ghiên cứu điều chỉnh cấu trúc và biến tính bề mặt vật liệu nano silica cấu trúc xốp rỗng cho ứng dụng phân phối thuốc điều trị ung thư.ghiên cứu điều chỉnh cấu trúc và biến tính bề mặt vật liệu nano silica cấu trúc xốp rỗng cho ứng dụng phân phối thuốc điều trị ung thư.ghiên cứu điều chỉnh cấu trúc và biến tính bề mặt vật liệu nano silica cấu trúc xốp rỗng cho ứng dụng phân phối thuốc điều trị ung thư.ghiên cứu điều chỉnh cấu trúc và biến tính bề mặt vật liệu nano silica cấu trúc xốp rỗng cho ứng dụng phân phối thuốc điều trị ung thư.ghiên cứu điều chỉnh cấu trúc và biến tính bề mặt vật liệu nano silica cấu trúc xốp rỗng cho ứng dụng phân phối thuốc điều trị ung thư.ghiên cứu điều chỉnh cấu trúc và biến tính bề mặt vật liệu nano silica cấu trúc xốp rỗng cho ứng dụng phân phối thuốc điều trị ung thư.ghiên cứu điều chỉnh cấu trúc và biến tính bề mặt vật liệu nano silica cấu trúc xốp rỗng cho ứng dụng phân phối thuốc điều trị ung thư.ghiên cứu điều chỉnh cấu trúc và biến tính bề mặt vật liệu nano silica cấu trúc xốp rỗng cho ứng dụng phân phối thuốc điều trị ung thư.ghiên cứu điều chỉnh cấu trúc và biến tính bề mặt vật liệu nano silica cấu trúc xốp rỗng cho ứng dụng phân phối thuốc điều trị ung thư.ghiên cứu điều chỉnh cấu trúc và biến tính bề mặt vật liệu nano silica cấu trúc xốp rỗng cho ứng dụng phân phối thuốc điều trị ung thư.
LITERATURE REVIEW
Overview of cancer and cancer treatment
Cancer ranks as the leading cause of death and detrimental to life expectancy in every country in the world According to the Global Cancer Registry (Globocan) in
In 2020, there were an estimated 19.3 million new cancer cases and nearly 10 million cancer-related deaths globally Female breast cancer has become the most commonly diagnosed cancer, with 2.3 million new cases (11.7%), followed closely by lung cancer (11.4%), colon cancer (10.0%), prostate cancer (7.3%), and stomach cancer (5.6%) Lung cancer remains the leading cause of cancer deaths, accounting for approximately 1.8 million fatalities (18%), followed by colorectal cancer (9.4%) and liver cancer (8.3%) By 2040, the global cancer burden is projected to rise to 28.4 million cases, marking a 47% increase from 2020, primarily driven by a surge in developing countries Therefore, establishing sustainable infrastructure for cancer prevention and care in these regions is essential for effective global cancer control.
In Vietnam, the leading cancers among men are liver, lung, stomach, colorectal, and prostate cancers, which together account for 65.8% of cases For women, the most prevalent cancers include breast, lung, colorectal, stomach, and liver cancers, making up 59.4% of diagnoses Both genders commonly face liver, lung, breast, stomach, and colorectal cancers According to Globocan's 2020 report, Vietnam ranks 91st out of 185 countries for new cancer incidences and 50th for cancer mortality rates per 100,000 people, showing improvement from 99th and 56th in 2018 The country estimates 182,563 new cancer cases and 122,690 cancer-related deaths, translating to 159 new diagnoses and 106 deaths per 100,000 individuals.
Thus, it can be seen that the figures for the new cases and the deaths of cancer in Vietnam are increasing rapidly
Figure 1.1 Global cancer data in 2020: a) Female, b) Male [1]
A tumor is an abnormal mass of tissue resulting from excessive cell division or failure to die, as defined by the US National Cancer Institute Tumors can be classified as benign (non-cancerous) or malignant (cancerous), with the key distinction being their impact on surrounding cells and organs Malignant tumors grow rapidly, invade blood vessels, and can spread to other tissues and organs through a process known as metastasis, complicating treatment and increasing the risk of recurrence In contrast, benign tumors do not spread and can typically be removed without the need for additional treatment.
Cancer arises from genetic mutations that disrupt normal cell functions by activating proto-oncogenes and inactivating tumor suppressor genes Proto-oncogenes are crucial as they can transform healthy cells into cancerous ones when mutated An increase in the expression of these genes leads to their conversion into oncogenes, which produce proteins that stimulate cell division, inhibit differentiation, and reduce apoptosis While these processes are essential for normal development and tissue maintenance, the overactivity of oncogenes results in uncontrolled cell division and the promotion of cancer cell characteristics Consequently, oncogenes are identified as promising molecular targets for the development of anticancer therapies.
Cancer treatment varies based on the type and origin of the disease, with common options including surgery, radiation therapy, immunotherapy, chemotherapy, and targeted therapy Emerging therapeutic methods such as hormone therapy, stem cell transplantation, and precision medicine are also gaining traction Notably, hormone therapy is closely linked to breast cancer, which was among the first cancers identified as hormone-dependent Tamoxifen, a selective estrogen receptor modulator (SERM), has been shown to enhance 10-year survival rates by 11% in patients with estrogen-positive (ER+) breast cancer.
Non-metastatic solid tumors, like skin tumors, are highly treatable through surgery, which boasts a cure rate nearing 100% due to the complete removal of tumor cells However, this surgical approach is only applicable to solid tumors and is ineffective for diffuse types, such as leukemia While surgery is the most invasive cancer treatment, its effectiveness in excising entire tumor tissue significantly reduces the risk of recurrence.
Figure 1.2 Common treatments for cancers [2]
For tumor tissues that metastasize to other tissues or organs, immunotherapy is used to utilize the body's immune system to defeat the cancer
Radiation therapy is a crucial treatment option that employs targeted doses of radiation to eliminate cancer cells and reduce tumor size Approximately 45% of newly diagnosed cancer patients undergo this therapy, particularly for accessible cancers such as prostate, neck, breast, cervical, and thyroid cancers However, due to potential side effects that can damage surrounding healthy tissue, radiation therapy is frequently combined with other cancer treatment modalities for optimal patient outcomes.
Chemotherapy is the leading treatment for cancer, utilizing small molecules to target and destroy rapidly dividing cells in the stroma This method can be administered through various routes, including oral and intravenous, making it one of the least invasive cancer treatments available While chemotherapy is effective for all cancer types and boasts a high success rate, it is associated with side effects such as damage to healthy cells, fatigue, and hair loss.
Targeted therapy represents the latest advancement in cancer treatment, focusing on the unique characteristics of cancer cells By utilizing drugs that specifically target these properties, this approach minimizes harm to surrounding healthy tissue, leading to reduced side effects.
Cancer research primarily focuses on discovering more effective drugs that target cancer cells, as well as enhancing drug delivery systems These advancements aim to minimize side effects on healthy cells while maximizing the therapeutic impact on cancerous tissues.
Nanomaterials in cancer treatment
1.2.1 Nanomaterials in anti-cancer drug delivery applications
Nanotechnology has emerged as a key platform in the development of drug delivery systems, with nanomaterials, often called nanomedicines, serving as effective drug delivery agents Defined as materials ranging from 1 to 100 nanometers in size, these nanomaterials can include nanodrugs with diameters reaching several hundred nanometers The journey of nanodrug development began in the early 1960s with the introduction of liposomes as carriers, paving the way for the creation of various innovative carriers aimed at enhancing treatment efficacy.
One of the advantages of nanodrugs is their ability to passively accumulate in solid tumor tissue due to their Enhanced Permeability and Retention (EPR) effects
In healthy tissues, the endothelial lining typically features gaps smaller than 2 nm, whereas tumor growth necessitates angiogenesis, resulting in new blood vessels with gaps ranging from 100 to 800 nm This size disparity allows free drug molecules to infiltrate these larger gaps, potentially harming healthy cells Conversely, drug-carrying nanosystems are designed to be too large to penetrate the endothelial gaps in healthy tissues, enabling them to effectively target tumor tissues These nanosystems accumulate in the intercellular fluid surrounding cancer cells, delivering therapeutic effects specifically to the tumor while sparing healthy cells.
Recent research has focused on various nanomaterials for drug delivery applications, highlighting a range of nano-carriers of different sizes These include inorganic nano-carriers such as gold nanoparticles, mesoporous silica, carbon nanotubes, and calcium phosphate, as well as polymer nano-carriers like nano gels, solid lipid nanoparticles, micelles, and dendrimers Additionally, vesicular carriers, including liposomes and niosomes, play a significant role in enhancing drug delivery efficiency.
Figure 1.3 Popular nanomaterials applied in drug delivery [6]
1.2.2 Silica nanomaterials in anti-cancer drug delivery applications
Silica nanoparticles, particularly mesoporous silica nanoparticles (MSN), are widely used in the development of chemotherapeutic agent delivery systems These amorphous white powders consist of siloxane groups (Si-O-Si) internally and silanol groups (Si-OH) on their surfaces MSN specifically refers to silica nanoparticles that feature pores with diameters ranging from 2 to 50 nm, enhancing their effectiveness in drug delivery applications.
In the 1990s, a researcher from Mobil Oil discovered the first mesoporous silica material, known as M41S This family of materials includes three primary members, notably the Mobil Composition of Matter No 41 (MCM-41).
MCM-48 and MCM-50 are two distinct materials characterized by their unique pore geometries While MCM-41 features a hexagonal pore structure, MCM-48 is defined by its cubic shape and an interwoven, continuous three-dimensional pore system.
MCM-41 features a lamellar structure composed of silica sheets or porous aluminosilicate layers interspersed with surfactant layers It is the most extensively researched among the MCM series, as MCM-48 and MCM-50 present challenges in synthesis and exhibit thermal instability.
Figure 1.4 Members of the M41S family [8]
Mesoporous silica nanoparticles (MSN) have been developed in various forms due to their flexible synthesis methods These can be categorized into several types based on their structure, including conventional mesoporous particles, hollow mesoporous silica nanoparticles, core-shell mesoporous silica nanoparticles, and yolk-shell mesoporous silica nanoparticles.
Figure 1.5 Structural classification of Mesoporous Silica Nanoparticles [9]
In 2001, Vallet-Regi and colleagues successfully utilized MSN as an ibuprofen carrier, marking a significant advancement in drug delivery systems Silica has been recognized by the FDA as "generally recognized as safe" (GRAS) for over 50 years and is commonly used as an excipient in pharmaceutical formulations Recently, the FDA has approved silica nanoparticles as imaging agents for clinical trials in humans, paving the way for the potential application of MSNs as drug delivery agents in clinical practice.
Mesoporous silica nanoparticles (MSNs) are gaining popularity in drug delivery systems due to their straightforward synthesis and customizable properties, including particle morphology, size, and pore diameter The ability to modify the surface of the particles and pores with functional groups enhances their functionality Additionally, the porous structure of MSNs significantly increases the loading capacity for poorly soluble drugs while protecting them from enzymatic degradation The adjustable pore diameter allows for selective drug loading, making MSNs an effective option for targeted therapies Studies have demonstrated that MSNs exhibit excellent biocompatibility in vitro at doses below 100 µg/mL and in vivo at doses under 200 mg/kg, highlighting their safety and compatibility for drug delivery applications.
Hollow mesoporous silica nanoparticles (HMSN), part of the MSN family, offer enhanced drug loading capabilities due to their large internal cavities, surpassing those of traditional non-hollow particles Consequently, there has been a growing interest in the use of HMSN-based systems for drug delivery applications.
Research situation of nano silica particles in drug delivery
Since the introduction of mesoporous silica nanoparticles (MSNs) as drug delivery systems in 2001, researchers have been dedicated to developing the ideal MSN system for this purpose The number of studies focusing on MSN drug-carrying applications has steadily risen, with 6,538 scientific publications reported by November 2017, highlighting the ongoing interest and potential of MSNs as effective drug delivery materials.
