Thermo-responsive magnetic Fe3O4@P(MEO2MAX-OEGMA100-X) NPs and their applications as drug delivery systems

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Inorganic/organic core/shell NPs

Core/shell NPs made from inorganic core and organic polymer shell represent a new class of materials that exhibit improved performance as the grafting of the inorganic core surface with organic compounds improve their dispersion stability in various liquid media and enhance their chemical functionality and biocompatibility for bio-applications [7]. Inorganic/organic core/shell NPs are often made by grafting synthetic polymers or a shell of any other high density organic material on inorganic particles or by adding modified NPs into polymer matrices and their further use as diagnostic tools.

Inorganic NPs

The inorganic core could be of metal (e.g. Au, Ag, and Fe), metalloid oxide (e.g. SiO2), magnetic oxide (e.g. Fe2O3 and Fe3O4) or semiconductors called quantum dots (e.g. CdSe, CdTe, PbS, ZnO, TiO2). In this thesis we will focus on the core/shell NPs obtained from an iron oxide or a quantum dot core. These two types of inorganic materials have attracted a great deal of attention due to the unique magnetic properties of the iron oxide, electrical/optical properties as well as photoluminescence of the semiconductors quantum dots and the ability for the two of being easily chemically modified for improved biocompatibility and dispersity.


Quantum dots (QDs) have received extensive attention during the past years owing to their advantages over the organic dyes and fluorescent proteins [42]. QDs are nanometer-sized crystals (2-10 nm in diameter) made of semiconductor materials. They are highly luminescent and are extremely resistant to photo bleaching. Moreover, they present a very broad absorption spectrum and a very narrow fluorescence emission spectrum in the visible and near infrared.
QDs exhibit novel optical and electronic properties due to the quantum confinement of excitons and phonons. The phenomenon of quantum confinement arises once the diameter of the particle is smaller than the Bohr radius. Quantum confinement generally results in widening of the band gap and it is the reason when the QDs are dispersed in a solution, the solution of same material in term of chemical composition but with varied diameter, exhibit various colors upon the same excitation energy (Figure 1.1.1) [43].
When a QD is excited with light of specific energy (energy equal or greater than the band gap), the QD absorbs a photon resulting in the formation of an exciton (electron-hole pair). Then the electron falls back down across the band gap towards the valence band, via a radiative electron-hole recombination, generating fluorescence (Figure 1.1.2) and this strongly depends on the size of the band gap, which can be altered by changing the size of the QD by changing the surface chemistry [44].
The strong size-dependence of QDs offers many advantages in use of their optical properties. Depending on the application, CdSe QDs of different sizes ranging from 2 to 8 nm emit photons across almost all the visible band. This unique property also attracts a lot of interest for applications of QDs in biological labeling and optical/electronic devices such as light emitting diodes and photovoltaic devices [45].
Nano-crystals of CdSe, CdTe, CdTe/CdSe and CdTe/ZnTe are reported as popular bio-labels in imaging and detection [46-48]. CdTe/ZnTe QDs can serve as excellent ultrasensitive probes for in vivo targeted bio-imaging. Figure 1.1.3 shows an image of a mouse injected with three different sizes emitting CdTe/ZnTe QDs through subcutaneous and tail vein injections. The treated spots are highly luminescence where the synthesized QDs possessed a high quantum yield (52 %) [49].
However, the acute toxicity of Cadmium based core materials on biological systems due to the release of toxic heavy metal ions, and formation of reactive oxygen species (ROS) limited their uses for health care [50]. Therefore, the development of non-toxic alternatives is currently increasing for health care applications [51]. As a result, zinc oxide (ZnO) is one of the most excellent semiconductors with great potential for replacing the traditional Cd-related species applied in the optical and biological fields [52].

