Diatoms: a biotemplating approach to fabricating drug delivery reservoirs

Abstract

Biotemplating is a rapidly expanding subfield that utilizes nature-inspired systems and structures to create novel functional materials, and it is through these methods that the limitations of current engineering practices may be advanced. The diatom is an exceptional template for drug delivery applications, owing largely to its highly-ordered pores, large surface area, species-specific architecture, and flexibility for surface modifications. Diatoms have been studied in a wide range of biomedical applications and their potential as the next frontier of drug delivery has yet to be fully exploited. In this editorial, the authors aim to review the use of diatoms in the delivery of poorly water-soluble drugs as reported in the literature, discuss the progress and advancements that have been made thus far, identify the shortcomings and limitations in the field, and, lastly, present their expert opinion and convey the future outlook on biotemplating approaches for drug delivery.

Keywords::

• biotemplating
• diatom
• frustule
• marine resources
• skeleton

1. Introduction

In the evolutionary tale between humans and nature, a reoccurring theme is seen wherein humans derive inspiration from nature to meet their needs. Likewise, in the field of biomedical engineering, the use of natural systems as templates – termed ‘biotemplating’ – has been argued to provide many biomimetic advantages over conventional biomaterials and drug delivery platforms [1]. Some examples of biotemplating include the intercalation of metal complexes in DNA strands to form metal nanowires [2], the viral template of tobacco mosaic virus to fabricate nanocrystals and inorganic films [3] and the coating of pollen grains to create ferrimagnetic pollen replicas tailored for multimodal adhesion [4]. There is an enormous amount of structural diversity in biological materials, and when coupled with the fact that many natural templates have repetitive topographical features and are highly amenable to a plethora of chemical functionalization techniques, biotemplating becomes a cost- and time-effective approach to fabricating the next generation of bottom-up biomaterials-based drug delivery systems.

In the field of drug delivery, the aim is to design vehicles that exhibit certain characteristics including low toxicity and safety risk, high amounts of drug incorporation and timed and targeted release profiles. However, due to the overwhelming use of combinatorial chemistry and high-throughput screening in the pharmaceutical industry, drugs have shifted toward more lipophilic and poorly water-soluble molecules, introducing yet another hurdle for various modes of delivery [5]. In an effort to overcome many of the problems with traditional methods for controlled drug release, a biotemplating approach has been undertaken where one such template of interest comes from the diatom – a unicellular, eukaryotic algae that is ubiquitous in nature and of which ∼ 100,000 different species have been identified [6]. Diatoms are microscale structures, ranging from a few microns to several hundreds of microns in size, and are surrounded by an outer cell wall, collectively known as the ‘frustule’, which is highly differentiated and almost always impregnated with amorphous silica (SiO₂) [7]. The siliceous frustule acts as the diatom skeleton – comprising the hypotheca and epitheca valves with nanoscale features – and it is this intricate structure that deems it an attractive template for drug delivery (Figure 1). The hierarchical structure of the diatom is species-specific and composed of unique and highly ordered pores on the nanometer scale, giving the frustule an extremely large surface area for biomolecule conjugation; furthermore, genetic manipulation and metabolic activity of the silicic acid transport within the diatom has been thoroughly investigated to provide a mechanistic understanding regarding silicic acid uptake [8-11]. Diatoms have been utilized extensively for a variety of applications, such as microfabrication, immunoisolation, optics, photonics, protein separation, water purification, filtration, biosensing and catalysis (Figure 2) [12-27]. Also, scale-up of diatoms is feasible based on asexual reproduction in an exponential manner, allowing it to be highly environmentally friendly compared with the manufacturing processes of synthetic (meso)porous silica-based materials currently being explored as targeted delivery systems [28]. In this editorial, the authors aim to review the occurrence of diatom skeletons in the context of drug delivery, discuss the potential advantages and shortcomings thus far and, lastly, identify the prospective future direction of this multifaceted field.

Figure 1. Topography of the marine diatom Thalassiosira weissflogii. (A – D) Scanning electron microscopy utilized to visualize diatom frustules. (A) Diatoms are abundant both marginal and amenable to scale-up (scale bar = 40 μm). (B) Arrow highlights girdle bands alongside diatom frustule (scale bar = 5 μm). (C) External view of valve face with arrows highlighting both marginal and central fultoportulae (scale bar = 3 μm). (D) Zoomed-in view of central fultoportulae (scale bar = 1 μm). (E) Transmission electron microscopy shows the highly-porous, hollow architecture with rib-like structure around central fultoportulae (scale bar = 500 nm). (F) Atomic force microscopy shows morphological analysis of diatom valve face (profile scale = 200 nm, x-/y-axis division: 1 μm, z-axis division: 200nm).

Figure 2. Various chemical functionalization strategies used on diatoms.