Figure 1.6 Number of “Mesoporous + Silica + Drug + Delivery” publications by year in the ISI Web of Science [21]
Adjustable mesoporous silica nanoparticles (MSNs) with various shapes (sphere, rod, oval), particle sizes (20 to 50 nm), and pore sizes (2 to 6 nm) have been successfully synthesized primarily using sol-gel methods Research by Ya-Dong et al demonstrated that the Taguchi statistical design method can effectively control MSN particle size, highlighting the significant impact of pH on particle size, while reaction time and the amount of Tetraethyl orthosilicate (TEOS) had a lesser effect Additionally, Naiara et al reported that the synthesis of MSNs from TEOS and Cetrimonium bromide (CTAB) led to a transformation in particle morphology from spheres to rods, with increased particle porosity correlating with higher CTAB concentrations Furthermore, Kusum et al utilized hexane/decane as pore expanders in their sol-gel method for MSN preparation.
The pore size of the obtained MSN increased from 2.5 to 5.2 nm, which was able to effectively deliver anticancer drug gemcitabine [25]
In addition, MSNs have been modified with a variety of ligands for better biocompatibility and effective delivery of different treating agents For example,
Recent advancements in the modification of mesoporous silica nanoparticles (MSNs) have significantly enhanced their efficacy in drug delivery for cancer treatment Chia-Hui et al improved Cisplatin effectiveness by modifying MSN surfaces with carboxylate groups through hydrazine bonds Additionally, Anna and colleagues successfully altered the pore walls of MSNs using hyperbranching polymerized poly(ethyleneimine), creating effective vectors for siRNA delivery Furthermore, Sahar et al employed dielectric barrier discharge plasma to directly modify MSN surfaces, enabling a dual-responsive delivery system for Doxorubicin (DOX) that reacts to both pH and temperature changes.
In 2004, the very first hollow versions of MSN have been introduced Zhu-Zhu et al successfully prepared hollow porous silica nanoparticles via sol-gel method
CaCO3 nanoparticles served as hard templates while Na2SiO3 acted as the silica precursor, enabling the successful loading of Brilliant Blue F within the hollow structures, which enhanced both loading capacity and release control This methodology was similarly utilized by Jian-Feng et al to produce porous hollow silica nanoparticles, with the hollow@shell structures depicted through Transmission Electron Microscopy (TEM) images Subsequently, extensive research has focused on developing an optimal system using Hollow Mesoporous Silica Nanoparticles (HMSN) for drug delivery applications.
In Vietnam, the research and development of silica nanomaterials has garnered significant attention, particularly from prominent research groups led by Prof Dr Phan Bach Thang, Assoc Prof Dr Ha Thuc Chi Nhan, Dr Vong Binh Long, and Dr Pham Dinh Dung.
Prof Dr Phan Bach Thang's research group has developed a biodegradable tetrasulfide-based organosilica nanomaterial, BPMO (Biodegradable Periodic Mesoporous Organosilica), for drug delivery in cancer treatment This innovative BPMO system effectively encapsulates daunorubicin (DNR), reduces particle size to improve curcumin loading efficiency, and features surface modifications that enhance the drug loading capacity and control the release of cordycepin.
Assoc Prof Dr Ha Thuc Chi Nhan and his team successfully synthesized nano silica from rice husks in 2013 using the sol-gel method, producing amorphous silica particles with a uniform size of approximately 3 nm These silica nanoparticles were effective in adsorbing Pb²⁺ and Cd²⁺ ions, achieving equilibrium adsorption in about 1.5 hours, with capacities of 21 mg/L and 24 mg/L, respectively Since 2014, Dr Vong Binh Long has been researching silica-containing redox nanoparticles (siRNP) for oral drug delivery and enhanced anti-inflammatory effects In 2017, the team advanced siRNPs to load BNS-22, a hydrophobic anti-cancer compound targeting reactive oxygen species (ROS) for treating colitis-associated colorectal cancer By 2020, they developed siRNPs with a diameter of 50-60 nm to improve the bioavailability of silymarin (SM@siRNP), demonstrating promising results in enhancing silymarin's anti-inflammatory activity and showing significant potential for treating inflammatory bowel disease.
Dr Pham Dinh Dung and his team have researched the use of nanosilica in antifungal and antibacterial formulations for plants Their 2016 study focused on the impact of nanosilica (10 - 30 nm) derived from rice husks on the growth of chili pepper plants (Capsicum frutescens L.) in a greenhouse setting The findings indicated that nanosilica significantly promoted the growth of chili pepper plants.
In 2017, Vinagamma Center developed oligochitosan-nano silica preparations (pH 5, oligochitosan MW 4-6 kDa, silica nanoparticle size 20-30 nm) to enhance the resistance of chili peppers against Colletotrichum gloeosporioides, the fungus responsible for anthracnose When applied at a concentration of 60 ppm, these preparations significantly improved the resistance of chili pepper plants from 37.8% to 88.8% and decreased the infection rate from 39.2% to 13.7%.
Since 2013, Assoc Prof Dr Nguyen Dai Hai's research team has been at the forefront of silica nanomaterials development for biomedicine in Vietnam, successfully synthesizing solid silica nanoparticles (dSiO2) and advancing mesoporous silica nanoparticles (MSN) and hollow mesoporous silica nanoparticles (HMSN) for anti-cancer drug delivery Funded by NAFOSTED (Grant No 104.03-2018.46), the team enhanced the stability and drug delivery capabilities of silica nanoparticles by modifying them with active groups and polymers such as PEG and chitosan-PEG Despite various strategies to utilize MSN in cancer drug delivery, challenges remain, particularly regarding the clinical application of MSN, as the open pore structure can lead to drug leakage and reduced delivery efficiency, alongside potential hemolysis due to interactions with red blood cell membranes Additionally, most research has primarily focused on the loading capacity and release profiles for single drugs, indicating a need for further exploration in this area.
This chapter highlights key techniques for synthesizing and enhancing hollow mesoporous silica nanoparticles (HMSNs) for the effective delivery of chemotherapeutic agents It will explore the tunable properties of HMSNs, including hybridized and multidrug-carrying variants Additionally, the discussion will address the achievements and challenges encountered during the research and development of the MSN carrier system.
Hollow mesoporous silica nanoparticles (HMSN)
Hollow mesoporous silica nanoparticles (HMSN), part of the MSN family, feature a unique structure comprising an outer mesoporous shell and an inner hollow cavity This design not only retains the specific properties of mesoporous silica nanoparticles (MSN) but also significantly enhances drug loading capacity, making HMSN a superior choice for drug delivery applications.
Figure 1.7 Structure of Hollow Mesoporous Silica Nanoparticle (HMSN): a) 2D radial section; b) 3D model; and c) Mesoporous structure of the shell
The synthesis of hollow nanoparticles involves using various templates such as carbon, polystyrene, ferromagnetic, or silica nanoparticles After the shell is formed, the template is removed through physical or chemical methods to create a hollow cavity The mesoporous shell, resembling the synthesis of mesoporous silica nanoparticles (MSN), consists of a silica precursor and surfactant Surfactant micelles serve as pore templates, while the silica precursor undergoes hydrolysis and condensation on the template surface and around the micelles, ultimately forming a porous shell once the surfactant is removed.
HMSN synthesis is generally followed a typical process:
(2) Coating the shell over the template surface and thus creating a core@shell structure;
(3) Remove the template to obtain a hollow structure
Hollow mesoporous silica nanoparticles (HMSNs) can be synthesized through three primary methods: hard template, soft template, and self-template methods These synthesis techniques categorize HMSNs into three distinct types: hard template HMSN, soft template HMSN, and self-template HMSN.
Figure 1.8 Synthesis methods of HMSN
Hard-template HMSN is created using inorganic compounds such as amorphous silica, metal carbonates, or polymer latex, offering advantages like narrow size distribution and a variety of sizes and configurations The shell formed on the template retains the shape and size of the original cavity, allowing for predictable final morphology and structure of the coated particles However, the use of hard templates involves a multi-step synthesis process and complex thermal or chemical removal, making it a time-consuming and labor-intensive approach.
The hard template method for synthesizing HMSN involves several key steps: first, a hard core that is compatible with the shell material is formed; next, a mesoporous shell is condensed around this core; and finally, the inner core is selectively removed to yield the final HMSN product.
Typical hard templates include inorganic compounds like amorphous silica, metal carbonates, and polymers (latex) that can be chemically etched Various methods, such as the sol-gel process, hydrothermal reaction, electrostatic assembly, and chimie douce route, are employed to agglomerate shell materials onto the template's surface Surface modification may be necessary to enhance compatibility between the template and shell material, often involving chemical changes to introduce specific functional groups or alter charge distribution The main techniques for template removal include chemical etching, heat treatment, and solvent dissolution, chosen based on the differences in composition between the template and shell Careful selection of experimental conditions is crucial to prevent shell collapse during the removal process, considering the properties of the hard template.
Polymer latex particles are an excellent choice for synthesizing hollow mesoporous silica nanoparticles (HMSNs) due to their uniform size and adjustable surface properties during synthesis As a widely available and cost-effective material, polymer latex facilitates the creation of silica shells, which can later be removed through heating or dissolution Various latex polymers, including polystyrene (PS), polyvinylpyrrolidone (PVP), poly(acrylic acid) (PAA), and polymethylmethacrylate (PMMA), have been successfully utilized as templates in the synthesis of HMSNs Additionally, hard template methods employing carbon and metal oxides offer alternative approaches for producing these advanced materials.
Metal oxide and carbon templates offer distinct advantages over polymer latex templates, including their polymorphic nature and the absence of the need for organic solvents during preparation Additionally, there is no requirement to modify surface properties prior to silica coating Fuji et al demonstrated that hollow silica shapes can be precisely controlled using CaCO3 templates, which can take on various forms such as cubes, rough-surfaced spheres, and rod-like particles Notably, the internal dimensions and configurations of the synthesized hollow silica particles closely mirror the external dimensions and shapes of the utilized templates.
Amorphous silica particles are popular as hard templates due to their high morphological uniformity and adjustable particle size distribution at an affordable cost The classical sol-gel method, also referred to as Stüber's method, is commonly used to synthesize single-dispersed SiO2 particles in the micrometer size range This process involves the hydrolysis and condensation of silicon alkoxides in a water and alcohol mixture, facilitated by a catalyst.
Homogeneous hollow mesoporous silica nanoparticles (HMSNs) can be synthesized using a silica-based hard-template method involving tetraethyl orthosilicate (TEOS) precursors The process includes three key steps: first, the synthesis of homogeneous dense silica (dSiO2) via an enhanced Stüber method; second, the creation of dSiO2@MSN, where cetyltrimethylammonium chloride (CTAC) acts as the porous template and triethanolamine (TEA) functions as the catalyst; and third, the etching of dSiO2@MSN with sodium carbonate (Na2CO3) to eliminate the solid template, followed by the removal of CTAC using a 1% sodium chloride solution in methanol to yield HMSNs.
Soft template hollow mesoporous silica nanoparticles (HMSNs) are created using liquid or gaseous templates like emulsions, micelles, and air bubbles These templates facilitate the incorporation of functional groups or the encapsulation of guest molecules during the shell formation process However, controlling the particle shape of soft-template HMSNs is more challenging than with hard-template HMSNs.
The soft template method utilizes amphiphilic molecules with both hydrophilic and hydrophobic components for direct synthesis, allowing for quick preparation of templates just before the silica coating process This in situ template method has gained popularity among researchers due to the ease of template preparation and removal However, it is important to note that hollow silica particles produced through this method often exhibit irregular shapes and a broad particle size distribution, attributed to the malleability of the soft template One application of this method involves using emulsions as the soft template.
The emulsion method is a traditional technique for preparing hollow silica, involving the creation of stabilized emulsion droplets from incompatible solvents with a stabilizer This process results in a dispersed phase and a continuous phase, separated by an interface To enhance stability, surfactants are employed to lower interfacial tension Emulsions can be classified as either oil in water or water in oil, depending on the phase composition Similar to the hard template method, hydrolysis and condensation of the precursor occur at the emulsion droplet interface, leading to a core@shell structure (emulsion@silica gel) Finally, the soft templates are removed to yield hollow silica spheres.
