VIETNAM NATIONAL UNIVERSITY HCMCUNIVERSITY OF SCIENCE MAI NGOC XUAN DAT SYNTHESIS OF BIODEGRADABLE POROUS SILICA NANOPARTICLES AND THE USE OF OPTICAL TECHNIQUES FOR CHARACTERIZING THE MA
LITERATURE REVIEW ĐẶT he 5 1.1 Nanoparticle-based drug delivery in cancer treafmenf .s sô 5 1.2 Application of biodegradable periodic mesoporous organosilicas as
Biodegradable periodic mesoporous organosilica nanoparticles as
nanocarriers Until now, various types of inorganic porous materials like mesoporous carbon NPs, magnetic colloidal NCs, mesoporous silica-based NPs, mesoporous TiO2 NPs, and others, have been synthesized for fabricating high drug-loading nanomedicines thanks to their essential characteristics, including porous surface, high surface: volume ratio and large hollow structure In particular, drug molecules interact with carrier materials through m—z stacking, hydrogen bond, noncovalent electrostatic, and hydrophobic, leading to high loading content In addition, to increase drug-loading content, drug molecules have been attached to the porous surface of nanocarriers and further ability to stimuli response (Figure 1.3).”Š: ?2 ri: e
Figure 1.3 Modern drug delivery systems response to internal stimuli conditions”?
A new class of hybrid materials, including organic groups in the inorganic framework, have emerged as promising materials thanks to the thermal stability of inorganic substrates and the functional versatility of organics Periodic mesoporous organosilica nanoparticles (PMOs), a new type of inorganic-organic hybrid materials, have attracted more attention as a promising vehicle in drug delivery.!' °°? PMOs have specific properties such as high hydrothermal, well-defined mesopores, large surface areas, mechanical stability, flexible physicochemical properties, and organic moieties in the inorganic frameworks.***° Furthermore, PMOs have more excellent biodegradability and higher hemocompatibility than MSNs.*° ô+ Advantages of periodic mesoporous organosilica nanoparticles
- High organic moiety contents (30-80%) thanks to the size of organic bridges.
- The hydrophobic silsesquioxane hybrid framework leads to higher drug loading capacities without leakage though no pore capping in the materials.
- Owing higher biocompatibility at the nanoscale: lower hemolytic than MSN, long-term blood circulation.
- Have outstanding biodegradability and more hemocompatibility than pure MSNs.
+ +° Synthesis of periodic mesoporous organosilica nanoparticles
MSNs and PMO NPs are generally synthesized through by sol-gel method This process regularly associates the hydrolysis and condensation of silanes and catalysts of basic or acidic solutions.
In PMOs, covalent siloxane bonds (Si—O—Si) and oligomers are formed through two main steps: the hydrolysis of silanes and organosilanes ((Si(OX)4, where X is typically OEt or OMe) and [(XO)3Si]n-R, where R is an organic group, n > 1), respectively) in basic media, followed by the condensation of reactive silanolates species
(=Si—O-) and other (organo)silanes By this time, silica (S1O2) or silsesquioxane (e.g.,
O15Si—R—SiO1.5) frameworks are produced by the sol-gel reaction (Figure 1.4a, b). Some surfactants as hexadecyltrimethylammonium bromide (CTAB), are used to produce mesoporous nanomaterials Briefly, separate CTAB molecules create positively charged micelles when CTAB concentration is above the critical micellar concentration (CMC) As a result, the hydrophobic tails of CTAB in the micelles “hide” from the aqueous solution When increasing the concentration continuously, spherical micelles turn into cylindrical and hexagonal packings with a few hundred nanometers In the aqueous medium, silanes and organosilanes react under a templating effect due to the attraction of positively charged CTAB ((—N*(CHs)3) micellar packings with negatively charged silanes (=Si—O’) MSNs will be formed when using only a silica precursor like tetraethoxysilane (TEOS) (Si(OEt)4) On the other hand, MONs will be created when applying a silane mixture, and an organosilane is used, and further PMOs will be formed while the sole presence of bridged organosilanes (Figure 1.4c).!>
Etể`Si(OEt)ạ ô——> HO-Si(OEt); ——~ 'O-Si(OEt); SiO;
(oEt);si $9 sị(OEt); =— (OEt);SĂ Ếẹệ si(oEt); ~Si- Si0;,5 4B Si0, 5
OEt OE ‘OH OEt OH +EtO' —= O° +EtOH
(oEt;sifẹsĂ(oE); ~(OH);SiỆẹé si(OH); - -
Figure 1.4 The formation of silica and silsesquioxane materials through the hydrolysis and condensation of silanes a) and organosilanes b), respectively The creation of MSNs,
MONs, and PMO NPs by templated sol-gel processes c)!>
Different from normal cells in our body, cancer cells exhibit a nearly 100—1000- fold higher redox capacity Glutathione (GSH) can destabilize the redox-mediated disulfide linkage Interestingly, the level of GSH in the tumor environment is nearly 500- fold higher than that in the blood, which can be applied to release drugs based on internal stimuli-responsive In the presence of the GSH enzyme, the disulfide bond-linked removable shell in the redox-responsive nanoparticle can be shed to cause drug release.*!:
* Diselenium and disulfide bonds, as redox-responsive bonds, are effectively incorporated in redox-responsive delivery systems Glutathione cleaves the disulfide bond, resulting in the formation of sulfhydryl groups followed by the polymer system breakdown, and the drugs immediately release It is noteworthy to mention that PMOs meet the requirements about safety issues in clinical application because of increased excretion and minimized side effects of anticancer drugs.