2. Uses of diatoms for drug delivery

The idea of exploiting the biological structure of the marine diatom to construct new materials was first proposed by Morse in 1999, where he argued that the biologically produced silica of diatoms exhibited a genetically controlled precision of nanoscale architecture that exceeded the current capabilities of human engineering [29]. Utilizing the diatom purposefully as a delivery system was then experimentally shown by Rosi et al. in 2004, when researchers controlled the release of gold nanoparticles from the surface of DNA-functionalized diatom surfaces [30]. More recently, the Losic research group has used the Aulacoseira species and have shown marked success in functionalizing the diatom skeleton with dopamine modified iron oxide nanoparticles as a method to creating magnetically guided drug microcarriers [31]. Using BET (Brunauer-Emmett-Teller) nitrogen adsorption analysis, the diatom surface area was quantified to be 18.5 ± 0.8 m²/g, significantly higher than traditional nano- and micro-scale drug delivery particulate systems [31]. Furthermore, they have also reported drug encapsulation and release of indomethacin (model water-insoluble drug) and gentamicin (model water-soluble drug) on both bare diatom structures [32,33] and diatoms modified with silanes and phosphonic acids to render the surface of the diatom either hydrophilic or hydrophobic, allowing them to investigate the effect of surface functionalization on controlling diffusion rates and drug delivery rates [34-36]. The aforementioned experiments observed drug release kinetics in a biphasic manner, with an initial burst release of 6 h (attributed to the surface-deposited drug) followed by near-zero-order sustained release over a prolonged 2-week period of time (attributed to the drug released from the internal hollow structures of the diatom). Another study by Zhang et al. investigated the cytotoxicity of diatoms loaded with mesalamine and prednisone – two commonly prescribed drugs for gastrointestinal diseases – and assessed cell viability and drug permeation efficiency using co-cultured cell monolayers, finding limited toxicity of diatoms up to concentrations of 1000 µg/ml [37]. Most recently, Kumeria et al. have fabricated a nano-hybrid material by attaching graphene oxide onto diatom frustules via 3-aminopropyltriethoxysilane surface functionalization and observed the release of indomethacin under different pH conditions [38]. The biphasic release kinetics observed from drug-encapsulated diatom frustules is extremely advantageous for orally administered therapeutics. While silica-based materials have been developed for implantable, intravenous and dermal drug delivery systems, oral drug delivery has always been the preferred route of administration for pharmaceutical products primarily because of its low medication expenses and high patient compliance rates when compared with more invasive delivery methods [39]. Furthermore, in order for oral delivery to be effective, the drug must not only remain bioactive but needs to dissociate from inert compounds of the oral formulation within the gastrointestinal tract and permeate across the intestinal wall. However, oral delivery is plagued with many disadvantages, such as the harsh conditions and variable pH of the gastrointestinal tract that could lead to degradation of the product, the poor water solubility of the therapeutic and the carrier, the lack of drug permeation across biological barriers, and the extensive first-pass metabolism that occurs before reaching systemic circulation [40]. As such, much effort has been undertaken to design materials that overcome these obstacles, one of which includes the use of porous microstructures with the capability of surface functionalization to improve the unfavorable pharmacokinetics of poorly water-soluble drugs – namely, the marine diatom. However, inherent toxicity arising from the biogenic silica of the diatom, similar to the immunogenic response of silica from intracrystalline proteins, has resulted in a focus on modifying the chemical composition of the frustule to create a diatom replica that does not induce an immunogenic response yet preserves the intricate porous architecture of the diatom [41].

Modifications to the diatom skeleton have been explored thoroughly, and there exists a wide range of various replicas with alterations in surface chemistry. Gold replicas of the diatom have been successfully fabricated by Losic et al. for various applications, proving it to be a feasible alternative to expensive lithographic procedures yet with the capability to create complex 3D metallic structures [42-44]. Similarly, other diatom replicas have been produced by Sandhage et al. via oxidation-reduction gas/solid displacement reactions to form various structural replicas composed of magnesium oxide, zirconium dioxide, titanium dioxide and silicon [45-49]. Furthermore, there are reports of successful generation of a negative replica of the diatom using an imprinting method with polydimethylsiloxane, which can act as a template to produce positive structural replicas in a wide range of materials [50,51]. However, while these inorganic methods have shown efficacy in replicating the diatom skeleton, there is currently a severe lack of organic replication techniques (i.e., oil-in-water emulsion, sol–gel synthesis, layer-by-layer assembly, etc.) and organic materials (i.e., polymers/peptides, extracellular matrix proteins, glycopolymers, etc.), which could provide the key for fabricating the ideal drug delivery reservoir for biological and cellular conditions, while offering significant scale-up capabilities.

