Ethical aspects of nanotechnology and synthetic biology in the framework of Nano3Bio

The Nano3Bio projects aims at the biotechnological production of chitosans, a family of naturally occurring functional biopolymers with enormous potential e.g. for purification of drinking water, for clearing of beverages, or for decontamination of waste waters, as environment-safe plant disease protectant, as a blood-stilling dressing promoting scar-free wound healing, as a probiotic food ingredient and animal feed additive replacing growth-promoting antibiotics, or as a consumer-safe stabilizer and preservative in cosmetics and personal care products. Since we find it essential to consider ethical issues from the outset, we provide this essay publicly. 

Today, chitosans are commercially produced through a simple chemical process from chitin, one of the most abundant natural biopolymers present e.g. in the discarded shells of shrimp and crabs. Chitosans are non-allergenic and biocompatible, i.e. they have no adverse effects on plant, animal or human cells, and they are fully biodegradable in the environment. However, the chemical process of converting chitin into chitosans yields mixtures of chitosan molecules differing in their chemical fine-structure and, thus, in their biological functionalities. But as only specific chitosans have the desired biological activities while others do not, such mixtures are unreliable in their performance, and this poor reproducibility has been a significant hurdle in the development of chitosan-based products. The Nano3Bio project, therefore, aims at the biotechnological production of defined chitosans with known chemical structure and predictable biological performance.

To this end, the Nano3Bio project develops and uses biotechnologial engineering tools to use nature’s own enzymatic machinery for the biosynthesis of chitosans, integrating them into micro-organisms or micro-algae suitable for large scale fermentation. Chitosans are rather poorly soluble in water, a property that is desirable for some applications but problematic for others. To overcome this solubility problem, the Nano3Bio projects aims to develop different nano-formulations of the well-defined, biotechnologically produced chitosans, e.g. as nanoparticles, nanocapsules, or nano-fibers. These nanoformulations will concomitantly convey additional properties to the chitosans, and they will allow using them e.g. for the development of drug carriers that can increase the bio-availability of poorly soluble drugs or their targeted delivery to specific organs such as the brain where diseases are difficult to treat because of the blood-brain barrier. Biotechnological production of defined chitosans in fermenters will not only solve the problem of today’s poorly defined chitosan mixtures with unreliable performance, it will also remove the possibility of contamination with allergens or viruses falsely assumed as a threat connected with conventional chitosans because of their animal origin from shrimp and crab chitin, and it will alleviate foreseeable pressures on natural shrimp and crab populations through over-fishing once chitosan-based products will be entering markets such as drinking or waste water purification which will require large amounts of bulk chitosans independent of their biological functionalities.

However, while biotechnological production and nano-scale formulation of chitosans will allow to exploit the enormous potential of these natural polymers in the interest of environmental and consumer safety, the involvement of nanotechnology and synthetic biology potentially raises safety and ethical issues which are being briefly discussed in this report.

Nanotechnology raises some general ethical issues, in particular safety issues because the proper-ties, and in particular the bioavailabilities and biocompatibilities of materials can change at the nano-scale. The Nano3Bio project deals with nanotechnologies of the functional biopolymer chitosan - or more correctly chitosans, as the term chitosan denotes a family of molecules that differ in details of their structures and, consequently, functions. Like other materials, biomaterials such as biopolymers do have specific properties that are governed by their nano-scale formulation. However, in contrast to e.g. silver nanoparticles, biopolymers are naturally nano-formulated - all biomolecules exert their biological activities on the molecular and cellular, i.e. nano- and micro-scale levels. Thus, no fundamentally new properties are expected when biopolymers are nano-formulated, and this is also what has been observed in systematic studies investigating the influence of nano-formulations on the bioactivities of biopolymers such as chitosans.

Perhaps the most prominent and most spectacular ethical issue raised by nanotechnologies - though not of the Nano3Bio project - is the theoretical possibility to design and build nano-machines. Construction of complex nano-machines will most likely need to involve the activity of more simple nano-machines, or molecular assemblers and disassemblers. This scenario invokes the vision of self-replicating nano-machines, and eventually even autonomously self-replicating nano-machines. This is clearly beyond the reach of today’s nanotechnologies, and it is also not a goal of the Nano3Bio project. But interestingly, this vision touches upon possible visions of synthetic biology which aims to improve the biotechnological performance of living organisms. Unlike genetic and metabolic engineering which recombine existing or optimised organic molecules, using them as bio-bricks in the framework of an existing organism, synthetic biology has the ambition to aim at the construction of a whole organism from scratch, possibly involving non-natural compo-nents. Eventually, thus, the visions of nanotechnology and synthetic biology may merge in the building of autonomously replicating nano-machines designed upon principles and concepts of living systems.

Such synthetic organisms and autonomous nano-machines may start competing with natural, biological organisms, as one of their properties inherited from their biological predecessors will be the ability to propagate and, in the process, to mutate and evolve. They would then behave like invasive species, possibly even out-competing and eventually replacing natural organisms or entire ecosystems. In the Nano3Bio project, the goals are less ambitious, and less threatening. We aim to engineer the metabolism of bacterial production strains to improve the purity and yield of the targeted chitosan biopolymers, without introducing new-to-nature components, and based on tried and tested biotechnological safety strains.

Biopolymers are among the most abundant natural resources, and among the most promising biomaterials. As renewable source for bioenergy as well as for biomaterials, they are currently very clearly at the forefront of developments in the transition from a petrol-based economy to a bio-based economy. But beyond these areas, functional biopolymers - with chitosans being the most prominent example - often combine superior material properties with versatile biological activities. Being biorenewable, biodegradable, and biocompatible, biopolymers as such raise no specific ethical questions beyond those of other biologically active natural compounds, e.g. in the context of the Nagoya protocol or issues of toxicity levels, reproducibility, and reliability.