Recently, a few sulfide-bridged-based PMOs have been utilized as nanocarriers for delivering and improving the anticancer drugs’ clinical performances, including curcumin, camptothecin, daunorubicin, and doxorubicin.** 43-46
Croissant et al synthesized BPMO nanospheres and nanorods by adjusting the ratio between two organosilica precursors, bis(3-triethoxysilylpropyl) disulfide and bis(triethoxysilyDethylene, through co-condensation (Figure 1.5) The particles displayed great compatibility and perfect biodegradability with physicological conditions Furthermore, the localization of the nanoparticles is also demonstrated after
24 h incubation.*” In this study, TEM and confocal microscopy were applied to evaluate morphology, and biodegradability and demonstrate the cellular uptake of the nanoparticles.
EPMO EDIS MPMO EDIS MPMO EDIS MPMO DIS BS
Figure 1.5 Obtained morphology and size of the BPMOs by adjusting the ratio between two organosilica precursors TEM images show various sizes and shapes of synthesized nanoparticles*’
Vu et al investigated the potential of a tetrasulfide-based BPMO for targeted delivery of doxorubicin (DOX) A chicken chorioallantoic membrane assay demonstrated the BPMO's efficacy in eliminating tumors formed by human ovarian cancer cells (OVCAR-8) Notably, DOX-loaded BPMO showed significantly reduced organ damage compared to free DOX, as observed through TEM and fluorescent stereomicroscope, indicating its enhanced delivery specificity and reduced systemic toxicity.
Moghaddam et al synthesized highly uniform redox-responsive polysulfide- based nanoparticles These biodegradable silica materials had a range of different porosity, size, and composition (Figure 1.6).** In these nanoparticles, disulfide or tetrasulfide bonds are incorporated into the structure They also evaluated the degradation and suggested that the mesoporous nanoparticle with the higher surface area can be greater degraded In addition, the cytotoxicity was also evaluated toward murine macrophage cells (RAW264.7) TEM and confocal laser scanning microscopy (CLSM) were also applied in this study.
\ ws s-S bì O 0 $ Ss ot su SẼ gs ` /
Tetraethyl orthosilicate (TEOS) Bis[3-(triethoxysilyl)propyl] disulfide (BTESPD) | Bis[3-(triethoxysilyl)propyl] tetrasulfide (BTESPT)
Figure 1.6 Degradable nanoparticles are synthesized by Moghaddam et al. Transmission electron microscope images of obtained nanoparticles synthesized by various combinations of inorganosilica precursor and organosilica precursors
Biodegradable mesoporous organosilica nanosheets were synthesized by Chen et al for the chemotherapy/mild thermotherapy of cancer The nanoparticles showed high cellular uptake and high drug loading with fast internalization.” In particular, polyethylene glycol-grafted copper monosulfide-coated mesoporous organosilica nanocapsules (PCMON) showed more effectiveness toward the uptake of doxorubicin compared to silica-based materials In addition, PCMON displayed excellent degradation They concluded that PCMONs could accumulate in the cancer cells and
15 then destroy the tumor cells based on triple stimulus-response including pH, GSH concentration, and laser irradiation) FT-IR, SEM, TEM, and CLSM were effectively utilized in this report.
Wu et al incorporated biodegradable disulfide bonds in hollow mesoporous organosilica nanocapsule (HMONs) framework.°° The biodegradation of HMON was evaluated on phosphate-buffered saline (PBS) solution with an addition of GSH (10 mM) TEM images showed a slight change after three days and a distinct structure collapse of HMONs after seven days (Figure 1.7) The significant acceleration of biodegradation is obtained after two weeks under reductive GSH-containing PBS The results demonstrated the high biodegradability of HMONs resulting from disulfide bonds in the framework In this study, FT-IR, DLS, TEM, and CLSM have been used for characterizing physicochemical and evaluating the cellular uptake and cytotoxicity of HMON:.
In addition, many reports can be found for synthesizing biodegradable periodic mesoporous organosilica nanoparticles, which is based on responsive to different conditions, including pH, enzyme, light, or ultrasound.>!>°
In Vietnam, few reports can be found for synthesizing biodegradable periodic mesoporous organosilica nanoparticles However, they almost focused on developed nanocarriers based on inorganic mesoporous silica nanoparticles like MCM-41.
TEM analysis revealed the progressive biodegradation of HMONs in PBS containing 10 mM GSH over 14 days Dynamic light scattering and mass spectrometry measurements confirmed the degradation process, with decreased particle size and silicon content observed A reaction scheme elucidated the degradation and biodegradation mechanisms, involving hydrolysis, glutathione conjugation, and subsequent degradation by enzymes.
Effective anticancer drugs in cancer freatmehI . ‹ô ôô++sx+sx++ 17 1.3 Efficient optical methods in the study of nanocarriers in drug delivery
Daunorubicin (DNR), an effective anticancer agent, faces limitations due to adverse effects such as cardiotoxicity, myelosuppression, and oral ulcers To overcome these challenges, researchers are exploring the development of novel nanoparticles that can encapsulate DNR, allowing for targeted and reduced toxicity This approach aims to enhance the antitumor efficacy of DNR while minimizing its harmful side effects in chemotherapy.
Figure 1.8 Structure of daunorubicin anticancer drug
Recently, the liposomal formulation has been developed and applied in AML therapy to deliver cytarabine and daunorubicin.°' Some liposomal formulations of daunorubicin have also been created to treat many cancers like glioma and non-small cell lung cancer.®” °' Admad et al synthesized surface-coated natural biodegradable macromolecule chitosan-based poly(lactic-co-glycolic acid) polymeric nanoparticles to enhance DNR oral bioavailability.©° Mohanta et al coated PEG-400 on the surface of iron oxide nanoparticles and then evaluated the DNR release profiles The results showed that the functionalized nanoparticles displayed pH-responsive drug release at low pH with a high drug-released amount Mesoporous core-shell silica nanoparticle was designed by coating polyethylenimine (PEI) on core-shell Fe304@SiOz and surface modified with a zwitterionic 2-methacryloyloxyethyl phosphorylcholine (MPC). Daunorubicin and siRNA were contemporarily loaded on this nanodevice They also demonstrated the increased cytotoxicity of daunorubicin in an ovarian cancer cell line
Cordycepin has been known as a nucleoside analog compound It shows the primary role in the pharmacologic effects of Cordyceps fungi (Figure 1.9) Cordycepin exhibits a
18 series of pharmacological properties, including anti-inflammatory, immunoregulatory,” antiviral,°* antifungal,” and notably antitumor activity”
Figure 1.9 Cordyceps fungi and cordycepin structure’!
