

After having optical density values at wavelengths of 492 nm and 620 nm (denoted as OD 492 and OD 620 ):

- Calculate OD value = OD 492 – OD 620 (1)

- Calculate OD 492 (or OD 620 ) = OD tb – OD blank (2)

- Calculate the cytotoxicity rate (%) according to the formula: With: %I = (I - OD TN / OD C ) x 100%

- OD tb : OD value of the well containing cells

- OD blank : OD value of the well (no cells)

- OD TN : OD value of the test sample calculated from formulas (1) and (2)

- OD C : OD value of control sample calculated from formulas (1) and (2)

IC 50 was determined using Prism software with multiparameter nonlinear regression method and R 2 > 0.9.

CHAPTER 3: RESULTS AND DISCUSSION

3.1. Characteristics of porous nano silica (PNS) materials

3.1.1. Survey of PNS particle size by solgel method

Particle size plays an important role in drug delivery systems. After intravenous injection, small carrier particles (10–20 nm) are mostly excreted through the kidneys, while larger particles (≥ 200 nm) are absorbed by the reticuloendothelial system (RES). Only nanoparticles with sizes ranging from 20–100 nm are able to pass through the microcapillaries and are not absorbed or selected by the reticuloendothelial system, resulting in a prolonged residence time of the drug carrier in the circulatory system and an enhanced efficacy of the targeted drug [59].

According to Bogush et al. [60, 61], the five important parameters affecting the particle size distribution are: (i) tetraethyl orthosilicate (TEOs) content, (ii) ethanol, (iii) ammonia, (iv) water and (v) temperature.

To produce porous silica nanoparticles with desired sizes suitable for drug delivery, the effects of TEOs, ammonia and ethanol concentrations were investigated. Cetyltrimethylamonium bromide (CTAB) as a surfactant used to shape the pores was kept constant at 2.6 (g) in all experiments.

(i) TEOs content:

According to Stober et al. [62], there was no effect of TEOs on the size.

seed.

Bogush et al. [63] showed that the particle size increased with increasing TEOs content.

Helden et al. [64] demonstrated the opposite, that when the amount of TEOs increased, the size

reduce.

To clarify the above issue, we conducted a survey of the amount of TEOs, the remaining substances ammonia and ethanol remained unchanged. The result was that when increasing the amount of TEOs from 6 to 10 mL, the size decreased from 92.8 nm to 56.6 nm. But when continuing to increase the amount of TEOs from 10 to 14 mL, the particle size increased from 56.6 to 153.8 nm (see Appendix 1 and Figure 3.1)).

Explain:

Hydrolyzed TEOs condense in an aqueous environment to form spherical silica nanoparticles surrounding CTAB. When the amount of TEOs increases but the amount of CTAB remains constant, CTAB will

Divide evenly to match the amount of TEOs to make the particles smaller because the less core, the smaller the particles. However, if we continue to increase TEOs, new nanoparticles will form around the previous nanoparticles, increasing the size.

Figure 3.1. Effect of TEOs on particle size

(i) Volume of ethanol

According to the study of Kota Sreenivasa Rao et al. [ 19], when the ethanol concentration was reduced from 8(M) to 4(M), the nanoparticle size decreased significantly. Continuing the above study, we investigated the volume of ethanol at NH 3 2.8% and TEOs 2.8 mL. The results showed that when ethanol increased, the particle size increased accordingly, the ethanol volume at 5.8 mL gave the smallest particle size (see Appendix 1 and Figure 3.2).

Figure 3.2. Effect of ethanol on particle size

(ii) Ammonia concentration

The hydrolysis reaction occurs very slowly, using ammonia as a catalyst for the hydrolysis and condensation of TEOs in ethanol. According to Matsoukas and Gulari [65], the size increase is achieved by increasing the concentration of ammonia and water. (see Appendix 1 and Figure 3.3).

Figure 3.3. Effect of ammonia concentration on particle size

From the above survey results, to achieve the desired size from 20-100 nm, we conducted this reaction at 60 o C, a mixture of 64 mL distilled water and 2.6 g cetyltrimethylamnonium bromide (CTAB); 11.25 mL ethanol and 550 µL NH 3 (2.8%), 8 mL tetraethyl orthosilicate (TEOS) stirred at a rotation speed of 300 rpm.

3.1.2. TEM image of porous silica nanomaterials (PNS) synthesized by sol-gel method

Through TEM measurement results, the synthesized PNS has a nano size ranging from 50 nm to 60 nm. Compared with the study, we see that the spherical silica nanoparticles have high uniformity, with such a structure we can conclude that the nano silica with controlled particle shape has been successfully synthesized. Solving the problem of amorphous and controlling the size and shape is one of the difficulties to put this material into practice. With a size of 58.93±2.42; PNS (porous nano silica) synthesized from TEOS by sol-gel is suitable for the size range of drug delivery nanosystems (20÷100 nm) [59].

Figure 3.4. TEM image and particle size distribution of porous nanosilica (PNS)

3.1.3. SEM image analysis results of porous silica nanomaterials (PNS) synthesized by precipitation method.

The process of synthesizing nano silica from rice husks by precipitation method when slowly adding HCl into sodium silicate solution (Na 2 SiO 3 ). Conducting many experiments changing the HCl concentration from 1.5 N; 1.0 N and 0.5 N, it was found that the particle size gradually decreased in the order of 30-60 nm; 25-35 nm and 10-15 nm. This is because with a fixed amount of sodium silicate (Na 2 SiO 3 ), when the HCl concentration decreases, the amount of acid used for precipitation is less, making the reaction occur slower, and the particles become smaller (Figure 3.5).