Because of the agglomeration, it is difficult to obtain uniform droplets less than
The conventional emulsion method produces silica spheres with a diameter of 100 nm, while the microemulsion method excels in creating homogeneous hollow silica spheres under 100 nm due to the thermodynamic stability of its microdroplets Additionally, the use of soft templates, such as micelles, enhances the production process.
The micelle soft template method involves the self-assembly of amphoteric molecules in a single-phase solvent, occurring when their concentration surpasses the critical micelle concentration (CMC) This technique enables the creation of hollow materials through direct assembly of precursors or chemical interactions with the template's surface Similar to the emulsion method, micelle templates can be categorized into water-in-oil or oil-in-water types, depending on the solvent used Various reaction parameters, including surfactant concentration, ionic strength, temperature, pH, and chemical additives, can be adjusted to produce micelles or particles with different shapes.
MATERIALS AND EXPERIMENTAL METHODS
Materials
The chemicals used in the study were analytical grade which are listed in Table 2.1
Table 2.1 List of used chemicals
No Chemicals’ names Origin Purity
22 Fetal bovine serum (FBS) Sigma ≤10 EU/mL endotoxin
The equipments used in the study were at the Institute of Applied Materials Science (IAMS) and other national research institutions which are listed in Table 2.2
Table 2.2 List of used equipments
Order No Equipments’ name Origin
1 Refrigerated Centrifuge Hermle Z 32 HK Germany
2 Bench Top Freeze Dryer Eyela FDU-1200 Japan
4 Transmission electron microscopy Jeol JEM-1400 Japan
5 Field Emission Scanning electron microscope with
Energy Dispersive X-ray microanalysis Japan
6 NMR Spectrometer Bruker 500 MHz Germany
7 X-ray Diffractometer Bruker D2 Advance Germany
9 Surface Area and Porosimetry Analyzer
Micromeritics TriStar II Plus (Version 3.03) USA
11 Bruker Equinox 55 FT-IR spectrometer Germany
Synthesis Methods
Hollow mesoporous silica nanoparticles (HMSN) were synthesized using a hard-template method that involves three key steps: first, the preparation of the hard template dSiO2 via the Stober method; second, the creation of a core@shell structure dSiO2@MSN through the sol-gel technique; and third, the selective etching of dSiO2@MSN to yield HMSN The synthesis process is illustrated in Diagrams 2.1, 2.2, and 2.3.
In the initial phase, dSiO2 hard templates were synthesized using a modified Stober method to ensure the HMSN particle diameter remained below 100 nm This was achieved by adjusting the concentrations of silica precursor TEOS, catalyst agent NH3, and ethanol Specifically, a mixture of 70.0 mL of absolute ethanol, 10.0 mL of deionized water, and 3.0 mL of ammonia was stirred for 15 minutes at 60 °C, followed by the addition of 6.0 mL of TEOS, allowing the reaction to proceed for 6 hours To optimize the mesoporous silica coating and hard template silica etching processes, reaction parameters such as temperature and catalyst concentration were modified.
Diagram 2.1 The preparation of the hard template dSiO2
In the second step, a mesoporous silica layer was applied to the hard templates using the sol-gel method A solution was prepared with 3.0 g of CTAB, 50.0 mg of TEA, and 20.0 mL of deionized water Following this, 10 mL of the dSiO2 solution from the first step was incorporated and stirred at room temperature for one hour The mixture was then alternately heated to 80 °C, after which 0.5 mL of TEOS was added and stirred for an additional hour to produce the dSiO2@MSN.
Diagram 2.2 The preparation of core@shell structure dSiO2@MSN
In the third step of the process, hard templates were eliminated through an etching procedure using an aqueous Na2CO3 solution The mixture from the previous step was cooled to 50 °C before adding Na2CO3 to achieve a concentration of 21.2 mg/mL, followed by continuous stirring to facilitate etching To monitor the etching of the silica hard-template over time, samples were collected every 30 minutes and labeled according to the etching duration, including HMSN 30, HMSN 60, HMSN 90, HMSN 120, HMSN 150, and HMSN 180 These samples underwent dialysis using 12 x 14 kDa membranes against ethanol and distilled water over three days before being lyophilized The particle size, morphology, and structure were subsequently analyzed using Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) to determine the optimal etching time The selected HMSN, characterized by appropriate etching duration, was further examined using FT-IR, Thermogravimetric Analysis (TGA), Brunauer-Emmett-Teller (BET) analysis, Barrett-Joyner-Halenda (BJH) method, Dynamic Light Scattering (DLS), zeta potential measurements, and X-ray diffraction.
Diffraction (XRD), Energy-dispersive X-ray spectroscopy (EDX) and MTT analytical methods
Diagram 2.3 The selective etching of dSiO2@MSN to form HMSN
2.2.2 Study the effect of PEG on the mesoporous shell thickness of HMSN
This study examines how polyethylene glycol (PEG) influences the mesoporous shell thickness of hollow mesoporous silica nanoparticles (HMSN) Various PEG molecular weights (1000, 2000, 4000, and 6000 g/mol) and weight percentages (ranging from 1% to 5%) were incorporated into the reaction mixture during the second stage of the HMSN synthesis process, as illustrated in Diagram 2.4.
The solution obtained after the second step was dialyzed using 12 x 14 kDa membranes against ethanol and distilled water over three days This process was followed by lyophilization to produce dSiO2@MSN Subsequently, the post-lyophilized dSiO2@MSN samples underwent calcination.
The dSiO2@MSN samples underwent a thermal treatment at 600 °C for 2 hours to eliminate residual organic components, preparing them for subsequent experiments The morphology and particle diameter were analyzed using Dynamic Light Scattering (DLS) and Scanning Electron Microscopy (SEM) The thickness of the mesoporous shell was calculated based on the DLS particle diameter measurements of both dSiO2 and dSiO2@MSN.
Where: TS: the thickness of mesoporous shell (nm) ddSiO2@MSN: diameter of dSiO2@MSN particles (nm) ddSiO2: diameter of dSiO2 particles (nm)
Diagram 2.4 Mesoporous silica layer coating step in HMSN synthesis process with the presence of PEG
The remaining dSiO2@MSN solution underwent a third step to synthesize HMSN The lyophilized HMSN samples were then calcined at 600 °C for two hours to ensure the complete removal of residual organic materials, preparing them for subsequent experiments.
To elucidate the role of PEG in the synthesis of HMSNs, we analyzed and compared the characteristics of synthesized HMSNs, designated as HMSN-0 (without PEG) and HMSN-P (with PEG), using various techniques including SEM, TEM, BET, FT-IR, EDX, DLS, and Zeta potential analysis.
The drug loading capacity (DLC) and drug loading efficiency (DLE) of HMSN-0 and HMSN-P were analyzed using doxorubicin (DOX) as a model drug, with its concentration measured via UV-Vis spectrometry at 480 nm Additionally, the cytotoxic effects of the synthesized HMSN-0 and HMSN-P were assessed on the MCF-7 cell line through the MTT assay.
2.2.3 Study the effect of non-ionic surfactants on the mesopore diameter of HMSN
This study explores the impact of non-ionic surfactants, specifically Brij S10 (BS10), Tween 20 (T20), and Tween 80 (T80), on the meso-pore diameter of HMSN Various molar ratios of these surfactants were tested alongside a constant CTAB ratio (0:1, 1:4, 1:3, 1:2, and 1:1) to create mixed micelles These micelles served as co-templates in the second stage of HMSN synthesis, facilitating the coating of a mesoporous shell onto the hard template dSiO2 The mesoporous shell coating process is illustrated in Diagram 2.5.
Diagram 2.5 Mesoporous silica layer coating step in HMSN synthesis process with the presence of non-ionic surfactants as co-templates
After the second step, the solution was dialyzed using 12 x 14 kDa membranes against ethanol and distilled water over three days, followed by lyophilization to produce dSiO2@MSN The resulting dSiO2@MSN samples underwent DLS size measurement, BET surface area analysis, and BJH pore size and volume analysis to assess particle morphology, diameter, and meso-pore dimensions.
The thickness of mesoporous structures was determined using the particle diameters of dSiO2 and dSiO2@MSN, as outlined in Equation (1) Through BET and BJH analysis, the mesopore diameters of dSiO2@MSN samples were assessed and compared The remaining dSiO2@MSN solution proceeded to the third step to yield HMSN, with the resulting products characterized using SEM, TEM, Zeta potential, XRD, and FT-IR analysis.
The DLC and DLE values as well as drug release profiles of HMSN and HMSN-
The study focused on the investigation of HMSN-BS10, HMSN-T20, and HMSN-T80 particles for their ability to load larger cargoes, using Rose Bengal (RB) as a model drug due to its molecular weight of 1017.64 g/mol, which is twice that of DOX The loading process was conducted through an equilibrium dialysis method To quantify the released drug, samples were analyzed using UV-Vis spectrophotometry at 546 nm Furthermore, the cytotoxicity of the synthesized HMSN and HMSN-S was assessed on the MCF-7 cell line using the MTT assay.
2.2.4 Surface Modification Method of HMSNs with Pluronics
To enhance the surface properties of HMSN using Pluronic, both HMSN and Pluronic molecules require activation This process involves modifying the HMSN nanoparticle surface with APTES to introduce a more reactive amine group, resulting in the formation of HMSN-NH2.
Physicochemical Analysis Methods
The particle size distribution was analyzed using Dynamic Light Scattering (DLS), while the zeta potential (ζ) was measured with a Helium-neon (He-Ne) laser at a detection angle of 90°, a temperature of 25 °C, and a wavelength of 532 nm This analysis was conducted using a Zetasizer Nano SZ (SZ-100, Horiba, Kyoto, Japan) at the Institute of Applied Materials Science.
To assess the particle size, morphology, and hollow-shell structure of the synthesized samples, Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) images were obtained using the FESEM S-4800 and JEM-1400 instruments, respectively Additionally, Energy Dispersive X-ray Spectroscopy (EDX) was employed for chemical characterization with the FESEM S-4800.
To study the components and evaluate the purity of the synthesized particles, FT-
Infrared (IR) and thermal gravimetric analysis (TGA) were conducted to investigate the properties of HMSN The FT-IR analysis utilized a Bruker Equinox 55 spectrometer, employing the KBr pellet method for sample preparation TGA was performed using a Mettler Toledo analyzer, operating under a nitrogen atmosphere with a temperature range from 30 to 800 °C, at a heating rate of 10 °C/min Additionally, X-ray diffraction (XRD) studies were carried out on a Bruker D2 Phaser diffractometer, utilizing mono-chromated CuKα radiation to examine the molecular structure of the material.
Brunauer-Emmett-Teller (BET) Surface Area Analysis and Barrett-Joyner- Halenda (BJH) Pore Size and Volume Analysis were operated on TriStar II Plus (Version 3.03, Micromeritics, GA, USA)
Drug amount including DOX, RB and QUE was determined by measuring the absorbance at 480, 546 and 380 nm, respectively, using a UV/Visible Scanning Spectrophotometer UV/Vis-1800 (Shimadzu, Japan).
Drug loading and in vitro release study
The study investigated the drug loading capacity (DLC), drug loading efficiency (DLE), and drug release profiles of nanoparticles A model drug was incorporated into the nanoparticles using an equilibrium dialysis method Specifically, 20 mg of nanocarriers was dispersed in 8 mL of a suitable solvent, such as distilled water, ethanol, methanol, or DMSO, while 2 mg of the drug was dissolved in an additional 2 mL of solvent The drug solution was then combined with the nanoparticle mixture and stirred at room temperature for 24 hours Following this, the loaded nanoparticles were separated by centrifugation at 10,000 rpm for 30 minutes, and the supernatant was analyzed for any unbound drug using UV-Vis spectroscopy.