Cordycepin exhibits significant antiproliferative activity against various cancer cell lines, including oral, liver, prostate, breast, and lung cancer cells However, its therapeutic application is hindered by its rapid deamination in vivo, resulting in the formation of an inactive metabolite and limited circulation due to its poor water solubility Additionally, its negative charge prevents diffusion across cell membranes To overcome these drawbacks, the development of nanocarriers is crucial to enhance the clinical efficacy of cordycepin.
Armwit et al studied to incorporate of cordycepin in two types of gelatin nanoparticles, type A (GA) and type B (GB), and then evaluated their sustained release profiles and compared the anti-proliferative and anti-migratory effects on A549 lung cancer cells.”” Marslin et al prepared cordycepin-loaded poly(lactic-co-glycolic acid)
Liposomes, such as cordycepin-loaded polymeric nanoparticles (CPNPs), have been explored to enhance the therapeutic efficacy of cordycepin CPNPs have been shown to improve cellular uptake, reduce hemolytic potential, and enhance cytotoxicity compared to free cordycepin In addition, liposome encapsulation techniques have been employed to increase the anti-colon cancer activity of cordycepin, demonstrating high encapsulation efficiency and effective treatment of HT-29 colon cancer cells.
1.3 Efficient optical methods in the study of nanocarriers in drug delivery
As mentioned above, BPMOs have been known as promising candidates for drug delivery and disease targeting Until now, many BPMOs-based drug delivery systems have been reported based on the redox conditions for biodegradation.*> Characterization of those formulations, especially the investigation of nanostructure during formulation, stability assessment, and biological transport through the body, remains an important aspect for the success of this drug delivery approach.°*°
To characterize these specific properties, advanced modern characterization techniques are usefully applied, especially optical techniques Two basic methods, including direct and indirect methods, are extensively used to determine the physicochemical of nanoparticles, such as morphology, composition, particle size, and particle size distribution The indirect method employs the relationship between particle size and its behavior On the other hand, the direct method investigates the particle and determines their actual dimensions.
Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) are the direct methods for evaluating particle size and morphology In addition, the size distribution or the surface charge of nanoparticles can be determined using dynamic light scattering (DLS) spectroscopy The nanoparticles’ elemental composition can be determined by energy dispersive spectroscopy (EDS) and/or wavelength
20 dispersive spectroscopy (WDS) The functional groups attached to the surface of the nanoparticles can be investigated through FTIR spectroscopy is used to investigate.*’ XPS to investigate the surface chemistry of materials and confocal laser scanning microscopy (CLSM) for monitoring the biodistribution of particles in biological models (Figure 1.10). ằ Scanning electron microscopy (SEM)
| Morphology and sizes [ˆ -., Transmission electron microscopy (TEM)
Optical techniques in Physicochemical characterizations x Fourier transform infrared spectroscopy (FT-IR)
Figure 1.10 Efficient optical methods applied in the study of nanocarriers in drug delivery
Fourier transform infrared spectrOSCOpy s:cesseceeseeseeeeeereeseeneeeseeeeeees 21 1.3.2 X-ray photoelectron spectrOSCOpy - Gv ng 23 1.3.3 Dynamic light MicrOSCOPY ceecesceeeseeeseessceceseceseesscecseeceaeeseeeeessaeesaes 24 1.3.4 Electron mICTOSCODY SH tt 25 1.3.5 Confocal laser scanning MICTOSCOPE - .- 56 1x +skESsekssseeskee 28 1.4 Biological models for evaluation of the cytotoxicity of nanoparticles
Fourier transform infrared spectroscopy (FTIR) is known as one of the most widely used methods to detect functional groups in materials This method relates to atoms or groups of atoms’ vibrational energy inside a material And the FTIR spectrum is seen as the molecule’s fingerprint due to the specific infrared spectrum of every compound.Š?
FTIR (Fourier transform infrared spectroscopy) is a vital technique for characterizing mesoporous organosilica nanomaterials, as evidenced by its extensive use in research studies For instance, Huang et al successfully synthesized functionalized MSNs (mesoporous silica nanoparticles) using a one-pot sol-gel reaction facilitated by a dual-templating micelle system To verify the composition of their synthesized MSNs, they employed FTIR analysis.
21 presence of vinyl in the mesoporous silica nanoparticles (MSNs) and DOX-conjugated polymer-grafted MSNs (Figure 1.10).8° He et al synthesized a mesoporous silica- calcium phosphate hybrid nanocarrier which can be degraded by pH FTIR was applied to determine chemical bonds and actual inorganic substances inside NPs, including phosphonate groups' the characteristic asymmetric stretching vibration.*° Nguyen-Thi et al synthesized and compared porous silica nanosilica and hollow mesoporous silica nanoparticles for Rhodamine loading In that study, the authors demonstrated the successful synthesis of two materials by means of FTIR.*? Gou et al doped strontium ion in MSNs to improve its specific surface area, cytocompatibility, and in vitro degradability The Si-O vibration band of amorphous materials was determined by
— DOX conjugated polymer grafted MSNs
Figure 1.11 Huang’s group performed FT-IR to confirm the presence of vinyl in the mesoporous silica nanoparticles and DOX-conjugated polymer grafted MSNs*®
X-ray photoelectron spectroscopy (XPS) has been known as a surface-sensitive analytical tool for examining the chemical compositions, electronic state, chemical state, and empirical formula of the surface of materials.Š
Many studies used X-ray photoelectron spectroscopy (XPS) to investigate the surface chemistry of silica materials Beaux II et al investigated the surface changes of silica nanowires by XPS after storing them in various pH solutions.?! Mekaru et al used
XPS to demonstrate the reduction of the disulfide bonds to thiol groups of biodegradability of disulfide-organosilica nanoparticles (Figure 1.11).? Karunakaran et al characterized the electronic states of metal species and organic of Ti-tailored silica particles by means of XPS The successful incorporation of Ti inside the mesoporous organosilica powder samples was demonstrated through the Ti XPS spectrum.” Picchetti et al used XPS to indicate the presence of the photolabile linker integrated into the light- responsive mesoporous organosilica particles.”