Figure 3.5. SEM image of porous nano silica (PNS) synthesized from rice husk by precipitation method at HCl concentrations of 1.5 N; 1.0 N and 0.5 N

Comparing the SEM images of porous nano silica (PNS) synthesized from rice husk with previous studies, it can be seen that the synthesized particles have a distinct spherical shape and relatively uniform size [55, 66, 67].

However, the nanoporous silica produced by the sol-gel method has a more uniform and beautiful round shape than the nanoporous silica produced by the precipitation method. Therefore, the nanoporous silica produced by sol-gel was chosen for the modification and drug-carrying processes of the thesis.

3.1.4. XRD analysis results of porous silica nanomaterials (PNS) synthesized by sol-gel method and precipitation method

As we know, X-ray diffraction spectrum provides important information about the shape and structure of materials. To confirm the porous structure of nanoparticles, we conducted wide-angle XRD analysis with scanning angles from 10 o to 80 o for 2 porous silica nanomaterial samples (PNS) synthesized by sol-gel method and precipitation method.

Recording conditions: CuK  x-ray tube with  = 1.54056 Å, voltage 40kV, powder sample, speed 0.005 o /sec.

In figure (3.6 a) is porous nano silica (PNS) synthesized by sol-gel method, we see the characteristic signal of silica at 2θ = 22 o , the peak is still very sharp showing that there is no collapse of the crystal structure of the particle. So the spectrum shape shows that the particle has a distinct structure and is no longer amorphous [68, 69].

In figure (3.6b) is a sample of porous silica nanomaterial (PNS) synthesized by the precipitation method, comparing with the wide-angle diffraction spectrum XRD of porous silica nanomaterial synthesized by the sol-gel method shows similarities. And comparing with other studies is also the same. Thereby, the synthesis of porous silica nanomaterial has been completed by both sol-gel and precipitation methods [67, 70].

Thus, we have created silica nanoparticles with a porous structure. The creation of pores plays a very important role in the drug-carrying ability of the material [71].

Figure 3.6. Comparison of XRD patterns of nanoporous silica synthesized by precipitation method (a) and by sol-gel method (b).

3.1.5. FT-IR results of porous silica nanomaterials (PNS) synthesized by sol-gel method and precipitation method

To determine the functional groups of the material, we analyze the FTIR spectrum and see the oscillations at the following wave numbers:

FTIR of PNS synthesized by Sol-Gel method[72, 73]:

- The stretching and bending vibrations absorbed at wavenumbers 3417 cm -1 and 1615 cm -1 respectively are characteristic of the –OH group.

- The three characteristic absorption peaks at 1093 cm -1 , 813 cm -1 and 466 cm -1 represent the stretching vibration of Si-O-Si.

- The absorption peak at wave number 813 cm -1 is characteristic of the bond vibration of Si-OH (silanol group).

FTIR of PNS synthesized by precipitation method

- The broad absorption band showing stretching vibrations at wavelengths 3436 cm -1 and 1627 cm -1 belongs to the –OH group (silanol, Si-OH) [71, 74].

- Siloxane absorbs strongly in the range 1130 cm -1 – 1000 cm -1 specifically 1067 cm -1 .

And the absorption peak with wavelength at 797 cm -1 is Si-O-Si.

So through the FTIR analysis results and comparison with studies [75, 76], we have initially successfully synthesized porous silica nanomaterials (PNS) by sol-gel method and precipitation method.

Figure 3.7. Comparison of FT-IR spectra of porous nano silica (PNS) synthesized by sol-gel method and by precipitation method

3.1.6. BET results of porous silica nanomaterials (PNS) synthesized by sol-gel method and precipitation method

BET of PNS synthesized by Sol-Gel method

To investigate the surface area as well as predict the structure of the material, BET is one of the powerful methods. From the above adsorption isotherm, we can see that this adsorption isotherm is characteristic of a structured material, which helps us conclude that the material after modification still has a porous structure. Moreover, the particle has a surface area of 129.818 m 2 /g (see Appendix 10), compared to other studies, the surface area of the material is not too large [47].

With a small surface area that limits drug loading, PNS is modified to increase chemical bonding and increase the adsorption of drugs and carriers. Modification may not increase the surface area and pore volume, but if the appropriate modifying agent is selected, chemical bonding will be created, increasing the adsorption when loading drugs, contributing to improving drug loading efficiency.

BET of PNS synthesized by precipitation method

Surveying the BET surface area of the PNS material synthesized by the precipitation method, we have the following results: the surface area is 117.047 m 2 /g, the pore volume is 0.137 cm 3 /g and the pore diameter is 12.037 A o . So the surface area is slightly smaller than the PNS material synthesized by the sol-gel method.

3.2. Modification of nano porous silica

3.2.1. Denaturation via GPTMS bridge

3.2.1.1. Modification by Hydrazine (synthesis of PNS-GPTMS-Hydrazine-drug carrier 1) Size of modified silicate nanoparticles by TEM imaging

According to some studies on the application of nanomaterials in the medical field, it is reported that the cellular uptake efficiency decreases with increasing particle size. Nanoparticles in the range of 100-200 nm have great potential to prolong the circulation time in the blood because they are large enough to avoid selective uptake in the liver, but small enough to be avoided by the filtration mechanism of the spleen. Besides, the small size (20-100 nm) allows the nanoparticles to passively concentrate around tumor cells through the permeation and retention effect, enhancing intracellular accumulation and keeping the nanoparticles in the tumor area [33].

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