Physical and optical properties

ZnO is a well-known n-type, direct wide-band-gap II–VI semiconductor. Wurtzite (B4 phase), zinc blende (B3) and Rocksalt (B1) are the three crystallographic phases of ZnO [53]. At ambient conditions, wurtzite crystalline ZnO is the thermodynamically stable crystal structure. The structure exhibits a hexagonal structure with two lattice parameters, a = 0.3296 and c = 0.52065 nm in the ratio of c /o =1.602 and belongs to the space group of or P63mc [54]. Each anion is surrounded by four cations at the corners of the tetrahedron, which shows the tetrahedral coordination and hence exhibits the sp3 covalent-bonding (Figure 1.1.4).
Much attention has been given to use ZnO for cell labeling applications because of their photoluminescence (PL) [51]. Wurtzite ZnO has normally two photoluminescent emission bands. One is centered in the UV region and the other is centered in the visible region. The origin of UV emission is well-known to be associated with the radiative electron-hole recombination, and because of their direct association with band gap, it is size dependent emission due to quantum confinement [55]. While for the visible emission, the origin and property of ZnO QDs have not been fully understood and it is believed that this emission is a result of many intrinsic defects such as oxygen vacancies, zinc interstitials, zinc vacancies, antisite oxygen, donor–acceptors pairs, and surface defects [56]. As discussed earlier, the wavelength of the fluorescent light is dependent on the size of the band gap. Bulk ZnO exhibits photoluminescence at about 495 nm excitation while this wavelength is decreased to 350 nm when ZnO is in the nanocrystalline form [57].
ZnO has unique properties compared to its counterparts such as the wide band gap of 3.37 eV, a large excitonic binding energy of 60 meV and a high thermal and mechanical stability at room temperature. ZnO NPs also show piezo- and pyroelectric properties plus its hardness, rigidity, low toxicity, biocompatibility and biodegradability [57]. Therefore, because of its exceptional physical and chemical characteristics, the ZnO based NPs have shown great potential for use in UV laser devices, bio-imaging, drug delivery, sunscreens, photo-catalysis, chemical sensors, biosensors, solar cells and piezoelectric devices [58].


