4.1. Diatoms Functionalized via Organosilane Coating
In 2008, Townley et al. reported the tethering of Coscinodiscus wailesii
, a large centric diatom, with different antibodies, using either the amino groups or the sugar moieties of the antibodies to bind the diatom surface [74
]. The authors demonstrated the potential application of the system in immunoprecipitation assays by tethering diatoms to tubulin antibodies and selectively isolating them from total Drosophila
homogenate. In 2012, Bariana et al. presented diatom microparticles functionalized with different hydrophilic or hydrophobic organosilanes and phosphoric acids (Figure 6
and Table 1
). Their studies focused on the loading and release capacities of the modified diatoms towards two common therapeutics, the NSAID Indomethacin and the antibiotic Gentamicin [64
]. They demonstrated that the hydrophilic coating of the diatoms leads to an increased loading capacity and slower release of the hydrophobic Indomethacin, whereas the hydrophobic coating of diatoms promotes the same behavior with the hydrophilic drug Gentamicin. In the latter case, however, a lower drug loading and faster release was observed.
This study further confirmed the great potential and the tunability of drug delivery systems made with the inexpensive and eco-friendly nanoporous biosilicas from diatoms. In 2013, Kumeria et al. reported the functionalization of diatoms with graphene oxide (GO) [65
], a modification that led to the enhancement of the diatoms’ photoluminescent (PL) properties. This interesting biomaterial, further exploitable in biosensing applications, shows improved Indomethacin loading capacities and a pH-dependent releasing behavior of the model drug. In 2014, Ruggiero and co-workers highlighted the well internalization of functionalized and size-reduced diatom nanoparticles (100 to 300 nm) into epidermoid cancer cells (H1355) [66
]. For this purpose, the authors treated the diatoms by crushing and sonication steps to reduce their size to the nanoscale before proceeding with their functionalization with organosilane and further tetramethylrhodamine isothiocyanate (TRITC) as a traceable dye. Confocal microscopy imaging of these nanoparticles demonstrated the well internalization into cytoplasm of epidermoid carcinoma cells, reinforcing the potential of this biomaterial in anticancer delivery applications. The same year, Aw et al. presented another study on the functionalization of diatoms with different hydrophilic or hydrophobic compounds, focusing on the assessment of the drug loading and release capacities of the modified diatoms towards the water-insoluble drug Indomethacin [67
]. They demonstrated that surface functionalization with N
-[3-(trimethoxysilyl)propyl]ethylenediamine increases the loading capacity of diatoms up to 60 wt% and highlighted the correlation between active polar functional groups at the diatom surface, showing both the increased loading capacity and prolonged release property of the drug (Table 1
). In 2014, Rea et al. used diatomite (earth made of the siliceous remains of diatoms) as a drug vector for the transport of siRNA inside human epidermoid cancer cells H1355 (Figure 7
]. To increase the cell uptake and internalization process of these diatoms, their mean size was decreased down to the nanometric scale (mean size of 200 nm). The biocompatibility and relative innocuity of the nonmetric-sized diatomite towards the H1355 cell line at a concentration up to 300 µg/mL exemplifies the potentiality of these particles in nanomedicines and drug delivery applications. Via the functionalization of diatomite with Poly D-arginine peptide/siRNA complex, Rea and co-workers showed, respectively, by fluorescence spectrometry and confocal microscopy, the in vitro sustained released and the efficient internalization of siRNA into the cytoplasm of H1355 cancer cells. Furthermore, gene silencing of specific targeted mRNA was demonstrated by western blot analysis, confirming the efficiency of their newly designed siRNA delivering system.
In 2015, Cicco et al. focused on the use of antibiotic-loaded diatom frustules modified with a reactive oxygen species (ROS) scavenger in regenerative medicine applications (Figure 8
]. The design was based on the idea of delivering the efficient antibiotic ciprofloxacin (CPFX), and preventing its adverse side effects, such as oxidative damage to cells, by functionalizing the diatoms with (2,2,6,6-tetramethylpiperidin-1
-yl)oxy (TEMPO) as an antioxidant and antiradical agent.