3. Conclusion

Herein, the authors have reviewed previous uses of diatom skeletons for drug delivery (Table 1). The literature has shown substantial preliminary success in utilizing the advantages of the intricate porous architecture of the diatom for delivery purposes. Various drugs of interest and surface functionalization techniques have been compared to pinpoint the ideal chemical modifications for different applications. Furthermore, the authors have presented current fabrication methods for altering the chemistry of the diatom and the available approaches for synthesizing numerous structural replicas. However, there are still unexplored avenues in this research field and these points will be identified in the next section, along with the future prospective and goals that need to be met.

Table 1. Diatoms in drug delivery as reported in literature.

Drug(s)	Surface functionalizations	Loading procedure	Loading capacity	Method of analysis	Results	Ref.
Indomethacin Gentamicin	Dopamine-modified iron oxide (Fe3O4) nanoparticles	Immersion	28 wt%	UV-Vis spectroscopic analysis*	• Functionalized diatom structures by dopamine modified magnetic nanoparticles and demonstrated their potential toward developing targeting drug delivery devices • Controlled motion of magnetic particles using external magnetic field • Observed sustained over 2 weeks of indomethacin from structures	[31]
Indomethacin Gentamicin	n/a	Incipient wetness method	14 – 22 wt%	UV-Vis spectroscopic analysis*	• Release kinetics indicated a characteristic two-step release where the first and rapid release (over 6 h) is attributed to the surface deposited drug and the second step with slow release is attributed to drug released from internal hollow structures	[32]
Indomethacin	n/a	Incipient wetness method	22 wt%	UV-Vis spectroscopic analysis*	• Investigated the morphological and structural characteristics of both diatom frustules and drug-loaded conjugates using zetasizer, SEM-EDX, TGA, XRPD, and BET analysis • Release kinetics demonstrated both initial first-order model burst release and slow zero-order prolonged release	[33]
Indomethacin	3-APTES N-(3-(trimethoxysilyl) propyl) ethylene diamine 2-CEPA 16-PHA	Incipient wetness method	15 – 24 wt%	UV-Vis spectroscopic analysis*	• Utilized ^29Si and ^13C solid-state NMR to investigate the interactions between the drug and the self-assembled organic monolayers on the diatom surface • Found that AEAPTMS-modified diatoms had superior increase of drug adsorption due to the high amount of amine groups on the surface • Observed that modifications with 16-PHA which creates a hydrophobic diatom surface caused quicker release and lower loading	[34]
Indomethacin Gentamicin	3-APTES mPEG-silane7-OTS 3-GPTMS 2-CEPA 16-PHA	Incipient wetness method	14 – 24 wt%	UV-Vis spectroscopic analysis^‡	• Observed that for indomethacin, hydrophilic surfaces (amino-rich APTES, epoxy-rich GPTMS, and carboxyl-rich 2-CEPA) improve the loading and provide extended drug release, whereas hydrophobic surfaces (16-PHA, mPEG-silane, and OTS) provide lower drug loading and faster release • Observed that for gentamicin, the trend is reversed where hydrophobic surfaces (16-PHA) favor improved loading and sustained releases, while hydrophilic surfaces (2-CEPA and APTES) give lower loading and shorter drug release	[35]
Indomethacin Gentamicin	3-APTES 2-CEPA 16-PHA	Immersion	Measured, not reported	UV-Vis spectroscopic analysis^‡	• Surface modification using the organosilane was performed via silanization and the selected phosphonic acids were performed using an annealing treatment • Release studies showed a biphasic pattern, comprising an initial burst release for 6 h, followed by near-zero order sustained release • Observed that diatom size, morphological regime, and surface hydrophobicity/hydrophilicity based on functionalization affected drug release profiles	[36]
Mesalamine Prednisone	n/a	Immersion	9.9 – 11.5 wt%	High-performance liquid chromatography^§	• Assessed drug permeation across Caco-2/HT-29 co-culture monolayers by measuring transepithelial electrical resistance • Very low toxicity of diatoms at concentrations up to 1000 mg/ml • Diatoms were able to act as permeation enhancers for orally administered drugs	[37]
Indomethacin	Graphene oxide attachment via 3-APTES	Immersion	28.5 wt%	UV-Vis spectroscopic analysis^¶	• Fabrication of nano-hybrid material coupling graphene oxide with APTES-functionalized diatom silica microparticles via covalent and electrostatic attachment strategies • Composite structure acted as a pH-sensitive micro-drug carrier and drug release data was fitted to zero-order and Korsmeyer-Peppas models • Observed interesting photoluminescent properties due to the silica-graphene synergy indicating opportunities for biosensing purposes	[38]

Drug release performed using: *PBS at pH 7.2, ^‡PBS at pH 7.4, ^§simulated GI conditions at 37°C, ^¶PBS at pH 3.5 & 7.4. All papers use centric Aulacoseira species found in diatomaceous earth.