Standard curves for free drugs were created by measuring the absorbance of known concentration drug solutions ranging from 5-50 ppm and 0-5 ppm at specific wavelengths using a UV/Vis-1800 spectrophotometer from Jasco Co., Tokyo, Japan The drug loading efficiency (DLE) and drug loading capacity (DLC) values for each drug were determined using the provided equations.
DLE (%) = 20 - W 20 non-D ×100% (2) DLC (%) = (W 20 - W non-D particles + 20 - W non-D )×100% (3) Where: 20 (mg) is the initial amount of drug used in the loading experiment
Wnon-D the total amount of unloaded drug detected in the dialysis solution
Wparticles is the dried weight of the carrier system
The drug release profiles of free drug and loaded drug were assessed in PBS buffer using dialysis method which was described in the previous study [45] First,
In a controlled experiment, 1 mL of either free or drug-loaded suspension was placed in a 12-14 kDa dialysis bag and submerged in a vial with 20 mL of release medium The vial was then subjected to continuous shaking at 100 rpm in an orbital shaker bath At specified intervals, 2 mL samples of the release medium were withdrawn, with an equal volume of fresh medium added to maintain the system's integrity The drug release was quantified using UV-Vis spectroscopy to analyze the collected samples.
Cell culture and MTT assay
The cytotoxicity of synthesized particles and their effectiveness in killing cancer cells were assessed using the MTT assay on human breast cancer MCF-7 and cervical cancer Hela cell lines The cells were grown in complete DMEM containing 10% FBS, 100 IU/mL penicillin, and 0.1 mg/mL streptomycin in a 5% CO2 humidified incubator Initially, 3×10^4 cells were seeded into a 96-well culture plate and incubated for 24 hours before being treated with the synthesized particles at varying concentrations.
In a study assessing cell viability, concentrations ranging from 10 àg/mL to 250 àg/mL were tested, along with free and loaded drugs at varying concentrations of 0.1 to 50 àg/mL over a 48-hour period After treatment, 20 μL of a 5 mg/mL MTT solution was added to each well and incubated for an additional 4 hours at 37 °C Following incubation, the culture medium was removed, and 100 μL of DMSO was added to each well, which was then shaken for 2 minutes The absorbance was measured at a wavelength of 570 nm using a microplate reader, and cell viability was calculated using a specified equation.
Cell viability (%)= Abs Abs sample -Abs blank control -Abs blank ×100% (4)
Statistical analysis
Each experiment was conducted in triplicate, and the results are expressed as mean ± standard deviation (SD) Significant differences between means were assessed using one-way ANOVA followed by Bonferroni's post-test, with a significance level set at p < 0.05.
A MODIFIED HARD-TEMPLATE METHOD FOR HOLLOW
Synthesis of silica hard-template
The dSiO2 particles were synthesized as part of the HMSN synthesis process, exhibiting a zeta potential of -45.8 ± 0.41 mV, aligning with previous silica nanoparticle studies The hydrodynamic size of the dSiO2 was measured at 96.8 ± 0.26 nm, with a low polydispersity index (PI) of 0.072, indicating a high uniformity in particle size.
The morphology of dSiO2 particles, depicted in SEM and TEM images (Figure 3.1 c and d), reveals that they are mono-disperse spheres with an average diameter of 65.27 ± 0.42 nm This represents a significant reduction from previous studies, where the hard template dSiO2 diameter exceeded 100 nm, now measuring approximately 65 nm [57, 72] Consequently, the decrease in hard template diameter is anticipated to result in a corresponding reduction in the synthesized HMSN.
Figure 3.1 Characterizations of the synthesized hard-template dSiO2: a) Zeta potential; b) DLS particle size distribution; c) SEM image; d) TEM image
Etching over time of silica hard-template in the synthesis of HMSN
HMSN particles were analyzed using SEM and TEM imaging after varying etching times from 30 to 180 minutes SEM images revealed that the particles exhibited a non-hollow structure at 30 and 60 minutes of etching A hollow structure became apparent after 90 minutes, with the most distinct hollow appearance observed at 120 minutes After 150 minutes, the shell showed signs of corrosion with some holes, though the particles remained spherical Ultimately, after 180 minutes of etching, the particles lost their spherical shape, and the shell collapsed.
TEM images reveal the status of the hard template, shedding light on the etching process Initially, the etching agent interacts with the template, producing rattle-type spheres with a double-shell structure (HMSN 30, HMSN 60) Subsequently, the cavity expands, leading to the formation of a hollow structure (HMSN 90, HMSN 120) Once the hard template is fully corroded, the mesoporous shell is also compromised by the etching agent (HMSN 150, HMSN 180), aligning with the selective etching mechanism reported earlier Notably, CTAB at a concentration of 21.2 mg/mL serves as a protector for the mesoporous shell, resulting in uniform HMSNs with distinct hollow cavities upon completion of the etching process, which is optimally set at 120 minutes However, extending the etching time beyond this (to 150 minutes) can lead to undesired effects.
Prolonged etching for 180 minutes can lead to over-etching, which damages the shells and produces aggregated particles with rough surfaces This process gradually thins the shell, ultimately resulting in its destruction and the collapse of the particle structure, as illustrated in Figure 3.2.
The findings indicated that an optimal etching duration of 120 minutes was determined, leading to the selection of HMSN 120 for subsequent experiments Notably, the synthesis time for HMSN in the primary reactions of this study was reduced.
The modified hard-template method significantly reduces the total preparation time from 21 hours to just 9 hours, with hard-template preparation cut to 6 hours, mesoporous shell coating to 1 hour, and hard-template etching to 2 hours This streamlined approach is beneficial for researchers focusing on silica nanoparticles and holds promise for enhancing efficiency in industrial-scale production.
Figure 3.2 SEM and TEM images of HMSN over etching time
Characterizations of synthesized HMSN
The synthesized HMSN particles were characterized via TEM, BET, BJH, FT-
IR, EDX, Zeta potential, DLS, XRD and TGA analysis whose results were showed in Figure 3.3 and Figure 3.4
TEM images of the synthesized dSiO2@MSN and HMSN 120, shown in Figure 3.3 (a and b), reveal distinct structural characteristics The dSiO2@MSN particles exhibit a core@shell structure, with a dark inner region representing the solid template and a brighter mesoporous shell surrounding it In contrast, the HMSN 120 particles demonstrate a hollow@shell structure, featuring a particle diameter of 80.15 ± 1.29 nm, a hollow diameter of 64.73 ± 0.95 nm, and a mesoporous shell thickness of approximately 7.5 nm Notably, the synthesized HMSN particles show a significant reduction in size from over 134 nm in previous studies to around 80 nm.
Recent advancements in nanomaterials involve surface modifications using various polymers and functional groups to enhance biocompatibility, drug loading capacity, and targeting ability As a result of these modifications, the particle diameter tends to increase Consequently, the resulting Hollow Mesoporous Silica Nanoparticles (HMSN) emerge as an ideal candidate for comprehensive nano-carrier development.
The N2 adsorption-desorption isotherms and pore size distributions of HMSN, illustrated in Figure 3.3 (c and d), reveal that the isotherms are classified as Langmuir type IV and exhibit a type H2 hysteresis loop according to IUPAC standards, confirming the mesoporous nature of the synthesized HMSN Furthermore, the surface area and pore diameter of HMSN were measured at approximately 767 m²/g and 2.5 nm, respectively, aligning with findings from previous studies.
Figure 3.3 a) TEM image of dSiO2@MSN; b) TEM image of HMSN, c) N2 adsorption-desorption isotherms of HMSN and d) Pore size distributions of HMSN
As seen from the FT-IR spectrum (Figure 3.4a), the strong absorption bands at
The absorption peaks at 1103 cm⁻¹, 814 cm⁻¹, and 467 cm⁻¹ are associated with the anti-symmetric stretching, symmetric stretching, and bending vibrations of Si-O-Si, respectively, while peaks at 3500 cm⁻¹ and 951 cm⁻¹ correspond to the bending vibration of Si-OH EDX analysis confirmed the elemental composition of HMSN's surface, revealing atomic percentages of 68.82% oxygen and 31.18% silicon, indicating the effective removal of CTAB and other reacted reagents.
Figure 3.4 Characterizations of the synthesized HMSN: a) FT-IR spectrum; b)
EDX parttern; c) Zeta potential; d) DLS particle size distribution; e) XRD pattern; and f) TGA graph
The synthesized HMSN exhibited a zeta potential of -46.6 ± 0.73 mV, which is effective in preventing agglomeration and maintaining particle dispersion Dynamic Light Scattering (DLS) analysis revealed that the hydrodynamic size of HMSN was 108.17 ± 1.83 nm, with a polydispersity index (PI) of 0.083 This low PI value, being less than 0.3, indicates that the HMSN particles are highly uniform in size.
The XRD pattern of HMSN, illustrated in Figure 3.4e, displays a broad peak at 2θ = 23.93°, confirming the amorphous nature of the silica particles Additionally, Figure 3.4f presents the TGA graph of HMSN, indicating an initial weight loss of approximately 11.5% at temperatures below 200°C, which is attributed to the presence of physically adsorbed water.
200 – 600 o C, the weight loss was about 7.0% This might be due to a small amount of CTAB remaining in the HMSN.
Cytotoxicity of synthesized HMSN
MTT assays conducted on the MCF-7 cell line assessed the cytotoxicity of synthesized HMSN, as illustrated in Figure 3.5 The results indicated that cell viability was 100% at a concentration of 10 µg/mL HMSN As the concentration increased to 250 µg/mL, cell viability gradually decreased but remained above 80% These findings suggest that the synthesized HMSN exhibits no significant toxicity to MCF-7 cells across the tested concentration range, confirming its potential as a biocompatible nanocarrier.
Summary
The HMSN particles were effectively synthesized using a modified hard-templating method, achieving optimal diameters in a significantly reduced time frame These particles exhibited a uniform spherical morphology with a hollow core and mesoporous shell structure, measuring approximately 80 nm in diameter, 65 nm for the hollow core, and 7.5 nm for the shell thickness SEM and TEM analyses revealed that the ideal etching time for the hollow mesoporous silica nanoparticle synthesis was 120 minutes, allowing for a reduction in overall synthetic time from 21 hours to just 9 hours Importantly, the synthesized HMSN demonstrated biocompatibility with no observed in vitro toxicity at concentrations up to 250 µg/mL This innovative method not only streamlines the synthesis process but also holds significant potential for both scientific research and industrial production.
Figure 3.5 a) Cell viability assay by MTT assay with variable concentrations of
HMSN on MCF-7 cells; b) Morphology of MCF-7 cells treated by HMSN at different concentrations
The research results of this content have been accepted for publication in Vietnam Journal of Science and Technology, ISSN 2525-2518, 0866-708X with the title “A
Modified Hard-Templating for Hollow Mesoporous Silica Nanoparticles Synthesis with Suitable Particle Size and Shortened Synthesis Time” Accepted notification in 20 August 2022.
SIMPLY AND EFFECTIVELY CONTROL THE SHELL
Effect of PEG molecular weight on the mesoporous shell thickness of
To investigate the effect of molecular weight of PEG on the mesoporous shell thickness of dSiO2@MSN, PEG with molecular weights of 1000, 2000, 4000 and
6000 g/mol was added 3% (w/v) into the reaction mixture The obtained samples named dSiO2@MSN-P1k, dSiO2@MSN-P2k, dSiO2@MSN-P4k and dSiO2@MSN- P6k were determined DLS size and FESEM images (Figure 4.1)
Figure 4.1 Size dispersion by DLS measurement and field-emission scanning
MSN
Effect of PEG weight percentage on the mesoporous shell thickness of
Hollow mesoporous silica nanoparticles (HMSNs) with a thicker mesoporous shell are anticipated to provide increased space for drug encapsulation and a more sustained drug release profile, especially when the cavity volume remains constant To maintain mechanical stability during handling and administration, HMSNs must possess a sufficiently thick mesoporous shell Research by Lasio et al demonstrated that the mechanical stability of HMSNs is directly related to shell thickness, with those ranging from 14 to 18 nm breaking under pressures of 103.0 to 123.6 MPa In the current study, HMSNs with a mesoporous shell thickness between 14.40 and 16.20 nm, produced using 3% (w/v) PEG 4000 and PEG 6000, are expected to exhibit these desirable characteristics, with PEG 6000 selected for further investigations.