The authors demonstrated the efficient functionalization of their diatom frustules with TEMPO (FT) by silanization, as well as the positive effect of FT-functionalized frustules on MG63 human osteosarcoma cell viability. Moreover, the enhancement of bone cells adhesion and proliferation on glass surface covered with FT was outlined. The same year, Vasani and co-workers designed thermo-responsive biosilica microcapsules and assessed the potential of this system by testing in vitro the release of Levoflaxin, an antibiotic used to treat various bacterial infections in humans [69
]. For this purpose, diatoms were functionalized with a derivatized silane-based atom transfer radical polymerization (ATRP) initiator. Alongside this, an oligo(ethylene glycol) methacrylate copolymer was prepared. Synthesis of the copolymers allowed producing compounds with unique reactivity to specific temperature changes in their environment. Activator regeneration by electron transfer based atom transfer radical polymerization (ARGET-ATRP) was the method selected to synthetize and graft the copolymers on the diatom surface. Drug release assessment above and below the lower critical solution temperature (LCST) of the grafted copolymers demonstrated a temperature-dependent release profile of the Levoflaxin in PBS at pH 7.4. To prove the retained activity of the antibiotic after its release, zone of inhibition (ZOI) studies were performed, and the results successfully confirmed this hypothesis. Terraciano et al. modified diatoms with a pegylated polymer ending with a cell-penetrating peptide (CPP) [37
]. They demonstrated enhanced drug loading capacity of the poorly water-soluble anticancer sorafenib (up to 22 wt% in diatoms) and improved releasing profiles in aqueous solution and haemocompatibility (assessed by red blood cell morphological study) after 48 h incubation at 200 µg/mL with erythrocytes. The authors also showed a considerable increase of cellular uptake of the CPP-modified diatoms in two different cancer cell lines, MCF-7 and MDA-MB-231 breast cancer cells. Martucci et al. conjugated diatoms with an idiotype-specific peptide (Id-peptide) able to target the antiapoptotic factor B-cell lymphoma/leukemia 2 (Bcl2) [38
]. They demonstrated a threefold increased uptake of these nanoparticles by Id-peptide recognition in A20 lymphoma cells compared to nonspecific 5T33MM myeloma cells. They also showed a more effective Bcl2 gene silencing using their modified nanoparticles as targeting vehicles when compared with the conventional lipofectamine transfection agent, suggesting the successful delivery of the siRNA into the host A20 lymphoma cells. In 2017, Managò et al. reported the internalization kinetics and the cytoplasmic localization analysis of siRNA-modified diatomite (Figure 7
) in human lung epidermoid carcinoma cell line (H1355) by Raman spectroscopy [75
]. Raman imaging of labelled-free siRNA-DNPs revealed efficient internalization of the nanoparticles within the first 40 h of incubation, as well as their co-localization in lipidic vesicles, leading to the conclusion that the internalization process takes place via endocytosis. The internalization kinetics and cytoplasmic localization were further confirmed by confocal fluorescence microscopy on cells treated with fluolabelled siRNA-DNPs and by PL measurements of supernatant from incubation wells. In 2017, Grommersch et al. reported the synthesis of diatomaceous earth (DE) modified with S-nitroso-N-acetyl-penicillamine (SNAP), a nitric oxide (NO)-releasing molecule (Figure 9
]. NO is a gasotransmitter that plays a crucial role in many different physiological processes. Its potential therapeutic effects in treating different diseases and pathophysiological processes, such as thrombosis, inflammation, vasodilation, inhibition of platelet adhesion and aggregation, neurotransmission, and immune system regulation, has been well recognized from many years [77
After optimizing the synthetic pathway by testing different organosilane linkers, Grommersch and co-workers confirmed, by chemiluminescence quantification, the ability of their SNAP-DE material to sustain NO release over a period of 24 h. SNAP-DE showed antibacterial activity when tested on S. aureus
, a common pathogen responsible for hospital-acquired infection, with bacterial reduction up to 92.95% ± 2.6%. Moreover, in vitro cytotoxicity assays on 3T3 mouse fibroblast cells revealed no toxicity of the SNAP-DE toward mammalian cells, making it a promising material in biomedical applications. Sasirekha et al. used the diatom Amphora subtropica
-modified with chitosan as a drug vehicle for doxorubicin, a chemotherapeutic belonging to the class of anthracyclines [72
]. Doxorubicin is known to provoke many side effects, and this dose-limiting toxicity encourages research of more efficient drug delivering systems. Through in vitro studies on immortalized lung cancer A549 cells, Sasirekha and co-workers demonstrated the enhanced cytotoxicity effect of doxorubicin due to sustained release from drug-loaded chitosan modified A. subtropica
diatoms ([email protected]
) when compared to the free drug. They also proved the haemocompatibility of their doxorubicin-loaded material [email protected]
at a particle concentration up to 50 µg/mL. Recently, Delasoie et al. presented the synthesis of vitamin B12
modified diatoms (DEMs-B12
-1) as a cancer cell-targeting delivery system for inorganic drugs (Figure 10
]. They highlighted the increased adherence of the B12
modified diatoms to MCF-7 breast cancer and HT29 colonic cancer cell lines when compared to unmodified diatoms. The authors also showed the resistance of the B12
coating to the intestinal tract fluids and the slow release of an experimental ruthenium anticancer complex in lipophilic environments at the cell membrane.
4.2. Diatoms Functionalized with Magnetic Coating and Antibodies
In early 2010, Losic and co-workers reported the functionalization of diatoms with a dopamine iron oxide (DOPA/Fe3
) composite [63
]. A simple electrostatically driven self-assembly approach was adopted and proved efficient in functionalizing diatoms with DOPA/Fe3
). The ability of these microcarriers to be magnetically driven in solution was demonstrated by simple tests in the presence of an external magnetic field. Moreover, the free ending amino group of DOPA resulted in availability for further functionalization with biomolecules of interest. For this purpose, fluorescein isothiocyanate (FITC) was used as a model fluorophore by coupling it to the free ending amino groups immobilized on the diatoms. Finally, the loading and release properties of DOPA/Fe3
-diatoms was assessed via the common nonsteroidal anti-inflammatory drug (NSAID) Indomethacin. This composite material is capable of partially sustaining the drug release over 2 weeks.