In general, NPs can be prepared by a variety of methods which are usually categorized in two main synthetic routes: the top down and the bottom up approaches. In the top down routes, the NPs are obtained from their bulk materials using different methods like lithography and laser ablation. While, in the bottom approach, the NPs are obtained from their basic building blocks (atoms or molecules) which react to generate the NPs of the desired shape and size [59].
As a versatile and multifunctional nanomaterial with very unique features, ZnO has attracted many researchers to develop a wide number of chemical and physical methods for its synthesis in different nanoscale as it provides one of the greatest assortments of varied particle structures among all known materials. Depending on the synthesis method we can obtain: particles, rods, belts, nanorings, needles, pellets, flowers, snowflakes, wires and other structures [60].
For NPs synthesis some examples are: vapor chemical deposition, precipitation in water solution, hydrothermal synthesis, electrochemical methods, sol-gel process, precipitation from microemulsions and mechanochemical processes [60]. Bio-routes have been also followed to obtain well-defined ZnO nanostructures using plant extracts and biotemplates, including macromolecules, bacteria, and bacteriophages [61]. A recent review reported these so-called green methods offering a good alternative choice over chemical and physical methods as they are environmentally friendly and of low-cost [62].
Chemical synthesis, found to be very simple, cost-effective, is one of the most important techniques which can be performed by using a range of precursors and different conditions like temperature, time, concentration of reactants, and other parameters. Variation of these parameters gives different structures and sizes of the produced NPs. Different chemical methods such as precipitation, hydrothermal and sol-gel were used for the synthesis of ZnO NPs [63]. Among these methods, the sol-gel is one of the most employed for the production of ZnO NPs in view of the simplicity, low cost, reliability, repeatability, smaller particle size and morphological control, better homogeneity and purity. Moreover, the synthesis is conducted in relatively mild conditions of synthesis, which enable during the process the surface modification of ZnO with selected organic compounds [64]. NPs as small as 2.5 nm could be indeed obtained since 1985 by Koch et al. [65].
In sol-gel method, monomers are converted into a colloidal solution (i.e. sol) that acts as the precursor for an integrated network (i.e. gel) of either discrete particles or network polymers
[66]. Figure 1.1.5 illustrates the main stages of ZnO thin films (Figure 1.1.5a) and particles (Figure 1.1.5b) preparation by the sol-gel process. For example, for the film preparation, the precursor solution is first prepared and then the colloidal solution obtained is deposited on the substrate by one of two techniques (spin or dip-coating). After, the gel is dried and the xerogel films are obtained. The xerogel is the dried gel at ambient pressure. In the case of particles preparation, the colloidal solution undergoes condensation to a gel and then the evaporation of the solvent from the gel gives the xerogels. In the final step, the heat treatment in both cases is necessary to obtain well-ordered structures [67].
The sol-gel method for the synthesis of ZnO NPs involves the alkaline hydrolysis of zinc salts in alcoholic or aqueous media. A slow and controlled hydrolysis generally leads to smaller particle sizes [69]. The preparation usually undergoes different stages: solvation (preparation of the precursor solution), hydrolysis (sol), polymerization (gel) and transformation into ZnO (NPs) (Figure 1.1.6).
First, the precursor which is a zinc salt (zinc acetate, zinc nitrate, or zinc acetylacetonate) is dissolved in an alcohol like ethanol, methanol or isopropanol (Equation 1). As a second stage, a colloidal–gel of zinc hydroxide is formed through the addition of a base solution like potassium hydroxide (KOH), sodium hydroxide (NaOH), lithium hydroxide (LiOH) or tetramethyl ammonium hydroxide (TMAH) hydrolyzing the precursor, followed by polymerization of hydroxyl complex to form “Zn–O–Zn” bridges and finally transformed into ZnO (Equation 2).
The choice of the solvent (size and activity) has obvious influence on the reacting progress and the final product [70]. Ethanol has smaller size and a more active OH- and therefore it can react more easily to form a polymer precursor with a higher polymerization degree, which is required to convert sol into gel [71]. Bari et al. had observed that when TMAH was used as the solvent for zinc acetate to synthesis ZnO NPs via sol-gel, the particles were spherical in shape (Figure 1.1.7a) and smaller (< 50 nm), while they were wire-shaped (Figure 1.1.7b) when NaOH was used [72].
The size of the synthesized ZnO QDs can be tuned by adjusting their synthesis time. ZnO QDs with tunable diameters were synthesized via sol-gel using self-made zinc-oleate complex as a precursor [73]. The synthesis time was varied from 5 min to 4 days obtaining NPs with diameters in a range of 2.2-7.8 nm. The results showed that the visible emission property of the ZnO QDs displays highly size-dependent behavior where with size decreasing, the visible emission peaks blue shifts to the positions with shorter wavelength. Figure 1.1.8 shows the normalized PL spectra of the different size ZnO QDs dispersed in ethanol.
On the other hand, the annealing temperature is an important factor to control the morphology of the produced ZnO NPs. Jurablu et al. found that with increasing temperature the morphology of the particles changes to the spherical shape and the NPs were less agglomerated. The average diameter was obtained through transmission electron microscopy (TEM) analysis and found to be about 28 nm [74].
The size of the ZnO NPs can be also controlled by varying the concentration of the precursor, the ratio between the zinc precursor and the base, and the addition of ligands [75]. ZnO NPs were readily synthesized through sol-gel method using zinc acetate as a precursor and the average particle size was found to be 58.3 nm [76]. Self-assembled ZnO NPs with a smaller size of 4 nm were also prepared earlier by the dissolution of zinc acetate dehydrate in ethanol without the aid of special organic additives or face-specific organic molecules [77].
Solid state reaction method is another easy process to obtain crystalline ZnO NPs. In this one, ZnO NPs are prepared by mixing the precursors together at a specific temperature in one step. It is nontoxic, simple, low-cost and high-yield synthetic method, but it has some disadvantages like inhomogeneity, bigger crystallite and phase impurity [78]. A comparison was made by producing ZnO NPs using sol gel and solid state reaction method. It was found that when prepared under the same ambient conditions (temperature, pressure …) and keeping all the parameters same (precursors, molarity, solvent …); the NPs prepared via sol-gel route were highly crystalline and had smaller crystallite size (≈ 24 nm) as compared to the one prepared by solid state reaction method (≈ 37 nm) [68].
After the formation of ZnO NPs, their PL exhibits a continuous red-shift as it was seen that the size continues to grow even after the synthesis is stopped and its quantum yield (QY) decreases gradually [79]. Therefore, protective layers must be employed to the ZnO surface to inhibit further crystal growth and aggregation. Silanes, for examples, are used to coordinate with Zn atoms on the surface of ZnO QDs to hinder the formation of bulk ZnO. This method among the different methods will be mentioned in the next part.


Surface modification

In recent years, obtaining well defined structures of ZnO QDs is not the only concern of researchers because the surface functionalization of these particles is very important to use them in different application. ZnO QDs require enhanced properties which are a result of their surface functionalization. For example, when using the ZnO QDs in bio-imaging, it is necessary that these dots are able to conserve their fluorescence at one point and to easily disperse in aqueous media.
Consequently, the functionalization of ZnO QDs is of high importance for some purposes. On one hand is to stabilize the dots, especially in aqueous solution, because stability toward water is strictly necessary for biological applications as the water molecules are able to degrade the ZnO QDs which lead to the loss of their photoluminescence properties [79]. On the other hand the grafting of specific functional groups onto ZnO surface for targeted analysis. On the third hand is to change or enhance ZnO luminescent properties by optimizing the surface structure of nanocrystals and minimizing the number of surface trap sites [43]. In order to stabilize ZnO QDs, various shells have been employed like silanes and polymers [80, 81]. In this context, during or after each ZnO QDs synthesis, a surface modification step is included [82-86]. Surface modification methods proposed by different groups are summarized in Figure 1.1.9. These methods are divided into inorganic (SiO2, Al2O3), organic compounds (organic acids or silanes) and polymer layers (methacrylates) mentioning some of the resultant effects of each type of modification on the ZnO QDs.