4.2 Effect of PEG weight percentage on the mesoporous shell thickness of dSiO 2 @MSN
This study examines the impact of varying PEG weight percentages (1% to 5% w/v) on the mesoporous shell thickness of dSiO2@MSN The resulting samples, labeled dSiO2@MSN-Py% (where y ranges from 1 to 5), were analyzed using DLS size measurements and FESEM imaging Consistent with previous findings regarding PEG molecular weight, the dSiO2@MSN-Py% samples exhibited spherical, monodisperse characteristics with uniform particle sizes (PI between 0.062 and 0.133) The mesoporous shell was effectively coated onto the dSiO2 hard template, which displayed indistinct boundaries and a slightly rough surface The mesoporous shell thickness was calculated based on the particle diameters of dSiO2, dSiO2@MSN, and dSiO2@MSN-Py%, as determined from FESEM images using ImageJ, with results summarized in Table 4.2.
Figure 4.2 Size dispersion by DLS measurement and field-emission scanning electron microscopy (FESEM) images of (a, a’) dSiO2@MSN-P1%, (b, b’) dSiO2@MSN-P2%, (c, c’) dSiO2@MSN-P3%, (d, d’) dSiO2@MSN-P4% and (e, e’) dSiO2@MSN-P5%
Increasing the concentration of PEG 6000 from 1% to 5% (w/v) resulted in a corresponding increase in mesoporous shell thickness from 10.45 nm to 18.25 nm, confirming that PEG enhances the shell thickness of HMSN particles However, ANOVA analysis and Bonferroni Correction revealed no statistically significant differences among the samples dSiO2@MSN-P2%, dSiO2@MSN-P3%, dSiO2@MSN-P4%, and dSiO2@MSN-P5%.
In aqueous solutions, the ether oxygen atoms of polyethylene glycol (PEG) form hydrogen bonds with water molecules, leading to the formation of an ordered net structure from separate zigzag chains of PEG As the molecular weight of PEG increases at a constant concentration, the horizontal expansion of the PEG net structure also increases Additionally, increasing the concentration of the same PEG in the reaction system results in a vertical expansion of its ordered net structure.
Table 4.2 presents the impact of varying weight percentages of PEG 6000 (ranging from 1% to 5%) on the particle diameter and mesoporous shell thickness of dSiO2@MSN samples, as analyzed through FESEM images The results indicate that means sharing the same letters (a, b, c) are statistically similar, with a significance level of p < 0.05.
Particles Diameter (nm) Shell thickness (nm) dSiO2 65.3 ± 1.99 - dSiO2@MSN 75.8 ± 1.83 5.25 dSiO2@MSN-P1% 86.2 ± 1.01 10.45 dSiO2@MSN-P2% 94.3 ± 0.87 a 14.50 dSiO2@MSN-P3% 97.7 ± 4.77 a 16.20 dSiO2@MSN-P4% 99.8 ± 2.46 a 17.25 dSiO2@MSN-P5% 101.8 ± 1.68 a 18.25
Exceeding the saturation adsorption of PEG in solution alters the growth kinetics of nanoparticles, enhancing nucleation and agglomeration Increasing PEG concentration or molecular weight promotes silica nanoparticle formation due to PEG's role as an inert polymer that reduces surface tension This reduction aids in the nucleation and condensation of silica nanoparticles, leading to thicker mesoporous shells with higher PEG concentrations or molecular weights However, beyond a certain threshold of TEOS precursors and specific reaction conditions, further increases in PEG molecular weight or concentration do not enhance nanoparticle nucleation and agglomeration, aligning with previous studies on PEG's influence on nanoparticle crystallization.
Figure 4.3 The structure of PEG changes from a) zigzag chains to b) ordered net structure in the solution
Based on the mesoporous shell thickness data and statistical analysis, PEG 6000 at a 2% (w/v) concentration was chosen for synthesizing HMSN for subsequent experiments To assess the impact of PEG on the properties of the synthesized HMSN materials, the resulting particles containing PEG, referred to as HMSN-P2%, will be characterized and compared with HMSN-0.
Characterizations of the synthesized HMSNs
The synthesized HMSN particles were characterized using various techniques, including TEM, Zeta potential, FT-IR, and EDX analysis, with results illustrated in Figures 4.4 and 4.5.
The morphological analysis of dSiO2@MSN and HMSN samples, both with and without PEG, revealed monodispersed spherical particles TEM image analysis indicated that HMSN-0 particles had an average diameter of 79.8 nm and a mesoporous shell thickness of 7.4 nm, while HMSN-P exhibited a larger diameter of 95.8 nm and a thicker mesoporous shell of 14.4 nm These findings align with previous results from section 3.2, derived from DLS size measurements Additionally, the zeta potential measurements for both HMSN-0 and HMSN-P showed negative values around -50 mV, confirming the effective removal of PEG from the synthesized HMSN-P samples.
The synthesized silica nanoparticles were characterized using various techniques, including TEM images of dSiO2@MSN-0 and dSiO2@MSN-P, as well as HMSN-0 and HMSN-P Additionally, the size distribution of HMSN-0 and HMSN-P was analyzed, along with the zeta potential measurements for both HMSN-0 and HMSN-P.
BET and BJH analyses were performed to assess the surface area and mesopore diameter of two samples, with results illustrated in Figure 4.5 Both dSiO2@MSN-0 and dSiO2@MSN-P exhibited Langmuir type IV isotherm curves and type H2 hysteresis loops, indicative of mesoporous materials, as per IUPAC classification This finding indicates that the inclusion of PEG in the reaction mixture during the mesoporous shell coating process did not alter the nitrogen adsorption-desorption isotherms of the original particles.
Figure 4.5 The N2 adsorption-desorption isotherms and pore size distributions of dSiO2@MSN (a and b) and dSiO2@MSN-P (a’ and b’)
The mesopore diameter of dSiO2@MSN-0 and dSiO2@MSN-P was measured at 2.5 nm, consistent with previous studies using CTAB micelle as a soft template for mesochannels Notably, dSiO2@MSN-P exhibited a higher N2 adsorption capacity and BJH Adsorption dV/dD pore volume compared to dSiO2@MSN-0, with surface areas of 76.1 m²/g and 67.2 m²/g, respectively This increase in N2 adsorption capacity, pore volume, and surface area for dSiO2@MSN-P is attributed to the enhanced mesoporous shell thickness Additionally, results from DLS, SEM, and TEM confirm that the incorporation of PEG during the shell coating process of HMSN synthesis contributed to the increased mesoporous shell thickness.
EDX analysis identified the elemental composition on the surfaces of HMSN-0 and HMSN-P, revealing oxygen at 0.53 keV and silicon at 1.75 keV, with atomic percentages of 70.6% oxygen and 29.4% silicon for HMSN-0, and 74.2% oxygen and 25.8% silicon for HMSN-P These findings align with the atomic ratio of silicon dioxide (SiO2), confirming the effective removal of CTAB, PEG, and other reagents Additionally, FT-IR spectra displayed strong absorption bands at 1103 cm -1, 814 cm -1, and 467 cm -1, corresponding to the anti-symmetric stretching, symmetric stretching, and bending vibrations of Si-O-Si, respectively, along with characteristic peaks for Si-OH bending vibrations at 3500 cm -1 and 951 cm -1.
Figure 4.6 Characterizations of the synthesized HMSN-0 (square dot) and HMSN-
P (solid): a) EDX patterns; b) FT-IR spectra
4.3.1 Drug loading and in vitro drug release study of the synthesized HMSN
The DLE and DLC values for DOX in HMSN-0 and HMSN-P were analyzed, revealing that HMSN-P had slightly lower values than HMSN-0 This difference can be attributed to the thicker shell of HMSN-P, despite both having the same hollow cavity volume Consequently, HMSN-P contained fewer particles for the same mass, resulting in less space for DOX loading compared to HMSN-0 However, statistical analysis indicated no significant difference in DLC and DLE between the two samples (p < 0.05) This suggests that while the mesoporous shell thickness increased with PEG synthesis, the drug loading capacity of the HMSN materials remained consistent.
Figure 4.7 DOX loading capacity (DLC - grey) and DOX loading efficiency (DLE
- black) of HMSN-0 and HMSN-P (a); In vitro release profile of Dox@HMSN-0 (empty circle) and Dox@HMSN-P (solid circle) (b) The marked points correspond to 0, 1, 3, 6, 9, 12, 24, 36 and 48 h, respectively
The drug release profiles of Dox@HMSN-0 and Dox@HMSN-P under microenvironmental conditions, illustrated in Figure 4.7b, demonstrated sustainable release characteristics Both formulations released no more than 35% of the initial DOX within 48 hours However, after the 12-hour mark, the drug release from HMSN-P was approximately 4-5% lower compared to HMSN-0.
0 This suggested that HMSN-P with similar loaded drug but longer meso-channels exhibited better controlled release pattern compared to HMSN-0 The reason for this might be that the drug took longer to pass through the longer meso-channels to the outside [73, 74].
Cytotoxicity of the synthesized HMSN
The cytotoxicity of synthesized HMSN-0 and HMSN-P was assessed using MTT assays on the MCF-7 cell line, as depicted in Figure 4.8 The results showed that cell viability remained at 100% with 10 µg/mL concentrations of both materials, and although there was a gradual decrease in viability with increasing concentrations up to 250 µg/mL, it still exceeded 80% These findings indicate that HMSN-0 and HMSN-P exhibit no cytotoxicity in the tested concentration range, highlighting their potential as biocompatible nanocarriers.
Summary
This study presents the synthesis of HMSN using a hard template method, incorporating PEG as a capping agent during the mesoporous shell coating process The findings reveal that varying the molecular weight and concentration of PEG significantly affects the size of HMSN Specifically, increasing PEG molecular weight from 1000 to 6000 g/mol at a 3% concentration resulted in a mesoporous shell thickness increase from 6.90 to 16.20 nm Additionally, raising the concentration of PEG 6000 from 1% to 5% (w/v) led to a thickness increase from 10.45 to 18.25 nm, as determined by FESEM imaging.
P synthesized with 2% PEG 6000 exhibited a monodispersed spherical morphology, measuring 95.40 nm in diameter and featuring a mesoporous shell thickness of 14.40 nm, which is 7.0 nm thicker than dSiO2@MSN-0 The pore size and surface area for dSiO2@MSN-0 and dSiO2@MSN-P were 2.5 nm with 67.2 m²/g and 2.5 nm with 76.9 m²/g, respectively Although HMSN-P demonstrated a slightly lower drug loading capacity, it provided a superior controlled drug release profile compared to HMSN-0 Importantly, both HMSN-0 and HMSN-P showed no in vitro cytotoxicity at concentrations up to 250 µg/mL, indicating their biocompatibility These findings offer valuable insights into the synthesis of hollow mesoporous silica nanoparticles for drug delivery applications.
Figure 4.8 Cell viability by MTT assay with variable concentrations of HMSN-0 and HMSN-P on MCF-7 cells (a); MCF-7 cells treated by HMSN-0 and HMSN-P at different concentrations (b)
The findings of this study have been published in the Journal of Applied Polymer Science, a reputable Q2 journal with an H index of 175 and an impact factor of 3.125 The article, titled “Simply and Effectively Control the Shell,” is available in both print (ISSN 0021-8995) and online (ISSN 1097-4628).
Thickness of Hollow Mesoporous Silica Nanoparticles by Polyethylene Glycol for Drug Delivery Applications” DOI: https://doi.org/10.1016/j.colsurfa.2022.130218.
NON-IONIC SURFACTANTS AS CO-TEMPLATES TO
Preparation of mixed micelles of non-ionic surfactants with CTAB
In this study, non-ionic surfactants Brij S10, Tween 20, and Tween 80 were utilized to create mixed micelles with a constant concentration of CTAB, resulting in a gradual increase in the molar ratio of surfactants to CTAB (nS:nCTAB) The viscosity and dynamic light scattering (DLS) diameter of the resulting mixed micelles were measured, with the findings illustrated in Figure 5.1.
