HALLOYSITE CLAY NANOTUBES FOR BIOMEDICAL AND INDUSTRIAL APPLICATIONS: OPTIMIZATION OF THEIR PHYSICO-CHEMICAL PROPERTIES.

Present PhD thesis aimed to investigate relatively unknown properties of halloysite nanoparticles, as well as to further examine HNTs as potential drug nanocarriers. NPs loading and release characteristics were studied using model active molecules: magnesium monoperoxyphthalate (MMPP), aspirin and epirubicin. The research was fulfilled with formation of complex multi-functional nanoarchitectures, which apart from ability to deliver incorporated drugs, showed the potential of controlled and sustain release of therapeutics, biocompatible and bioresorbable characteristics as well as potential targeting abilities. Great attention was dedicated to characterization of formed halloysite-based nanoarchitectures in qualitative as well as quantitative manner. Investigations performed in this thesis also faced the problem of exceeding dimensions of halloysite units, nanoparticles aggregation, poor loading capability and dose dumping effect. Subsequently, studies for trying to find a solution to these obstacles were undertaken. Fully characterized halloysite nanoconstructs were further examined in biological field, employing different cancer cell lines. Studies on pristine halloysite nanotubes: Physico-chemical and biological properties of halloysite nanoparticles were evaluated using microscopic techniques, spectroscopic analysis, surface studies regarding charge, porosity and wettability. The thermal and time-based examination of pristine halloysite was performed as well, showing stability of HNTs alumino-silicate skeletons up to ~400 ℃ and over a long period of time (2 years) at room temperature, however with a variable amount of incorporated water molecules. Biological performance of HNTs was determined in vitro in multiple cellular systems by toxicity, cellular uptake, colocalization and accumulation studies using [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] tetrazolium reduction (MTT) assay and set of microscopic techniques. Aiming to deeply characterize halloysite nanoparticles, the study proceeded with employment of non-standard techniques, as multiphoton microscopy that drove to discovery of novel NPs promising capabilities. It was revealed that halloysite is able to convert light to its second harmonic, at twice of the frequency (and therefore half of the wavelength) while using high intensity femtosecond pulsed laser. Halloysite Second Harmonic Generation (SHG) signal was detected over a broad wavelength range, showed stability over a long period of time, polarization properties and quadratic dependence on the intensity of incident light. The analysis also pointed out characteristic structure properties of the nanoparticle that is lack of the center of symmetry and the high crystalline structure organization. Among a wide spectrum of domains where discovered HNTs characteristics can be utilized (e.g. optoelectronics, biosensors), we have explored its application in alternative label-free bioimaging. The proposed multiphoton method of analysis showed advantages over the standard confocal microscopy, since e.g. nanoparticles did not have to be stained prior the analysis, thus no possible alterations of HNTs including size, surface chemistry and consequent cellular uptake were induced. Therefore, for the first time, halloysite nanotubes were exploited as imaging agents, taking advantage of their endogenous properties. Along the research it was revealed that the length of pristine HNTs and the strong aggregation limit their ability to pass intracellular membranes and thus minimize their effectiveness as drug nanocarriers. Therefore, efforts were devoted to the development of facile methodology to efficiently disperse and shorten HNTs units. Set of characterizations techniques, such as Scanning Electron Microscopy (SEM) analysis with size distribution profile and nitrogen adsorption Brunauer–Emmett–Teller (BET) method revealed that the applied ultrasonication procedure resulted with longest tubes breaking and favored obtaining HNTs below 300 nm in length (39.1 % to 76 % of the batch). The number of voids among the pristine nanoparticles when packing together (123–43 nm) greatly increased (total pore volume from 0.23 cm^3/g to 0.30 cm^3/g), meaning that the nanomaterial was efficiently disaggregated as well. In vitro internalization and colocalization studies by Scanning Electron and Multiphoton Microscopy demonstrated that the sonicated halloysite were preferentially internalized via macropinocytosis within 60 min and accumulated in the perinuclear