Table of contents :

Chapter 1: General Overview
1. Inorganic/organic core/shell NPs
1.1. Inorganic NPs
1.1.1. ZnO QDs
1.1.2. Iron oxide NPs
1.2. Thermo-responsive polymers
1.2.1. Properties
1.2.2. Oligo (ethylene glycol) methacrylates
1.2.3. Effect of salt on polymer behavior
1.3. Polymerization process of polymer on NPs surface
1.3.1. Grafting methods “grafting from”
1.3.2. Polymerization methods
2. Biological applications of inorganic/organic core/shell NPs
2.1. NPs engineering
2.2. In vitro and in vivo applications
3. Characterization techniques
3.1. High resolution Transmission electron microscopy (HR-TEM)
3.2. Dynamic Light Scattering (DLS)
3.3. X-ray diffraction (XRD)
3.4. Fourier transmission Infrared Spectroscopy (FT-IR)
3.5. Ultraviolet-Visible (UV-Vis) spectroscopy
3.6. Fluorescence spectroscopy
3.7. Thermal Gravimetric Analysis (TGA)
Chapter 2: Efficient synthetic access to thermo-responsive core/shell nanoparticles
2.1. Materials
2.2. Synthesis of ZnO QDs coated with the silane derivatives
2.2.1. Synthesis of hydrophobic ZnO@oleate QDs
2.2.2. Silanization of ZnO QDs
2.2.3. Synthesis of ZnO QDs coated with the P(MEO2MAX-OEGMA100-X)
2.2.4. Preparation of Fe3O4 NPs
2.2.5. Silanization of Fe3O4 NPs
2.2.6. Synthesis of Fe3O4 coated with the P(MEO2MAX-OEGMA100-X)
2.2.7. SiO2 NPs
2.3. Characterization Methods
3.1. Growth of PS from SiO2@Ph-Cl
3.2. Growth of P(MEO2MAX-OEGMA100-X) from ZnO and Fe3O4@Ph-Cl NPs
3.3. Physical characterization of the core/shell NPs
3.4. Amount of copolymer grafted at the surface of the NPs
3.5. Temperature responsive properties of the core/shell NPs
3.6. Fluorescence properties of the ZnO@copolymer samples
3.7. Magnetic properties of the Fe3O4@copolymers
Chapter 3: Synthesis, characterization and cytotoxicity of ZnO@P(MEO2MAX-OEGMA100-X) NPs
2.1. Materials
2.2. Synthesis of ZnO@P(MEO2MAX-OEGMA100-X) NPs
2.3. Cytotoxicity tests
2.3.1. Cell culture
2.3.2. Adding the NPs
2.3.3. Performing the MTT cell viability test
3.1. Characterization of ZnO and ZnO@P(MEO2MAX-OEGMA100-X) NPs
3.1.1. Chemical Characterization
3.1.2. Microstructural Characterization of the core/shell NPs
3.1.3. Amount of the grafted copolymer
3.1.4. Optical properties of the core/shell NPs
3.2. Thermo-responsive behavior of ZnO@P(MEO2MAX-OEGMA100-X) NPs
3.2.1. In water
3.2.2. In physiological media
3.3. Cytotoxicity Tests
3.3.1. Cytotoxicity of ZnO@P(MEO2MAX-OEGMA100-X) NPs
3.3.2. ZnO@P(MEO2MAX-OEGMA100-X) NPs loaded with DOX
Chapter 4: Thermo-responsive magnetic Fe3O4@P(MEO2MAX-OEGMA100-X) NPs and their applications as drug delivery systems
2.1. Materials
2.2. Synthesis process
2.3. Drug release
2.4. Cytotoxicity
2.5. Characterization Methods
3.1. Chemical characterization
3.2. Microstructural characterization of the NPs
3.3. Amount of grafted co-polymer
3.4. Magnetic properties
3.5. Thermo-responsive behavior of the core/shell NPs
3.6. Drug release from Fe3O4@P(MEO2MAX-OEGMA100-X) NPs
3.6.1. DOX release
3.6.2. In vitro cytotoxicity study


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