The viscosity of mixed micelle solutions was measured using a Brookfield DV III Rheometer at 250 rpm, revealing notable trends among different surfactant combinations Both T20-CTAB and T80-CTAB showed a decrease in viscosity upon addition, while the viscosity of polysorbate-CTAB solutions increased with higher molar ratios, reaching values between 3 to 30 cP, but remained significantly lower than the original CTAB micelle viscosity of 57.09 cP In contrast, BS10 notably elevated the viscosity of its mixed micelle solution from 57.09 to 484.02 cP as its molar ratio increased This variation in viscosity is crucial, as it reflects the mechanical and physical properties of the medium, potentially influencing the synthesis process.
Figure 5.1 a) Viscosity of mixed micelles versus molar ratio of non-ionic surfactants and CTAB Molar concentration of CTAB remained constantly at 0.02
M b) Hydrodynamic diameter of mixed micelles versus molar ratio Molar concentration of CTAB in each mixture was 50 mM in the presence of 1 mM KBr
At a concentration of 0.02 M, significantly exceeding the critical micelle concentration (CMC) of CTAB and its binary combinations with non-ionic surfactants, the mixed micelles exhibited cylindrical forms To assess the relationship between the diameters of these cylindrical micelles and the molar ratios of non-ionic surfactants to CTAB, solutions were diluted to a CTAB concentration of 50 mM, where spherical micelles form Measurements of the hydrodynamic size revealed that the mixed micelle diameters were considerably larger than those of CTAB micelles As the molar ratio of non-ionic surfactants to CTAB increased, the micelle diameter also increased for each binary combination Notably, the size changes in mixed micelles T20-CTAB and T80-CTAB showed similar trends, with diameters rising from 3.1 nm to 4.0 nm and from 3.2 nm to 4.2 nm, respectively, as the ratio shifted from 1:4 to 1:2 These findings on the spherical mixed micelle diameters are crucial for predicting the mesopore diameters of synthesized hollow mesoporous materials using mixed micelles as soft templates.
Effect of non-ionic surfactants on the mesoporous shell thickness of
This study examines the impact of Tween 20, Tween 80, and Brij S10 on the mesoporous shell thickness of dSiO2@MSN by utilizing mixed micelles of these non-ionic surfactants alongside CTAB at varying molar ratios during the shell coating process The resulting samples, labeled dSiO2@MSN-S, were characterized using Dynamic Light Scattering (DLS) to determine their size (Figure 5.2).
The DLS measurements reveal the size distribution of dSiO2@MSN-T20, dSiO2@MSN-T80, and dSiO2@MSN-BS10, indicating that the diameters of dSiO2@MSN and dSiO2@MSN-S are larger than that of dSiO2 This confirms the successful coating of the mesoporous shell onto the dSiO2 templates, resulting in spherical, monodisperse particles with a high degree of uniformity (PI values ranging from 0.062 to 0.133) Additionally, the mesoporous shell thickness was calculated based on the DLS diameter of dSiO2, previously reported as 96.8 ± 1.99 nm in Chapter 3.
Table 5.1 The mesoporous shell thickness (nm) of dSiO2@MSN particles versus the molar ratio of non-ionic surfactants with CTAB in mixed micelles
As the molar ratio of non-ionic surfactants to CTAB increased, the thickness of the mesoporous shell also increased, indicating that a higher concentration of non-ionic surfactants in the mixed micelle results in a larger micelle diameter and a thicker mesoporous shell This behavior can be illustrated by the model in Figure 5.3, which shows that as the diameter of the micelles used as co-templates for mesopores increases, the residual surface area and the space surrounding the dSiO2 templates for siloxane bridge condensation become limited, consequently leading to an increase in the mesoporous shell thickness.
To explore the impact of non-ionic surfactants on the mesopore diameter of HMSN, three synthesized dSiO2@MSN samples were analyzed These samples were created using mixed micelles of T20, T80, or BS10 combined with CTAB at a molar ratio of nS:nCTAB of 1:2 The study focused on determining their surface area and mesopore diameter.
Figure 5.3 Illustration of the effect of non-ionic surfactants in mixed micelles on the mesoporous shell thickness of dSiO2@MSN
Effect of non-ionic surfactants on the mesopore diameter of
The mesopore diameter can be predicted from the mixed micelle S-CTAB diameter; however, to accurately determine the mesopore size distribution, the BET method and BJH model are essential The nitrogen adsorption-desorption isotherms and pore size distributions of dSiO2@MSN samples, illustrated in Figure 5.4, reveal that the four samples exhibit a Langmuir type IV isotherm curve and type H2 hysteresis loop, as classified by IUPAC This indicates that the incorporation of non-ionic surfactants during the mesoporous shell coating did not alter the nitrogen adsorption-desorption characteristics of the original particles.
Figure 5.4 The N2 adsorption-desorption isotherms and pore size distributions of a) dSiO2@MSN, b) dSiO2@MSN-T20, c) dSiO2@MSN-T80 and d) dSiO2@MSN-
The study revealed that using micelles such as CTAB, T80-CTAB, T20-CTAB, and BS10-CTAB as soft templates significantly enhanced the N2 adsorption capacity and pore size of the samples The introduction of the non-ionic surfactant led to a notable increase in pore diameter from 2.5 nm to over 4.0 nm, with dSiO2@MSN-BS10 exhibiting the largest mesopores at 4.5 nm, while dSiO2@MSN-T20 and dSiO2@MSN-T80 showed similar pore sizes of 4.1 nm and 4.0 nm, respectively Correspondingly, the surface areas of the samples increased, measuring 67.2 m²/g for dSiO2@MSN, 91.8 m²/g for dSiO2@MSN-T20, 92.2 m²/g for dSiO2@MSN-T80, and 106.7 m²/g for dSiO2@MSN-BS10 These findings align with the mixed micelle diameter results, illustrating the significant impact of non-ionic surfactants on mesopore diameter and mesoporous shell thickness.
Characterizations of the synthesized HMSNs
The synthesized HMSN particles, both with and without non-ionic surfactants at a molar ratio of 1:2 (nS:nCTAB), were thoroughly characterized using various techniques including SEM, TEM, particle size distribution analysis, Zeta potential measurement, XRD, and FT-IR.
Figure 5.5 SEM images, TEM images, Size distribution and Zeta potential of
HMSN, HMSN-T20, HMSN-T80 and HMSN-BS10
The synthesized hollow mesoporous silica nanoparticles (HMSNs), both with and without non-ionic surfactants, exhibited a uniform spherical morphology and narrow size distribution, as confirmed by SEM images The particle diameters were measured at 77.5 nm for HMSN, 99.3 nm for HMSN-T20, 100.8 nm for HMSN-T80, and 110.4 nm for HMSN-BS10, aligning with TEM findings TEM analysis further revealed that the HMSNs maintained their hollow shell structure despite the presence of non-ionic surfactants Additionally, the zeta potential measurements for all samples showed negative values around -50 mV, indicating successful removal of CTAB and other non-ionic surfactants from the synthesized HMSNs.
The XRD patterns in Figure 5.6a displayed broad peaks around 2θ = 23°, indicating that the synthesized HMSN samples retained their amorphous silica structure, regardless of the presence of non-ionic surfactants.
Figure 5.6 a) XRD patterns and b) FT-IR spectra of HMSN, HMSN-T20, HMSN-
The synthesized HMSNs demonstrated high purity, as indicated by the absence of additional peaks in the XRD patterns The FT-IR spectra revealed key information about the functional groups present, with strong absorption bands at 1098-1105 cm⁻¹, 807-811 cm⁻¹, and 466-468 cm⁻¹ corresponding to the anti-symmetric stretching, symmetric stretching, and bending vibrations of Si-O-Si, respectively Additionally, two notable absorption peaks for the bending vibration of Si-OH were observed at wavelengths of 3430-3440 cm⁻¹ and 955-963 cm⁻¹.
Drug loading and in vitro drug release study of the synthesized HMSNs 90 5.6 Cytotoxicity of the synthesized HMSNs
The DLE and DLC values for RB in HMSN, HMSN-T20, HMSN-T80, and HMSN-BS10 were significantly lower than those of HMSN-S, likely due to differences in mesopore diameter and cargo size Previous studies indicated that HMSN, with a mesopore diameter of 2.5 nm, achieved DLE and DLC values of 5.8% and 11.09% using Doxorubicin (DOX), which has a molecular weight of 543.52 g/mol In contrast, the RB used in this study has a molecular weight of 1017.64 g/mol, making it more challenging to pass through the original HMSN's mesopores, resulting in low DLE and DLC values of 1.95% and 3.82%, respectively However, the introduction of non-ionic surfactants enlarged the mesopore diameters of HMSN-T20, HMSN-T80, and HMSN-BS10 to over 4.0 nm, facilitating the passage of RB and increasing cargo loading The similar DLE and DLC values of HMSN-T20 and HMSN-T80 were attributed to their comparable mesopore sizes, while HMSN-BS10 exhibited the highest DLE and DLC due to its largest mesoporous channels, as indicated by BET and BJH analyses.
The drug release profiles indicate that RB@HMSN-S exhibited similar cumulative drug release over time, demonstrating superior control compared to RB@HMSN The burst release observed in RB@HMSN is attributed to the absorption of RB on the surface, while the drug remains trapped inside the particles In contrast, RB@HMSN-S requires more time for the loaded RB to be released through its longer meso-channels Among the three systems, RB@HMSN-BS10 displayed a slightly faster release profile, which aligns with its larger mesopore diameter as noted in previous findings.
The loading capacity (DLC) and loading efficiency (DLE) of various HMSN formulations, including HMSN, HMSN-T20, HMSN-T80, and HMSN-BS10, are illustrated in Figure 5.7(a) Additionally, the in vitro release profile of Rose bengal (RB) from these formulations is presented in Figure 5.7(b), with marked time points at 0, 1, 3, 6, 9, 12, 24, 36, 48, and 60 hours.
5.6 Cytotoxicity of the synthesized HMSNs
The cytotoxicity of synthesized HMSN and HMSN-S was assessed using MTT assays on the MCF-7 cell line, as shown in Figure 5.8 The results indicated that cell viability remained at 100% with a treatment concentration of 10 µg/mL for both materials Although there was a gradual decrease in cell viability with increasing concentrations, it stayed above 80% even at 250 µg/mL This suggests that the synthesized nanocarriers exhibited no cytotoxicity on MCF-7 cells within the tested range of 10 to 250 µg/mL, highlighting the biocompatibility of HMSN, whether synthesized with or without non-ionic surfactants.
Figure 5.8 a) Cell viability by MTT assay on MCF-7 cells; and b) MCF-7 cells treated by HMSN, HMSN-T20, HMSN-T80 and HMSN-BS10 at different concentrations
Summary
In a recent study, three non-ionic surfactants—Tween 20, Tween 80, and Brij S10—were effectively utilized as co-templates with CTAB in varying molar ratios to modify the mesopore diameter during the synthesis of HMSN via the hard template method The resulting HMSN-S, synthesized with mixed micelles S-CTAB, exhibited significantly larger mesopores, with HMSN-T20 and HMSN-T80 achieving meso-channel diameters of 4.0 nm at a molar ratio of 1:2, while HMSN-BS10 reached 4.5 nm In contrast, the original HMSN featured a smaller mesopore diameter of 2.5 nm.
The study demonstrated that as the mesopore diameter of hollow mesoporous silica nanoparticles (HMSNs) increased, there was a corresponding increase in mesoporous shell thickness Furthermore, all synthesized HMSNs were characterized and confirmed to be biocompatible materials These findings provide valuable insights into the fundamental theory of HMSN synthesis, particularly for drug delivery applications.