region within 24 h. Halloysite application in nanomedicine: To study halloysite potential as a carrier for drugs, we set up the preparation and characterization of hybrid nanoconstructs with model molecules such as magnesium monoperoxyphthalate hexahydrate (MMPP), a negatively charged oxidizing agent, aspirin, an anti-inflammatory drug and epirubicin, a chemotherapeutic. Chosen molecules were incorporated with 3.5 %wt, 1.1 %wt and 5.1 %wt capacity, respectively for MMPP, aspirin and epirubicin. Loading efficiency (LE) improvement was achieved through the choice of the right solvent (1), enhancement of electrostatic forces between nanoparticle and the drug, via functionalization of HNT surfaces with an active linker (2), as well as NPs structure modification leading to increase of inner lumen volume (3). Specifically, the use of water: EtOH (7:3 v/v) as a solvent instead of water, increased MMPP loading capacity up to 6.1 %wt. Poor incorporation of aspirin was improved by enhancing electrostatic forces between deprotonated aspirin molecules and modified HNTs inner walls with amine-rich organosilane. It was also demonstrated that by enlarging volume of the NPs cavities, more molecules could be loaded. To do that, pristine HNTs were treated with 0.1M aqueous solution of NaOH, which resulted in an exfoliation of bilayers located inside the lumen. At the same time, outer surface of the halloysite tubules was preserved. As a consequence of the base treatment, halloysite cylinders gained more volume in the inner cavity as concluded from Transmission Electron Microscopy (TEM) and nitrogen adsorption BET analysis. The actual test on loading capacity using model MMPP molecule revealed increased MMPP incorporation from 6.1 % wt to 11.7 %wt. To evaluate if the activity of MMPP as an oxidizing agent remained unchanged upon incorporation and release from halloysite, and therefore to demonstrate the inactivity of the inorganic skeleton towards carried molecule, we tested HNT-MMPP nanoconstruct with selective fluorescent 1,3-diphenylisobenzofuran (DPBF) probe. Among available modifications of halloysite nanoparticles via covalent bond, the surface silanization is commonly recognized as one of the most efficient and widespread reaction while HNT manipulation. Up to date, the halloysite nanotubes functionalized with silanes have been used as a support for versatile applications in diverse scientific domains, including enzymes immobilization and biosensing. Willing to explore the halloysite functionalization with those active linkers, we have performed grafting reactions with representative organosilanes carrying the same backbone, while varying in the content of terminal groups, namely (3-aminopropyl)triethoxysilane (APTES), 3-(2-aminoethylamino)propyldimethoxymethylsilane (AEAPS), (3-mercaptopropyl)trimethoxysilane (MPS). Successful HNTs surfaces functionalization with organosilanes was demonstrated by means of quantitative thermogravimetric analysis (TGA) that allowed to estimate the loading capacity of organosilanes to be of 5.7 %wt for APTES, 7.4 %wt for AEAPS and 0.7 %wt for MPS. In addition, particular attention was dedicated to further quantify incorporated organosilane (APTES), since only one method has been so far reported, that is the destructive thermogravimetric analysis (TGA). For this reason, we set up and optimized a Fmoc based method by performing the following three reactions: (i) synthesis of “APTES-Fmoc” molecule; (ii) halloysite functionalization with “APTES-Fmoc”; and (iii) time-dependent Fmoc deprotection reaction in piperidine: EtOH (20 %) solution, resulting in dibenzofulvenepiperidine adduct (DBF-pip) formation. The UV-visible spectroscopic analysis of supernatant solutions demonstrated that the DBF-pip deprotection from halloysite support needs 5 h to be completed. Therefore, it was evidenced that HNT Fmoc-method showed strong coherence with already existing TGA method (± 2 % measurement error) and stood out as a valuable complementary technique for quantification of silane grafting on HNTs surface with additional low-cost and nondestructive advantages. The possibility of using halloysite nanotubes as a non-viral gene delivery nanosystem for therapeutic treatments was studied as well. Aiming to immobilize plasmid DNA (pDNA) based on the Green Fluorescent Protein (GFP) on HNTs support, the layer-by-layer (LbL) adsorption technique was applied. Obtained multi-component assembly was characterized qualitatively by monitoring variation in nanoparticle physico-chemical properties including surface charge, mass weight, presence of functional groups at each step of hybrid