C-SO, S-S S-CS2p;/; S-H \ \99 foe O-C=O O-C \os oe Si-O Si-C
S2p— on C1s | i Phi O1is ~~N Aa Si 2p Nj¿
Binding Energy (eV) Binding Energy (eV) Binding Energy (eV) Binding Energy (eV)
Figure 1.12 Mekaru et al reported the reduction of the disulfide bonds to thiol groups of biodegradability of disulfide-organosilica nanoparticles by means of XPS including
Dynamic light scattering (DLS) or photon correlation spectroscopy (PCS) is a standard method to determine the particle size distribution of a powder sample We also have information relating to particle size and aggregates.*”
Many researchers applied dynamic light microscopy (DLS) to demonstrate the consistency between DLS with scanning electron microscopy (SEM) or transmission electron microscopy measurements (TEM) Gu et al showed the similarity in mean hydrodynamic diameter by DLS and TEM of the polyethylene glycol-luminescent nanoparticles made of porous silicon formulation.®> Sully et al used DLS to investigate the average size of the pristine and FTIC-loaded silica nanoparticles DLS is also applied for determining the surface charge of these nanoparticles in this research.?5 Zyuzin et al. developed silica-based biodegradable submicrometric particles, and their particle size is investigated by means of DLS.’ Qian et al synthesized biodegradable mesoporous silica
24 with incorporated carbon nanodots in the silica framework applied in debris-mediated photothermal synergistic immunotherapy The biodegradation of particles was conducted in simulated physiological solutions, and evaluated the products by different measurements including DLS.”*
Electron microscopy enables the characterization of nanomaterial morphology, with SEM and TEM as two widely used techniques SEM offers advantages such as substantial depth of focus, wide field-of-view, and 3D topographic imaging capabilities It is often coupled with energy-dispersive X-ray (EDX) spectroscopy for elemental composition analysis, providing a comprehensive understanding of the nanomaterial's structure and composition.
Most publications used scanning electron microscopy (SEM) to investigate the morphology of synthesized nanoparticles Croissant et al synthesized biodegradable oxamide-phenylene-based mesoporous organosilica nanoparticles with a remarkably high organic content They used SEM imaging to confirm the monodisperse spherical
100 nm particles.°* Maggini et al also applied SEM to determine the morphological characterization of disulfide-doped silica nanoparticles (Figure 1.12) SEM results displayed the homogeneous spherical morphology of obtained materials with an average diameter of 88.9 + 10.9 nm.”? Zyuzin et al developed hollow silica-based biodegradable submicrometric carriers for loading various bioactive molecules like proteins, nucleic acids, and dextran Their structures were also characterized using SEM (Figure 1.13).”
Sully et al synthesized biodegradable FITC-doped silica nanoparticles and characterized the shape and size of the fabricated nanoparticles using SEM.” It is clear that SEM is a
25 powerful method to evaluate the morphology of nanomaterials and thus is utilized to perform characterization of PMO nanoparticles in this study.
Figure 1.13 Morphology of disulfide-doped silica nanoparticles was reported by
Figure 1.14 SEM images of submicrometric capsules synthesized by Zyuzin et al., including SiOz capsules with (A) low and (B) high amounts of tetraethyl orthosilicate,
(C) (DEXS/PARG), and (PSS/PAH), capsules”
The nanocrystallites, nanoparticles size, and the interfaces in nanocomposites can be determined directly via TEM Through TEM analysis, we can have information on the size distribution, particle size, and morphology of the nanoparticles Especially the individual particles are directly observed and measured using TEM The dispersion or
26 aggregation degree of the nanoparticles can also be determined via TEM However, this technique requires complicated sample preparation.Š”
In developing drug delivery systems, transmission electron microscopy (TEM) is considered an indispensable method to explore the possible alterations in the morphology of the nanoparticles Many authors reported well-defined SEM images for the physical characterization of biological and biomaterial samples and especially biodegradability evaluation of NPs Biodegradable oxamide-phenylene-based mesoporous organosilica nanoparticles were reported by Croissant et al They used the TEM method to confirm the monodisperse spherical 100 nm particles In addition, TEM is also performed to analyze the degradation of hollow nanoparticles in phosphate- buffered saline (PBS) with the addition of trypsin (Figure 1.14).>4
Figure 1.15 Croissant et al applied TEM images to evaluate the degradation of oxamide-phenylene-based mesoporous organosilica nanoparticles in PBS with trypsin at various times ((B) 24 h and (C) 48 h)**
Maggini et al synthesized disulfide-doped silica nanoparticles and performed TEM to check the morphology of homogeneous spherical nanoparticles Similarly, TEM results showed a structural breakdown of disulfide-based nanoparticles in the reducing solution.”? Sully et al reported biodegradable FITC-doped silica nanoparticles and performed TEM to identify the size and shape of the fabricated nanoparticles (Figure
1.15) All the reports indicated that TEM is extensive and effectively utilized for characterizing the morphology and further evaluating the biodegradability of nanoparticles, especially biodegradable nanocarriers like periodic mesoporous organosilica.
Figure 1.16 TEM images of (a) pristine, (b) FITC-doped silica nanoparticles, and (c-f) individual anticancer drugs silica nanoparticles were reported by Sully et al.?