The research results of this content have been published in the journal Colloids and Surfaces A: Physicochemical and Engineering Aspects, SCIE/SSCI, Q2, H index
179, IF 5.518, ISSN 0927-7757 with the title “Non-ionic Surfactants as Co-
Templates to Control the Mesopore Diameter of Hollow Mesoporous Silica Nanoparticles for Drug Delivery Applications” DOI: https://doi.org/10.1016/j.colsurfa.2022.130218.
SURFACE MODIFICATION OF HOLLOW MESOPOROUS
Activation Pluronic with NPC
The Fourier FT-IR spectra of activated NPC-Plu-OH samples displayed absorption signals at 2878 cm⁻¹, 1468 cm⁻¹, and 1109 cm⁻¹, corresponding to the characteristic oscillations of -OCH₂-CH₂-, -NO₂, and C-O, respectively These findings confirm the successful activation of the three Pluronic compounds with NPC.
Figure 6.1 FT-IR spectra of NPC-Plu-OH
The 1 H-NMR analysis of NPC-Plu-OH samples revealed distinct chemical shift signals, confirming the successful activation of Pluronics by NPC Key signals included δH = 3.34 ppm for HC–O– (d), δH = 3.80 – 3.35 ppm for –O–CH2–CH– (OCH2–)(CH3) (e), δH = 3.83 – 3.82 ppm for –O–CH2–CH2–OCOO– (c), δH = 4.46 – 4.44 ppm for –O–CH2–CH2–OCOO– (c', c"), δH = 7.47 – 7.45 ppm (b), and δH = 8.33 – 8.31 ppm (a) These results are consistent with the expected structure of NPC-Plu-OH.
Figure 6.2 1 H-NMR spectra of NPC-Plu-OH: a) NPC-L64-OH, b) NPC-F68-OH, c) NPC-F127-OH, d) Annotation the molecular structure of NPC-Plu-OH
Amination of HMSNs’ surface
The HMSN particles after being surface modified with APTES were characterized to demonstrate successful amination through zeta potential, total amount of amine groups, DLS, FT-IR and EDX analysis
The surface charge of HMSNs was measured at -46.6 mV, attributed to the hydroxyl groups present on their surface Following amination, the amine groups became protonated to form NH3+, resulting in a positive charge of 49.6 mV This transition from a negative to a positive surface charge indicates the successful functionalization of HMSNs with APTES.
The quantity of amine groups on the surface of HMSN-NH2 was assessed using the Kaiser test, which revealed a characteristic pink complex formed from the reaction between ninhydrin and -NH2 This complex was subsequently measured for its UV absorbance.
570 nm and calculated against a standard curve to quantify the surface -NH2 groups
[44, 157] The result of the analysis was identified 78.62 àg per 100 mg of HMSN-
NH2, which was consistent with the previous publication [44]
The hydrodynamic diameters of HMSN and HMSN-NH2 were measured at 108.1 nm and 109.6 nm, respectively, indicating their similarity This resemblance is attributed to the low molecular weight of APTES, which is 221.37 g/mol.
Figure 6.3 Characterizations of HMSN and HMSN-NH2: a) Zeta potential; b) Hydrodynamic particle diameter; c) FT-IR spectra; and d) EDX patterns
The FT-IR spectra of HMSN and HMSN-NH2 both showed adsorption bands at
The observed peaks at 1090 cm⁻¹, 952 cm⁻¹, and 801 cm⁻¹ are attributed to the stretching vibrations of Si-O-Si and the asymmetric bending and stretching of Si-OH Additionally, broad bands in the range of 3300 - 3500 cm⁻¹ indicate the OH stretching of silanol groups, while the peak at 1629 cm⁻¹ corresponds to the OH stretching of water These signals confirm the presence of silicon in the two samples.
IR spectrum of HMSN-NH2 appeared new signals at wavenumbers of 2925 cm -1 and
The FT-IR analysis revealed successful amination of the HMSN surface, indicated by C-H tensile and bending vibrations of the aminopropyl group at 1416 cm-1 Additionally, a weak absorption peak at 1547 cm-1 confirmed the bending vibration of the NH2 group, further supporting the presence of the amine group.
The EDX pattern of HMSNs revealed the presence of only silica and oxygen, with atomic percentages of 68.82% and 31.18%, respectively, indicating the effective removal of CTAB and other reagents In contrast, the EDX pattern of the HMSN-NH2 sample showed the presence of carbon and nitrogen, with atomic percentages of Si, O, C, and N at 22.96%, 53.85%, 16.19%, and 7.00%, respectively, confirming the successful modification of APTES on the surface of HMSNs.
Modification of HMSNs’ surface with Pluronics via amine intermediate 97 6.4 Dual drugs loading capacity and in vitro release behavior of HMSN-
The surface modification of HMSNs was achieved using three types of Pluronics: L64, F68, and F127, through reactions between HMSN-NH2 and activated NPC-Plu-OH The resulting samples, HMSN-L64, HMSN-F68, and HMSN-F127, were characterized by zeta potential, dynamic light scattering (DLS) size, Fourier-transform infrared spectroscopy (FT-IR), and thermogravimetric analysis (TGA), with results illustrated in Figure 6.4.
The zeta potential values for the samples HMSN-L64, HMSN-F68, and HMSN-F127 were measured at 37.8 mV, 30.2 mV, and 20.2 mV, respectively, indicating all samples were positively charged but lower than HMSN-NH2 This reduction in zeta potential may result from the reaction between activated Pluronics and the amine groups on the HMSN-NH2 surface, which decreases the number of amine groups on the nanoparticles Additionally, with zeta potential values exceeding 15 mV, the HMSN-Plu particles are anticipated to exhibit stability based on electrostatic considerations.
The hydrodynamic diameter of HMSN-Plu samples, as shown in Figure 6.4b, demonstrated a gradual increase in dynamic light scattering (DLS) size from 124.5 nm to 142.1 nm and 172.7 nm with the conjugation of Pluronics of increasing molecular weight (2900 kDa to 8400 kDa and 12600 kDa) This increase is attributed to the corresponding elongation of Pluronic molecule chains Additionally, the zeta potential and DLS results indicate that Pluronics were effectively denatured on the surface of HMSNs.
Figure 6.4 Characterizations of HMSN-L64, HMSN-F68 and HMSN-F127: a)
Zeta potential; b) Hydrodynamic particle diameter; c) FT-IR spectra; and d) TGA graphs
To better confirm the successful Pluronics denaturation on HMSN surface, FT-
IR and TGA experiments were conducted, revealing the FT-IR spectra of HMSN-Plu, which displayed absorption signals from both the naked HMSN particles and activated NPC-Plu-OH The absorption peaks at 3300-3500 cm -1 and 1089 cm -1 were associated with the -OH and Si-O-Si groups of the naked HMSN Additionally, the FT-IR spectra of HMSN-Plu exhibited absorption peaks at 2888 cm -1 and 1111 cm -1, attributed to the -OCH2-CH2- groups of the Pluronics.
The TGA graphs of HMSN and HMSN-Plu, as illustrated in Figure 6.4d, indicate that initial mass loss below 200 °C is attributed to the removal of humidity and the condensation of surface silanols In contrast, the weight loss observed above 200 °C is directly linked to the degree of surface functionalization.
Thermogravimetric analysis (TGA) data revealed the weight loss values of HMSN and HMSN-Plu across temperature ranges of 0-200 °C and 200-800 °C, as detailed in Table 6.1 Specifically, the weight loss percentages for HMSN-L64, HMSN-F68, and HMSN-F127 in the 200-800 °C range were found to be 14.56%, 17.61%, and 19.05%, respectively, indicating a correlation with the amount of Pluronic modifying agents on the HMSNs' surface The TGA results, in conjunction with FT-IR, zeta potential, and dynamic light scattering (DLS) analyses, confirmed the successful modification of the HMSNs' surface with the three Pluronics.
Table 6.1 Weight loss (%) of HMSN and HMSN-Plu by temperature ranges through thermogravimetric analysis
6.4 Dual drugs loading capacity and in vitro release behavior of HMSN-Plu
DOX and QUE were respectively encapsulated into the conventional HMSN and the modified HMSN-Plu Their loading capacity and loading efficiency were presented in Table 6.2 as follow:
Table 6.2 Loading capacity (DLC) and loading efficiency (DLE) for Doxorubicin
(DOX) and Quecertin (QUE) of HMSN and HMSN-Plu
The loading capacity of DOX in the HMSN-Plu samples showed a slight increase compared to HMSN, likely due to the ability of DOX to interact with the remaining amine groups on the HMSN-Plu surface through an imine formation reaction.
The study revealed significant variations in the loading capacity of QUE among different samples HMSN exhibited the lowest drug loading capacity (DLC) at 2.34%, while HMSN-L64 demonstrated an improved DLC of 9.80% In contrast, HMSN-F68 and HMSN-F127 achieved the highest DLC values, measuring 18.83% and 17.80%, respectively.
The similar DOX loading capacity among the samples can be attributed to the initial encapsulation of DOX in the empty cavities and pores of the materials, allowing the hydrophilic drug to occupy these spaces without competition Subsequently, QUE was loaded into the DOX-encapsulated materials, but the available space for QUE was limited compared to DOX, resulting in the lowest DLC value for QUE in HMSN However, the presence of hydrophobic PPO blocks in the Pluronic molecules played a beneficial role in this process.
HMSN-Plu's surface effectively encapsulates the poorly soluble drug QUE through physical interactions, significantly enhancing its loading capacity An analysis of the structures of L64, F68, and F127 reveals a correlation between QUE loading capacity and the number of PPO blocks; specifically, an increase in PPO units within the Pluronic results in a higher QUE loading capacity for HMSN-Plu.
In vitro drug release behavior of HMSN-Plu
The in vitro drug release behavior of both free and loaded drugs was examined under various conditions: [37 °C, pH 7.4], [18 °C, pH 7.4], and [37 °C, pH 5.5] The findings, illustrated in Figure 6.5, reveal that comparing cumulative drug release at [37 °C, pH 7.4] and [18 °C, pH 7.4] allows for an assessment of the temperature's impact on release behavior Additionally, analyzing the cumulative drug release at [18 °C, pH 7.4] versus [37 °C, pH 5.5] provides insights into how pH values influence drug release dynamics.
In the study of DOX release, free DOX exhibited the fastest release rate, followed by DOX from HMSN, while HMSN-Plu demonstrated superior control over DOX release profiles This indicates that the modified Pluronics on the surface of HMSNs functioned effectively as caps, reducing the opening of mesoporous channels and slowing the release of DOX from the hollow cavity into the surrounding environment.
After 72 h, although the released DOX amount from HMSN-Plu samples increased when the temperature reduced from 37 to 18 o C (at the same pH value of 7.4), the differences were not significant Meanwhile, there were notable increases in the amount of cumulative DOX release after 72 h at pH 5.5 This phenomenon was attributed to the dimerization of DOX in PBS pH 7.4 where DOX interacted with the buffer and formed covalently bonded DOX dimers [165] The results suggested that the three HMSN-Plu systems released DOX in response to pH, in which HMSN-Plu systems performed slow DOX release rates in physiological condition and higher rates in tumor microenvironment condition
Figure 6.5 In vitro release behaviour of free drugs and loaded drugs in different conditions of temperatures and pH values
In the study of QUE release, HMSN exhibited burst release profiles across all tested conditions, primarily because the loaded QUE was largely adsorbed onto the surface of HMSNs Although the drug release percentage was high, indicating a significant cumulative release, the absolute mass of the drug released remained minimal Notably, both free QUE and QUE loaded in HMSN-Plu were released more slowly compared to those from HMSN, likely due to the high initial concentration of QUE and its poor solubility This characteristic of the HMSN-Plu system is advantageous, as it helps minimize QUE leakage while circulating in the body under physiological conditions.
HMSN-Plu demonstrated enhanced drug release rates at lower temperatures (18 °C), attributed to the stretching of Pluronic chains, which facilitates the easier release of encapsulated QUE molecules from the hydrophobic PPO blocks due to concentration differences The findings indicated that the three HMSN-Plu systems exhibited temperature-responsive release behavior, with slower QUE release rates at physiological temperatures and accelerated rates in cooler conditions The release dynamics of HMSN-Plu under varying conditions are depicted in Figure 6.6.