formation, which confirmed the successful nanoarchitecture formation. In order to additionally demonstrate the presence of GFP encoding plasmid (pGFP) on HNTs, the nanoarchitecture was treated with the bovine pancreatic deoxyribonuclease (DNase) enzyme, which induced the pGFP degradation through hydrolytic cleavage of phosphodiester linkages in DNA backbone. Thus, as expected, such nanoform with deposed genetic material varied in physico-chemical properties, expressing similar ones of the nanoconstruct without pGFP plasmid attached. The biological efficiency of HNTs-pGFP nanosystem was checked by means of Multiphoton microscopy. Successful pGFP plasmid transportation into cells was verified by detection of GFP expression, which yielded fluorescence emission. The interesting and innovative aspect of this case study was the simultaneous observation of GFP expression via fluorescence detection, and colocalization of halloysite nanoparticles by their SHG signal. This study proved that halloysite can act as an efficient carrier of genetic material, since free pGFP cannot be internalized by same cells, due to its large size and significant charge. Drug-loaded halloysite nanoconstructs (HNT-MMPP, HNT-APTES-aspirin) were also examined on the drug release kinetics, demonstrating long-term MMPP leakage taking 18 days and aspirin over 60 min. However, great drug liberation into the solvent of release was observed in the first minutes, followed by desired sustained drug release. The initial molecule liberation (dose dumping effect) is known to entail local toxicity. Herein, trying to find a solution to this problem, the coating of HNTs with the natural collagen polymer was investigated. Two strategies for the loading with this biopolymer were studied: (i) formation of a covalent bond between collagen and APTES-modified HNTs using glutaraldehyde cross-linker or (ii) noncovalent adsorption of collagen into pores of NPs. Immobilization of collagen on the surface of HNTs was estimated to be 3.7 %wt (i) and 1.8 %wt (ii). Other supplementary characterization techniques, such as water contact angle, ζ–potential analysis, Kaiser test, ultraviolet and visible (UV-vis) spectroscopy and Fourier Transform Infrared Spectroscopy (FTIR) were in accordance and proved nanoarchitectures formation. For the visualization purpose of HNTs encapsulated in collagen shell, the innovative characterization technique was implemented, namely 3D Multiphoton microscopy. It revealed that the biopolymer coating blocked the entrances of the hollow tubes thus, entrapping the drug in NPs. Mimicking tumor microenvironment (TME), the pH and/or enzyme triggered release was performed. LC-Mass analysis revealed that the collagen coating slowed down the release of aspirin from HNTs. Studies on cells showed that the collagen coating on HNTs is biocompatible and cell viability assay performed on 5637 urinary bladder and HeLa cervical cancer cell lines demonstrated the sustained release of the entrapped epirubicin chemotherapeutic agent in the biological context. Industrial application of halloysite: During a stage in BASF SE (USA), validation and properties enhancement of halloysite-based products potentially manufacturable in the company on an industrial scale were studied. In particular, the research was dedicated to aspects such as the pH-dependent dispersion behavior of halloysite nanotubes and iron coarse impurities removal from bulk samples. Applied methodologies and set of physico-chemical characterization techniques generated and revealed decreased percentage of present aggregates, maintained low shear viscosity under the threshold value and increased solids loading capacity in final halloysite-based products. Conclusions: In conclusion, PhD studies here reported contributed to the exploration of halloysite nanotubes for their application in the nanomedical and industrial fields. The investigations suggest a facile manipulation and functionalization of HNTs, useful for properties modification and improved NPs performance. Specifically, the study was directed toward formation of multi-functional nanocarriers with controlled drug delivery and release properties, together with targeting and imaging abilities. Moreover, the research was completed with halloysite-related technology transfer to the BASF SE, for the purpose of knowledge increase in the halloysite-field and bringing forward placement of halloysite-based products on the market. The systematic study on HNTs characterization and application performed in this PhD thesis will contribute to the development of HNTs as a high performance structural and functional material.