There are several reports of the application of a confocal laser scanning microscope (CLSM) in drug delivery studies Zyuzin et al developed the silica-based biodegradable submicrometric particles and investigated the cellular uptake of particles by HeLa cells and the transfection efficiency T of Hela cells by particles by means of CLSM (Figure
1.16) ”” Lyles et al have studied biodegradable silica-based nanoparticles to deliver
Spheroids - Jn vitro three-dimensional (3D) cell model 30 1.4.2 Tumor-bearing chicken embryo model - ô+ sôÊ+sÊ++eexsseeesees 32
Currently, in vitro cytotoxicity tests for anticancer drugs are mainly conducted by evaluating tumor cell reactions using a 2D monolayer model Because of its advantages,
2D in vitro models often precede in vivo testing for drug evaluation Despite providing valuable insights, 2D models exhibit limitations They lack the intricate 3D tissue architecture present in vivo, which restricts interactions between cells and the extracellular matrix (ECM) This architectural disparity leads to discrepancies in drug responses observed in 2D models compared to clinical trials in cancer patients Consequently, 2D models fail to fully replicate the pathophysiology of tumor cells and accurately predict drug efficacy in vivo or in patients.
On the other hand, 3D tumor cell culture techniques have been expanded In this model, the culture environment fully exhibits the characteristic of the ECM and spatial
30 organization of the cancer cell As a result, a biomimetic 3D multicellular tumor model is restructured as an amalgamation of the 2D in vitro and the in vivo animal models. Compared to in vivo models, the similar physiological properties of tumor cells in 3D models makes it become a powerful tool for researching tumor and related anticancer drug.!2
3D cell cultures are extensively utilized in the examination of biological properties, e.g., cancer cells, cell diffractions, or intracellular interactions, and the investigation of toxicity and efficacy of materials, substances, or drugs Compared to 2D cultured cells, cells in 3D models show higher tissue-specific marker levels, retrieve tissue-specific functions as well as express a variety of gene profiles.
The results of comparison between 2D and 3D models of human breast cancer cells (MCF-7) indicated the reduced expression of the mesenchymal marker vimentin and basal marker keratin 14 and the increased mRNA expression of the luminal epithelial markers keratin 8 and keratin 19 of cells in 3D systems In 3D spheroids, external agents or substances can penetrate through permeability barriers The comparison of 2D and 3D culture techniques is shown in Table 1.1.
Table 1.1 Differences between two cell culture models !%
2D 3D e Limiting cell-cell contact e Dominating cell-cell contact e Dominating cell-flat and _ plastic e surface contact e Contact with ECM only on one e Cells remain in contact with ECM surface e No gradient e Diffusion gradient of nutrient, waste, oxygen and drugs e A microenvironment does not obtain s® Mimic microenvironment obtained via co-culture e No resistance for anticancer drug e Resistant to anticancer drugs (mimic tumor morphology)
Figure 1.19 Spheroids formation on human breast cancer cell (MCF-7)!
Spheroids are formed by the aggregation and self-assembling cells to avoid attachment with a flat surface in an environment (Figure 1.18).!°4 It can be done thanks to extracellular matrix proteins and membrane proteins A series of methods are applied to create spheroids, such as bioprinting, spheroids-based co-cultures, microfluidic systems, magnetic levitation method, cell suspension with the addition of nanofibers, rotary cell cultures, hanging drop, and hydrogels !3
1.4.2 Tumor-bearing chicken embryo model
Chick embryo models, as an alternative to murine cancer models, are gaining prominence for cancer research and microsurgical advancements The chicken egg chorioallantoic membrane (CAM) serves as the site for tumor transplantation, offering advantages over murine models One notable superiority is the incomplete development of the lymphoid system in chick embryos, resulting in a natural immune deficiency This deficiency enhances the survival and growth of transplanted tumors, providing a valuable tool for studying tumor behavior and treatment strategies.
Xenotransplantation, the transfer of cells or tissues between different species, has been advanced through the use of chicken embryo assays These assays have enabled the study of early embryo development (32 embryo), overcoming species-specific limitations Additionally, the abundant blood vessel network in chicken embryo assays allows for the formation of primary tumors and large blood vessels, facilitating research in these areas.
Another important aspect is the legal or ethical concerns when using in vivo animal experiments The CAM model system does not violate the above categories and could replace in vivo animal models.
The CAM model is also cheaper and maintained easily with the formation of an excellent for evaluating biodistribution and cytotoxicity of labeled agents and compounds (Figure 1.19).37, 105
Pre-incubation Depositcells Sacrifice Eggs Êf————————ơ———————t| ‡
Figure 1.20 The experiment timeline of the CAM model using chicken embryo*’
Table 2.1 listed chemicals used in the experiment section Milli-Q water was used for prepare all solutions.