Also in 2013, Todd and co-workers reported the fabrication of magnetically guidable diatom materials [81
]. Human serum albumin (HSA)-coated iron oxide nanoparticles (IONPs) were loaded onto diatoms (DTMs) to confer them their magnetic properties. The 8 wt% Fe loading, determined by inductively coupled plasma (ICP) analysis, explained the important magnetic susceptibility of the IONP-DTMs. Cytotoxicity study on 4T1 murine breast cancer cell line showed good biocompatibility up to 625 µg·mL−1
with 80% of the cell population kept viable after 24 h exposure. Furthermore, in vivo testing was conducted on ZW800-loaded IONP-DTMs injected in animals equipped with or without a magnetic bar fixed to the skin on the site of their tumor (ZW800 is a fluorescent dye used as a drug model in the study). One hour after the intravenous injection of ZW800-loaded IONP-DTMs into 4T1 tumor xenografts, the animals were imaged by T2-weighted MR and by fluorescence. Both tests revealed a specific tumor accumulation in the magnet-treated group region of interest (ROI) analysis. After ex vivo fluorescence imaging on dissected tumors, IONP-DTMs showed 6.4 times higher accumulation in the magnet-treated animals (Figure 12
). These results demonstrated the potential use of magnetically guided diatom microcarriers for drug delivery applications.
In 2015, Javalkote et al. reported the fabrication of magnetically responsive diatoms by two different techniques [82
]. The first technique was a simple mixing of pure diatoms with both a drug solution and a ferrofluid (a fluid containing ferromagnetic nanoparticles) leading to the loading of both items in the diatoms in one pot. For the second technique, prior to the drug loading, the diatoms were soaked in a solution containing different ferrous salts followed by addition of ammonia, which promoted the growth of iron oxide nanoparticles directly inside the diatoms. The magnetically active diatoms were demonstrated as able to be guided by the application of an external magnetic field. The magnetically active diatoms were loaded with curcumin as a water insoluble drug model and tested on human cervical cancer (HeLa) cells. In 2015, Delalat et al. reported the use of genetically engineered diatom biosilica as a form of targeting drug-delivery vehicles (Figure 13
]. By incorporating the gene domain of protein GB1 in the genome of diatom Thalassiosira pseudonana
, the authors designed a biosilica material able to bind immunoglobulin G (IgG) antibodies on their surface. The authors labelled these new functionalizable diatoms with rituximab— an antibody specific for the antigen CD20—and demonstrated the specific targeting of cancer cells expressing antigen CD20 both in the case of surface-attached and suspended cells. To overcome the problem of antibody denaturation in organic solvent, the drug was loaded in positively charged nanocapsules as liposomes or micelles before adsorbing them on the negatively charged surfaces of antibody-labelled biosilica. It was shown that both drug-loaded nanocapsules could deliver their cargo (camptothecin and its derivative 7-ethyl-10-hydroxy-camptothecin, also called SN38) at higher levels than the minimum toxic dose after 16 h in DMEM medium. In vitro tests assessed the specific cytotoxicity of SN38 micelle-loaded anti-p75NTR-GB-biosilica towards SH-SY5Y neuroblastoma cells (only 10% of which remained viable after 2 days) with BSR cells as control (95% remaining viable after 2 days). In vivo tests in nude mice further confirmed the efficiency and the reduction of tumor growth after a single dose injection.
Maher et al. reported the magnesiothermic reduction process of silica diatom frustules to silicon replicas (Figure 14
]. The process improves the specific surface area of the material 13-fold. Moreover, silicon replicas (SiNPs) showed increased biodegradability compared to the silica diatom precursors. While less than 1% of the silica diatoms degraded after 30 days, 20% of the SiNPs were dissolved after the same time in PBS at pH 7.4, 37 °C. Drug loading and release from silica diatoms or SiNPs using daunorubicin as a drug model (an anticancer also used in the treatment of vitreoretinopathy [83
]) indicates a better penetration of the drug in the SiNPs, in which the pores are slightly bigger than in the silica diatoms. The study showed that the drug release from SiNPs was partially due to degradation of the particles and not only due to the concentration gradient. The authors also demonstrated for the first time that diatoms could act as self-reporting nanocarriers for both luminescent and non-luminescent drugs by following the photoluminescence (PL) spectrum of the characteristic red band at 682 nm belonging to SiNPs. These were shown to not be toxic towards Raw 264.7 murine macrophage and MDA-MB-231 breast cancer cells [56
]. When loaded with doxorubicin, they sustain its release over a period of 30 days and enhance drug cytotoxicity when compared with equivalent free drug concentration, further validating the applications of SiNPs as drug nanocarriers for chemotherapeutics.
More recently, Janićijević and co-workers reported a study on diatomite inorganically modified with aluminum salt that showed potentiation of the ibuprofen antihyperalgesic effect and demonstrated a greater effectiveness of the modified diatomite-ibuprofen composite than the equivalent doses of pure ibuprofen in pain suppression in rats [71