Figure 6.6 Illustration of release behavior of HMSN-Plu in different conditions
Cytotoxicity of HMSN-Plu
MTT assays conducted on the HeLa cell line demonstrated that the nanocarriers HMSN-Plu exhibited no significant cytotoxicity Specifically, the viability of HeLa cells treated with HMSN-L64, HMSN-F68, and HMSN-F127 remained unaffected at concentrations up to 250 µg/mL Consequently, HMSN-Plu can be considered biocompatible nanocarriers.
HMSN-L64, HMSN-F68, and HMSN-F127 demonstrate favorable zeta potential values, DLS sizes, and biocompatibility, making them suitable for drug delivery systems Among these, HMSN-F127 emerged as the most promising candidate for encapsulating dual drugs with varying solubility, exhibiting equivalent DLC values for DOX and the highest for QUE Additionally, HMSN-F127 displayed excellent drug release controllability and effective responsiveness to temperature and pH changes, leading to its selection for further studies.
Figure 6.7 a) Cytotoxicity by MTT assay of HMSN-Plu on Hela cells; b)
Morphology of Hela cells treated by HMSN at different concentrations
Characterizations of the HMSN-F127
The synthesized HMSN-F127 has been previously analyzed for zeta potential, DLS size, FT-IR, TGA, loading capacity, and release profile Selected as a suitable nanocarrier for dual drug delivery, HMSN-F127 will undergo further characterization, including morphology assessment via TEM images, porosity evaluation through BJH analysis, structural analysis using XRD, and cytotoxicity testing with the MTT assay.
TEM images revealed that both HMSN and HMSN-F127 are spherical, monodisperse particles, with diameters of 77.16 nm and 119.75 nm, respectively The HMSN-F127 particles, with their optimal size, are capable of loading a significant amount of drug while also extending circulation time during administration.
Figure 6.8 TEM images and Size distribution of a), a’) HMSN and b), b’) HMSN-
The nitrogen adsorption-desorption isotherms of HMSN and HMSN-F127, classified as Langmuir type IV according to IUPAC, indicate their characterization as mesoporous materials The presence of large hysteresis loops in the relative pressure range of 0.5–1.0 further suggests a hollow structure in both particle systems Notably, HMSN exhibits a higher BET surface area of 767 m²/g compared to 446 m²/g for HMSN-F127 Despite similar pore diameters of 2.5 nm for HMSN and 2.2 nm for HMSN-F127, the dV/dD value for HMSN is significantly higher, highlighting its enhanced pore volume characteristics.
HMSN-F127 was due to the fact that F127 molecules presented on HMSNs’ surface and acted as capping agents of the open pores
Figure 6.9 The N2 adsorption-desorption isotherms and pore size distributions of
Figure 6.10a displays the XRD patterns of naked HMSN and HMSN-F127, analyzed with OriginPro software, achieving an accuracy of ± 0.02° across the full measurement range and a peak width of less than 0.05° (as shown in Figures 6.10b and 6.10c).
Figure 6.10 (a) XRD patterns of HMSN (dash line) and HMSN-F127 (solid line);
(b) Fitting XRD peaks of HMSN (XRD pattern – Dash; Cumulative fit peak – Solid line); and (c) Fitting XRD peaks of HMSN-F127 (XRD pattern – Dash; Cumulative fit peak – Solid line)
The XRD patterns of HMSN and HMSN-F127 exhibited broad peaks at 2θ values of 23.5° and 13.9°, indicating their amorphous nature In the HMSN diffractogram, a fitting peak at 2θ 23.18° corresponds to amorphous silica, while the HMSN-F127 pattern shows a reduced intensity peak at 2θ 23.35°, likely due to the intercalation of silane moieties after APTES activation Additionally, a peak at 2θ 13.08° is attributed to APTES, and a peak at 2θ 15.89° is linked to F127 Overall, these XRD patterns confirm the amorphous characteristics of the silica particles and highlight the effects of surface modifications.
F127 had no impact on the structure of the silica particles [142].
Cancer cell killing ability of DOX.QUE@HMSN-Plu
In vitro cancer cell killing ability of free DOX, free QUE and
DOX.QUE@HMSN-F127 against Hela cells was evaluated and presented in Figure
Figure 6.11 a) Cell viability and b) Mophorlogy of Hela cells treated by Free
DOX, Free QUE and DOX.QUE@HMSN-F127
The study demonstrated that free DOX, free QUE, and DOX.QUE@HMSN-F127 samples induced cell death in a concentration-dependent manner Free QUE exhibited the lowest cytotoxicity on Hela cells, with an IC50 value of 3.29 µg/mL, while free DOX achieved 50% cell death at a concentration of 0.07 µg/mL Notably, DOX.QUE@HMSN-F127 exhibited the most potent effect, with an IC50 for DOX at 0.03 µg/mL, half that of free DOX Although DOX was released more slowly from the nanocarrier HMSN-F127, the concurrent release of QUE contributed to a reduced required dose and minimized DOX's side effects Consequently, the DOX.QUE@HMSN-F127 system demonstrated superior cancer-killing efficacy against Hela cells.
Summary
Three nanocarrier systems utilizing HMSN modified with Pluronics L64, F68, and F127 have been successfully developed for dual drug delivery applications Characterization techniques confirmed that HMSN-Plu samples exhibit promising drug delivery potential, featuring zeta potentials exceeding 20 mV and DLS diameters under 120 nm, while demonstrating no toxicity at concentrations up to specified limits.
HMSN-Plu significantly improved the loading capacity and release controllability of DOX and QUE, with HMSN-F127 emerging as the most promising candidate due to its exceptional loading capabilities, particularly for QUE This nanocarrier exhibited temperature-responsive release for QUE and pH-responsive release for DOX, ensuring effective drug delivery Notably, DOX.QUE@HMSN-F127 demonstrated a strong cytotoxic effect against the Hela cell line, indicating its potential as an effective dual drug delivery system for cancer treatment.
In conclusion, this thesis successfully achieved its objectives by synthesizing HMSN materials through hard-templating, which reduced the synthetic time and optimized particle size Additionally, it involved the modulation of mesoporous shell thickness and mesopore diameter, followed by surface modification using Pluronics to enhance the delivery of anti-cancer drugs.
The synthesis method of Hollow Mesoporous Silica Nanoparticles (HMSN) has been successfully optimized, resulting in a significant reduction of the synthetic time from 21 hours to just 9 hours Additionally, this modified process has led to a decrease in HMSN particle size to approximately 80 nm.
The mesoporous shell thickness of HMSN was effectively regulated using PEG during the shell coating process Increasing the molecular weight of PEG (1000, 2000, 4000, and 6000 Da) or its concentration (1, 2, 3, 4, and 5%) resulted in a corresponding increase in shell thickness Notably, HMSN-P synthesized with 2% PEG 6000 exhibited a particle diameter of 95.40 nm and a mesoporous shell thickness of 14.40 nm, demonstrating a slightly lower DOX loading capacity while offering a more controlled DOX release profile compared to the original HMSN.
The mesopore diameter of HMSN was effectively regulated using non-ionic surfactants (Tween 20, Tween 80, and Brij S10) as co-templates during the shell coating process As the molar ratio of surfactant to CTAB increased, the thickness of the mesoporous shell also increased Specifically, HMSN-T20, HMSN-T80, and HMSN-BS10, synthesized at a molar ratio of 1:2, exhibited enlarged mesopore diameters of 4.1, 4.0, and 4.5 nm, respectively These modifications resulted in significantly enhanced loading capacity and improved release controllability for Rose Bengal, a high molecular weight agent, when compared to the original HMSN.
Three types of Pluronics—L64, F68, and F127—were successfully modified on the surface of HMSNs, resulting in enhanced loading capacity and controlled release of DOX and QUE Notably, HMSN-F127 demonstrated superior loading capacities, achieving 8.47% for DOX and 17.80% for QUE, compared to HMSN's 7.90% for DOX and 8.89% for QUE Additionally, HMSN-F127 exhibited effective release controllability, with temperature-responsive release for QUE and pH-responsive release for DOX.
HMSN, HMSN-P, HMSN-S, and HMSN-Plu are biocompatible nanocarriers that demonstrated no cytotoxicity at concentrations up to 250 µg/mL, as confirmed by MTT assays Additionally, the dual drug loading system DOX.QUE@HSMN-F127 exhibited significant inhibition activity against the human cervical cancer HeLa cell line, with an IC50 value for DOX of 0.03 µg/mL, which is half the potency of free DOX.
The hard-template synthesis method of HMSN was successfully modified to reduce the particle size below 100 nm with shortened synthetic time
PEG with different molecular weights and concentrations were investigated and used as capping agent in the shell coating step to control the mesoporous shell thickness of HMSN
Mixed micelles of non-ionic surfactants and CTAB were investigated and used as soft templates in the shell coating step to enlarged the mesopore diameter of HMSN
Pluronic copolymers with varying PPO and PEO unit compositions were utilized to enhance the surface properties of HMSN particles for dual drug loading applications The study assessed the loading capacities of two drugs with different solubility profiles, doxorubicin (DOX) and quercetin (QUE), along with their release behaviors under varying temperature and pH conditions This evaluation aimed to identify the most effective HMSN-Pluronic system for dual drug delivery.
Ongoing research into effective cancer treatments has highlighted HMSN as a promising carrier capable of addressing the limitations of existing therapies Future studies aim to enhance the HMSN-Plu system by incorporating ligands like folic acid for targeted delivery Additionally, modifying HMSN with various stimulus-responsive agents is expected to improve the system's delivery capabilities It is also essential to verify and expand the understanding of the in vitro and in vivo activities of these modified HMSN systems.
1 Ngoc Hoi Nguyen, Cuu Khoa Nguyen, Dai Hai Nguyen, “A Modified Hard-
Templating for Hollow Mesoporous Silica Nanoparticles Synthesis with Suitable Particle Size and Shortened Synthesis Time”, Vietnam Journal of
Science and Technology, ISSN 2525-2518, 0866-708X, Accepted 20 August
2 Ngoc Hoi Nguyen, Dieu Linh Tran, Ngoc-Hang Truong-Thi, Cuu Khoa
Nguyen and Dai Hai Nguyen present a method for controlling the shell thickness of hollow mesoporous silica nanoparticles using polyethylene glycol, aimed at enhancing drug delivery applications This research, published in the Journal of Applied Polymer Science, demonstrates a simple and effective approach to optimize nanoparticle properties for improved therapeutic efficacy.
H index 175, IF 3.125, ISSN 0021-8995 (print); 1097-4628 (web) First published: 17 September 2022 DOI: http://doi.org/10.1002/app.53126
3 Ngoc Hoi Nguyen, Ngoc-Hang Truong-Thi, Dinh Tien Dung Nguyen, Yern
Chee Ching, Ngoc Trinh Huynh, Dai Hai Nguyen, “Non-ionic Surfactants as
Co-Templates to Control the Mesopore Diameter of Hollow Mesoporous Silica Nanoparticles for Drug Delivery Applications”, Colloids and Surfaces A:
Physicochemical and Engineering Aspects, SCIE/SSCI, Q2, H index 179, IF 5.518, ISSN 0927-7757, Received 27 July 2022, Revised 30 August 2022, Accepted 19 September 2022, Available online 21 September 2022, Version of
Record 22 September 2022 DOI: https://doi.org/10.1016/j.colsurfa.2022.130218
4 Ngoc Hoi Nguyen, Dai Hai Nguyen, “A Research on Drug Delivery System based on Mesoporous Silica Nanoparticles for Anti-Cancer”, The International
Chemistry and Its Applications Conference 2022
1 Sung, H., et al., Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries CA: a cancer journal for clinicians, 2021 71(3): p 209-249
2 Bidram, E., et al., A concise review on cancer treatment methods and delivery systems Journal of Drug Delivery Science and Technology, 2019 54: p
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