Table 2.1 Chemicals used for experiments
Chemicals Purity Company e Cetyltrimethylammonium bromide (CTAB) > 98% Sigma-Aldrich e 1,2-Bis(triethoxysilyl)ethane 96% Sigma-Aldrich e Bis[3-(triethoxysilyl) propyl] tetrasulfide = 90% Sigma-Aldrich e Bis(triethoxysilyl)benzene 96% Sigma-Aldrich e Tetraethyl orthosilicate (TEOS) 98% Acros e 3-(Trihydroxysilyl)propyl methylphosphonate 50% Sigma-Aldrich e N-(2-aminoethyl)-3-
Sigma-Aldrich aminopropyltrimethoxysilane 297.0% e Rhodamine B isothiocyanate (RBITC) Sigma-Aldrich e Glutathione (GSH) >97.0% Sigma-Aldrich e Cordycepin (3’-deoxyadenosine) 98% Alfa Aesar e Daunorubicin hydrochloride > 90% Sigma-Aldrich e Ammonium nitrate 99% Wako e 3-(4,5-dimethylthiazol-2-yl)-2,5- Roche diphenyltetrazolium bromide (MTT) = 90% Diagnostics e Phosphate-buffered saline (PBS) Sigma-Aldrich e Ethanol >99.5% Fisher Scientific
Synthesis of biodegradable periodic mesoporous organosilica nanoparticles 35 1 Synthesis of ethane-containing tetrasulfide-based biodegradable periodic
2.2.1 Synthesis of ethane-containing tetrasulfide-based biodegradable periodic mesoporous organosilica nanoparticles (E4S)
Mix CTAB (250 mg) + water (120 mL) + NaOH 1M and heat to 80 °C
Add dropwise 1,2-bis(triethoxysilyl)ethane (300 pL) Ỷ
Add dropwise bis[3-(triethoxysilyl) propyl] tetrasulfide (100 pL) v
Add 3-(trihydroxysilyl) propyl methylphosphonate (315 uL)
Keep condensation for two hours at 80 °C Ỳ
Collect by centrifugation (16 000 rpm/30 minutes) and washed twice with EtOH Ỷ
Reflux overnight with an alcoholic ammonium nitrate solution nt
Collect by centrifugation (16 000 rpm/30 minutes) and wash with EtOH, water, and then EtOH
Ethane-containing tetrasulfide- based BPMOs
Figure 2.1 Procedure for synthesis of ethane-containing tetrasulfide-based biodegradable periodic mesoporous organosilica nanoparticles
Briefly, CTAB (250 mg), water (120 mL), and 1 M NaOH (1200 pL) were mixed and heated to 80 °C The 1,2-bis(triethoxysilyl)ethane (300 uL) was added and followed by
35 the immediate addition of bis[3-(triethoxysilyl) propyl] tetrasulfide (100 uL) After 15 minutes, 315 HL of 3-(trihydroxysilyl) propyl methylphosphonate was added to negatively charge the surface of NPs The condensation was complete at 80 °C for two hours Nanoparticles were collected by centrifugation (16 000 rpm/30 minutes) and washed twice with EtOH The residual CTAB inside the materials was eliminated by reflux overnight with an alcoholic ammonium nitrate solution The post-synthesized materials were purified by washing with EtOH, water, and then EtOH, and the final product dried at 60 °C (Figure 2.1).
2.2.2 Synthesis of fluorescent ethane-containing tetrasulfide-based biodegradable periodic mesoporous organosilica nanoparticles (RITC-E4S)
The synthesis of RITC-E4S was slightly modified from a reported procedure.*’ In particular, RBITC was attached to the framework by co-synthesis method: 2.5 mg of
RBITC was dissolved in ethanol (EtOH, 5 mL), and then 3-aminotriethoxysilane
(APTES, 6 uL, 2.6x10 mmol) was added After stirring this solution for 30 minutes,
1,2-bis(triethoxysilyl)ethane (300 wL, 0.8 mmol) was added for further 5 minutes. Simultaneously, a mixture of CTAB (250 mg, 0.7 mmol), distilled water (120 ml), and NaOH (8M, 219 uL) were stirred vigorously and heated to 80°C Once the temperature of the CTAB solution reached stability, the silane-containing solution was added dropwise into the flask Then bis[3-(triethoxysilyl) propyl] tetrasulfide (100 uL, 0.2 mmol) was added immediately To modify the surface of the nanoparticle, 3- (trihydroxysilyl) propyl methyl phosphonate (2 M, 315 HL) was added to the solution after 15 minutes (zeta potential -31.27 mV) In the case of positively charged E4S, 120 uL of N-(2-aminoethyl)-3-aminopropyltrimethoxysilane was added instead of phosphonate compound to get a 22.53 mV zeta potential value After condensation at 80 °C for 2 hours, the PMO material was recovered by centrifugation (30 min at 14k rpm) and washed twice with EtOH To remove CTAB from the pores, the particles were refluxed overnight in ethanolic solution of ammonium nitrate (0.3 g in 50 mL) The
36 particles were then purified by washing with EtOH (3 times), followed by desiccation. The material was stored at room temperature for further characterization and activity evaluation (Figure 2.2).
Mixture A: mix RBITC + EtOH + APTES + 1,2-
Bis(triethoxysilyl) ethane and stir at RT Mixture B: Mix CTAB + water + NaOH 8M and heat to 80 °C
| Add dropwise mixture A into mixture B and keep stirring at 80 °C |
| Keep condensation for two hours at 80 °C | ¥
Collect by centrifugation (16 000 rpm/30 minutes) and wash twice with EtOH | yv ¥
Reflux overnight in NH,NO,/EtOH (0,3 mg/ 50 mL)
Collect by centrifugation (16 000 rpm/30 minutes) and wash with EtOH, water, and then EtOH
Fluorescent ethane-containing tetrasulfide-based BPMOs
Figure 2.2 Experimental procedure for the synthesis of fluorescent ethane-containing tetrasulfide-based biodegradable periodic mesoporous organosilica nanoparticles
2.2.3 Synthesis of phenylene-containing tetrasulfide-based biodegradable periodic mesoporous organosilica nanoparticles (P4S)
The primary aqueous mixture was obtained by continuously stirring CTAB (250 mg) and | M NaOH (1752 uL) in water (120 mL) The temperature of the mixture was held at 80 °C, and 1,4-Bis(triethoxysilyl)benzene (300 HL) was added, followed by the immediate addition of bis[3-(triethoxysilyl) propyl] tetrasulfide (100 pL) After 15 minutes, 315 HL of 3-(trihydroxysilyl) propyl methylphosphonate was added to negatively charge the surface of the P4S NPs The condensation was conducted within two hours, and the pre-synthesized product was collected by centrifugation (16 000 rpm/30 minutes) The synthesized NPs were refluxed overnight to remove CTAB residue with an alcoholic ammonium nitrate solution (dissolved 300 mg of NH4aNO3 in 50 mL of ethanol) Finally, the post-synthesized nanoparticles were washed with EtOH, water, and then EtOH and dried by desiccation at 60 °C (Figure 2.3).