APTES: Aminopropyltriethoxysilane; BET: Brunauer-Emmett-Teller; CEPA: Carboxyethyl-phosphonic acid; GPTMS: 3-(glycidyloxypropyl)trimethoxysilane; mPEG-silane: Methoxy-poly-(ethylene-glycol)-silane; NMR: Nuclear magnetic resonance; OTS: 7-Octadecyltrichlorosilane; PHA: Phosphono-hexadecanoic acid; SEM-EDX: Scanning electron microscopy coupled with energy-dispersive; UV: Ultraviolet; X-ray spectroscopy; XRPD: X-ray powder diffraction.

4. Expert opinion

Based on the work done in this field so far, there remains significant unharnessed potential in utilizing the diatom template for developing the next generation of delivery reservoirs, through the presence of species-specific porous architecture, large surface area and flexibility for surface modifications and scale-up. Critically, studies to date have shown a high potential for organic and nonorganic loading, and drug-encapsulated diatoms have demonstrated a biphasic pattern with both burst and prolonged release profiles. These results hold great promise for biotemplate-inspired drug delivery; however, key tenets of diatom functionalization still need to be addressed.

First and foremost, there is still a lack of understanding on a molecular level concerning the exact biomineralization processes that are attributed to diatom valve formation and silicic acid deposition. Significant progress has been made over the past decade in understanding the mechanisms associated with the silica deposition vesicle [8-11] and the associated proteins involved in the valve synthesis pathway [52-54]. Unraveling these processes further will provide a deeper understanding of diatom development on a mechanistic level, which in turn provides mechanistic insight for chemical modifications during frustule synthesis [55,56].

Second, with regard to the application of diatoms as drug delivery systems, comparative studies are needed to investigate the effects of local versus systemic delivery. The topographical advantages of the diatom structure are well established and preliminary drug delivery experiments have confirmed favorable release kinetics [31-38]; however, only a single study has looked at the efficacy of diatoms as drug delivery reservoirs in vitro [37]. Furthermore, there is a need to evaluate the efficacy of diatom drug delivery in vivo and a comparison of the delivery route of administration and regulatory path will decide the clinical application and implementation of this novel drug delivery platform. Critically, biotemplate augmentation may add synergistic advantages to function and incorporation of functionalized diatom particles into a bioactive scaffold may significantly enhance local delivery through focused delivery of diatoms to a site of injury. Scaffolds can also be combined to complement the response of the drug itself or to provide stimuli to the cellular microenvironment, and thus are extremely beneficial in providing a dual release therapeutic effect. Indeed, there is considerable work to be done in investigating the biological response of diatoms as a pharmaceutical. While diatomaceous earth is approved by the EPA, USDA and FDA as an anti-caking agent in livestock feed and use in chemical pesticides, the use of diatom materials in pharmaceuticals does not have regulatory approval and it will only be through extensive biological characterization for diatoms to make the journey along the regulatory pathway for approval.

Lastly, while the diatom frustule is highly tunable to a variety of modification and functionalization techniques, there still lies the inherent problem with regard to the immunogenicity from the biogenic silica. As such, there is a need to develop a system that retains the intricate architecture of the natural diatom while circumventing the problems associated with biogenic silica, and overcoming this challenge would represent a major breakthrough in the field. In order to address this issue, a method of replication followed by dissolution needs to be achieved to remove the biogenic silica while retaining a replica diatom structure. Ultimately, elucidating these areas will allow for enhanced biofunctionalization, mechanistic understanding of the diatom skeleton and the potential for drug delivery.

In the coming years, marine resources and bioinspired templates will emerge as key technologies in biomedical design, drawing upon the ever-increasing emphasis on sustainability and renewability. An abundance of biological templates exists in our ecosystem with much potential waiting to be exploited; the discovery of which will be driven by the demand for smaller, greener hierarchical materials in biomedical engineering. Indeed, modern-day technology and new strategies are constantly pushing the boundaries of human engineering capabilities. The area of micro- and nano-level fabrication is ever-progressing with the rise of three-dimensional nanoprinting and various nanolithography techniques; however, the high operating and instrument costs associated with these processes fail to compete with the versatility and fidelity of economical bioinspired templates. Ultimately, the level of precision of the nanoscale features seen in biology are incredible tools for biotemplating, and exploiting these design advantages will usher in the next age of drug delivery systems.

Declaration of interest

The authors gratefully acknowledge the financial support of Science Foundation Ireland (Grant 07/SRC/B1163 and 11/SIRG/B2135) and the Atlantic Area Transnational Programme (Grant MARMED 2011-1/164). The authors wish to thank Dr Éadaoin Timmins for microscopy support, Mr Maciek Doczyk and Ms Marie Keely for graphical illustration support, and Dr Yvonne Lang for the insightful discussions and editorial assistance. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Notes