Mix CTAB (250 mg) + water (120 mL) + NaOH 1M (1752 uL) and heat to 80 °C j
Add dropwise 1,4-Bis(triethoxysilyl)benzene (300 uL) ¥
Add dropwise bis[3-(triethoxysilyl) propyl] tetrasulfide (100 uL) y
Add 3-(trihydroxysilyl) propyl methylphosphonate (315 uL ) Ỷ
Keep condensation for two hours at 80 °C v
Collect by centrifugation (16 000 rpm/30 minutes) and washed twice with EtOH Ỷ
Reflux overnight with an alcoholic ammonium nitrate solution
Figure 2.3 Experimental procedure for the synthesis of phenylene-containing tetrasulfide-based biodegradable periodic mesoporous organosilica nanoparticles
2.2.4 Synthesis of inorganic mesoporous silica nanoparticle
The inorganic mesoporous silica nanoparticle, MSN, was synthesized based on our previous report with slight modifications An aqueous mixture was obtained by a continuous stirring of CTAB (250 mg) and 1 M NaOH (1752 pL) in 120 mL of water and heated to 80 °C Next, 1250 uL of TEOS was added dropwise, and after 15 minutes,
315 uL of 3-(trihydroxysilyl) propyl methylphosphonate was added for surface modification of the nanoparticles After two hours of condensation, the nanoparticles
Following centrifugation at 16,000 rpm for 30 minutes, the materials were washed twice with EtOH To remove CTAB, the materials were refluxed overnight in an alcoholic hydrochloric acid solution The purification process involved subsequent washing steps with EtOH, water, and finally EtOH The materials were then dried at 60 °C.
Mix CTAB (250 mg) + water (120 mL) + NaOH 1M (1752 pL) and heat to 80 °C
Add dropwise Tetraethyl orthsilicate (1250 HL)
| Add 3-(trihydroxysilyl) propyl methylphosphonate (315 uL) |
Keep condensation for two hours at 80 °C
| Collect by centrifugation (16 000 rpm/30 minutes) and washed twice with EtOH Y
| Reflux overnight with an alcoholic hydrochloric acid solution | v
Collect by centrifugation (16 000 rpm/30 minutes) and wash with EtOH, water, and then EtOH
Figure 2.4 Experimental procedure for the synthesis of inorganic mesoporous silica nanoparticles
Physicochemical characterization by optical techniques sôô 40 2.4 Degradation and drug loading behavior experiments .scscscssssssseseees 42
A S4800 Hitachi machine and a JEOL JSM-75FCT 200 instrument are used to record scanning electron microscope (SEM) images.
A JEOL JEM-2100F machine is utilized to obtain transmission electron microscope (TEM) images.
A Zetasizer 1 V Malvern apparatus is applied for DLS analysis.
A Quantachrome Autosorb iQ2 is used to analyze the nitrogen adsorption isotherms Ultrahigh purity He and N2 are supplied, and liquid nitrogen is used to maintain the sample at 77 K.
Low-pressure Na adsorption measurements were carried out on the Micromeritics volumetric gas adsorption analyser (3-FLEX Surface Characterization) A liquid nitrogen bath was used for measurements at 77 K Helium was used as an estimation of dead space Ultrahigh-purity-grade ẹ› and He (99.999% purity) were used throughout adsorption experiments.
To assess the thermal stability of materials, a TA Instrument (Q-500) was employed This instrument measures thermal stability under a steady airflow, with a heating rate of 5 °C per minute The temperature range evaluated spanned from room temperature to 800 °C.
Fourier transform infrared (FT-IR) spectra were observed by a Bruker Vertex 70 FT-IR spectrometer.
The chemical states of materials were examined using X-ray photoelectron spectroscopy (XPS, AXIS Supra, Kratos) with monochromatic Al Ka-1486.6 eV, pass energy of 20.0 eV and 0.1 eV/step All the spectra were calibrated using C-C bond (C 1s
Confocal laser microscopy images were observed on a Nikon AIR confocal laser microscope.
2.4 Degradation and drug loading behavior experiments
The in vitro degradation behavior of nanoparticles was assessed in both PBS and SBF solutions Specifically, pristine nanoparticles were dispersed into two kinds of PBS solutions: pure PBS (pH 7.4) and reducing PBS (pH 7.4, 10 mM) at 0.1 mg.mL"! The higher GSH concentration in the cytosol (2-10 mM) than in the extracellular condition
(2-10 uM) generates a reducing intracellular microenvironment.!°° The degradation behavior of E4S in SBF solution was also evaluated under identical experimental conditions to that above at a concentration of 0.1 mg.mL"! All the solutions were mixed at 37 °C under magnetic stirring Small fractions of degraded medium were sampled for TEM observations at a given period Jn vitro degradation profiles and microstructure changes in nanoparticles were assessed by TEM (Figure 2.5).
Disperse NPs (1 mg) in 10 mL of PBS or
Figure 2.5 Experimental procedure for evaluating the degradation of nanoparticles in various conditions
Daunorubicin (DNR) was chosen as an anti-cancer drug in the experiments 3 mg of E4S-250 NPs were added into 0.1 M NaHCO3 aqueous solution (400 uL) and stirred overnight at 4 °C in a cold room Activated E4S-250 NPs were centrifuged at 14000 rpm for 30 minutes and then suspended in 355 uL of Milli-Q water before adding 45 uL of
DNR solution (10 mg.mL!) The suspension was stirred overnight at 4 °C DNR-loaded
EAS NPs were then collected by centrifugation (14000 rpm for 30 min), washed with Milli-Q water, and stored for further experiments The supernatant was transferred to an Eppendorf tube and filled to 1 mL with water for fluorescence measurement The loading efficiency was analysed by fluorescence measurements of the supernatants to determine the mass of encapsulated DNR A calibration curve of free DNR was prepared under the same conditions to determine the drug encapsulation rate Measurements were taken at
480 nm (excitation) and 560 nm (emission) (Figure 2.6) The calculated drug loading capacity was 12.04 wt%.
Disperse E4S (10 mg) in 0.1 NaHCO, (400 pL) y
Centrifuge at 14000 rpm for 30 min and suspend in water (355 pL) | Ỷ
Centrifuge at 14000 rpm for 30 min
Figure 2.6 Experimental procedure for daunorubicin loading
The loading of cordycepin was measured by an adsorption equilibrium method.!°’
The mixture of nanomaterials (10 mg) was stirred in a cordycepin solution for 24 hours at room temperature The influence of cordycepin on loading capacity was evaluated using varying concentrations of cordycepin (1-10 mg.mL*!) The mixture was collected by centrifuging after loading (16 000 rpm/30 minutes) Cordycepin-loaded NPs were dried and stored for further experiments while the supernatant was separated and checked the concentration (Figure 2.7) Unloaded-cordycepin was checked by high-performance
44 liquid chromatography (HPLC) analysis using an Agilent Technologies 1200 series HPLC The equipment was connected with a ZORBAX SB-C18 (Agilent, 5 um, 4.6 x
250 mm) column and UV detector to measure cordycepin at 260 nm The mobile phase was water:acetonitrile (90:10 v/v) at a flow rate of 1.0 mL.min' with an injection volume of 5 uL The separation was performed at 30 °C, and the following formula was used for calculating the loading capacity of NPs.
Loading capacity (mg.gTM!) = (mo - m;)/mnps (1)
Here mo (mg) is the initial mass of cordycepin added into NPs, m; (mg) is the residual mass of cordycepin in the loading solution, and mnps (g) is the mass of NPs.
| Centrifuge at 16000 rpm for 30 min | Ỷ
Analyze supernatants by HPLC (UV detector at 260 nm)
Figure 2.7 Experimental procedure for cordycepin loading
A dialysis bag diffusion technique was applied to evaluate drug release profiles.'%
The loaded materials were evenly dispersed in solutions of pH 5.5 and 7.4 The mixtures were transferred into dialysis bags and then shaken in the respective solutions (30 mL) at 37 °C The solution was withdrawn at the predetermined time intervals for HPLC
45 analysis Free cordycepin was evaluated with the same procedure as a control (Figure 2.8).
Disperse Cordycepin-loaded materials (3 mg) in
Sonicate and transfer to dialysis bag
Withdraw solution at time intervals
Analysis HPLC (UV detector at 260 nm)
Figure 2.8 Experimental procedure for evaluating the in vitro release profile of cordycepin from nanoparticles
2.5 Cytotoxicity of materials in biological models
The human cell lines AGS (gastric carcinoma), 293T (embryonic kidney), and A549 (lung carcinoma) were acquired from the American Type Culture Collection and the Center of Molecular Biomedicine, respectively A549 and 293T cells were cultured in DMEM medium, whereas AGS cells were maintained in RPMI 1640 medium (Sigma-Aldrich) Supplementation of all culture media was crucial for optimal cell growth.
46 with 10% fetal bovine serum and 1% penicillin-streptomycin, and cells were cultured in a humidified incubator containing 5% CO; at 37 °C.
The viability of control and treated cells were evaluated using Cell Counting Kit-
8 (CCK-8, Sigma-Aldrich, St Louis, MO, USA) in triplicate Briefly, cells were seeded in 96-well plates at a density of 3 x 10° cells per well and treated with different concentrations of test compounds (E4S or Cordycepin-loaded E4S (Cor@E4S) nanoparticles at 0, 5, 10, 20, 40, and 80 ug mL!, Cordycepin at 0, 5, 10, 20, 40, and 80 ug mL!) After a 24-hour incubation, cells were washed, refreshed in complete media, and further incubation for 48 h CCK-8 solution was added to the well and incubated for another 2 h The absorbance was measured using a microplate reader IRE96 (SFRI, France) at 450 nm (Figure 2.9) The absorbance of untreated cells (the control) was considered as 100% Cell viability ratio (CVR%) is the percentage of OD sample divided by OD control.
Cell morphology: Before performing CCK-8 assay, cells were observed under a phase-contrast microscopy (x200) (Nikon Eclipse TS 100, Japan).
Cell experiments were conducted at University of Medicine and Pharmacy at Ho Chi Minh City.
47 co Add E4S / Cor@E4S / Free Cor in well of cell >
Wash with PBS and refresh in complete media
Measure the absorbance at 450 nm
Figure 2.9 Experimental procedure for evaluating cytotoxicity of nanoparticles
Human ovarian cancer cell line OVCAR-8 was cultured on a 100 mm? culture dish in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C For spheroid formation, 1.0x10* of OVCAR-S8 cells were inoculated on a PrimeSurface96U culture plate (MS-9096U, Sumitomo Bakelite Co., LTD., Japan) and cultured for 7 days Tumor spheroids with a diameter of around
2.5.4 Uptake of nanoparticles by 3D tumor spheroid
Rhodamine B-labelled E4S NPs were added to tumor spheroids and incubated for
24 h The spheroids were collected into an Eppendorf tube and centrifuged The supernatant was removed, and spheroids were washed with ice-cold PBS They were then centrifuged at 1500 rpm for 5 min and fixed overnight with 4% paraformaldehyde at 4 °C Spheroids were then washed with ice-cold PBS and treated with 99.8% methanol for 30 min at -80 °C After washing, spheroids were stained using Hoechst 33258 solution for 30 minutes in the dark to show nuclei The E4S NPs biodistribution was finally observed using a confocal microscope (Figure 2.10).
Add Fluorescent E4S BPMOs to tumor spheroids
Wash with ice-cold PBS and Treat with MeOH 99,8% for 30 min at -80 °C |
| Stain with Hoechest 33258 for 30 min ¥
Uptake result in 3D tumor spheroid
Figure 2.10 Experimental procedure for evaluating the uptake of E4S NPs by 3D tumor spheroids
2.5.5 Evaluation of 3D tumor spheroid growth