Biopolymers also offer excellent opportunities for soft nanotechnologies. Biopolymers such as chitosans can easily be used for the production of nano-particles, nano-complexes, nano-capsules, etc. Such soft nano-structures combine excellent biocompatibilities with often improved solubility or bioavailability, and they can also be used as ideal carriers for other, e.g. poorly soluble drugs or biologics. Chitosans in particular, due to their polycationic nature in slightly acidic environments, are easily interacting with negatively charged biomolecules or supramolecular and cellular structures, such as proteins, nucleic acids, and phospholipid membranes. In a biomedical setting, chitosans can also be regarded as biomimetics as they resemble the glycosaminoglycans of the human proteoglycan-based extracellular matrix, and chitosan-based hydrogels are, therefore, almost ideally suited for human cell culture and tissue engineering.

On the other hand, the many bioactivities reported for chitosans and chitosan-based nano-formula-tions need to be taken into account when considering biosafety issues. Clearly, while chitosans are regarded as biocompatible, they are not biologically inert. And given that detailed structure-function relationships of partially acetylated chitosans are not yet fully understood, the development of chi-tosan-based biomedical and pharmaceutical applications necessitates comprehensive safety studies. The situation here again is not different from other biologically active ingredients which require careful pre-clinical and clinical trials before being approved for medical uses. Chitosan nano-formulations may cause a potential health threat when inhaled as current evidence suggests that they tend to be endocytosed by e.g. lung epithelial cells, with uncertain metabolic fate.

While such considerations are warranted, it needs to be emphasized that biopolymers and biopoly-mer-based soft nano-formulations can be considered safer both for human health and for the en-vironment when compared to inorganic nano-formulations. A prominent case currently much debat-ed are silver nanoparticles which are already used widely for antimicrobial finishings of e.g. textiles or medical devices. It is becoming increasingly apparent that such metal nanoparticles accumulate in the body and in the environment where they are not degraded, with unknown consequences. This is much less a concern with biopolymers, in particular in their unmodified natural form lacking chemical substitutions. Most natural biopolymers are easily biodegradable. This is particularly true for chitosans which are biodegradable by a range of ubiquitous enzymes such as chitinases and chitosanases. Chitinases are very widespread in nature, being produced by archaea, bacteria, fungi, plants, invertebrates and even vertebrates, including humans. This is not surprising given the widespread occurrence of chitin in nature - chitin is sometimes considered to be the second-most abundant biopolymer on Earth, second only to cellulose. Chitosanases are less ubiquitous, they appear not be produced by plants and vertebrates, but their occurrence still far exceeds what would be expected given the rather scarce occurrence of chitosan in nature. However, it is import-ant to note that chitosans (except for fully deacetylated polyglucosamine) can be depolymerized by both chitinases and chitosanases, yielding partially acetylated chitosan oligomers which in turn are degraded to the safe monosaccharide building blocks glucosamine and N-acetylglucosamine by the action of glucosaminidases and N-acetylglucosaminidases which are again very widespread, possibly being produced by all organisms.

For biomedical and pharmaceutical applications, it is relevant that humans possess three different enzymes with chitinase activity, namely acidic mammalian chitinase, chitotriosidase, and lysozyme, but no chitosanase. However, human chitotriosidase does have a good chitosan hydrolytic activity, degrading even chitosans with low degree of acetylation, even if only slowly. The subsite specificity of chitotriosidase is partially known now so that in principle, the chitotriosidase-catalyzed degrada-tion of a chitosan polymer with a known degree and pattern of acetylation can be predicted. Thus, if a chitosan with a defined pattern of acetylation could be engineered, its half-life in a human tissue as well as the oligomeric degradation products generated could be programmed. However, chitosans produced using today’s physical or chemical technologies - partial de-N-acetylation of chitin or partial re-N-acetylation of polyglucosamine - invariably yield partially acetylated chitosans with random patterns of acetylation. It is one of the goals of the Nano3Bio project to develop bio-technological tools and processes to produce chitosans with defined, non-random patterns of ace-tylation.

Biotechnological production ways for chitosans from simple bacterial growth media have other potential advantages over current chemical techniques converting chitin extracted from nature into chitosans - with further ethical implications. Firstly, biotechnological production which unlike alka-line deacetylation does not require high temperatures may save energy, and it may also save fresh water and reduce the output of waste water which are considerable with current production ways. However, as a biotechnological process for chitosan production is not yet established, it is difficult to predict with any degree of certainty that it will be less energy demanding and less environmentally burdening than today’s processes. This is why the Nano3Bio project has a strong focus on full Life Cycle Assessment, comparing the conventional to potential novel processes.

Today’s practice of producing chitosans from chitin extracted from e.g. shrimp or crab shells also potentially threatens to lead to overfishing of these crustaceans, or to a further extension of the environmentally damaging shrimp farming practices, should the demand for chitosans rise further. In fact, we have today for the first time a situation in which the demand for high quality chitosan exceeds that of the current global production facilities. Here again, biotechnological production of chitosans as developed in the Nano3Bio project offers a potential remedy for this ethically challen-ging situation.

Finally, chitosans may in future partially replace toxic agro-chemicals for plant disease protection, and may reduce the health threatening use of growth-promoting antibiotics in animal feed, leading to more sustainable agricultural practices. It is becoming increasingly evident that these beneficial effects of chitosans are strongly dependent on their molecular or nano-scale fine structure. Bio-technological production ways using specific chitin synthesizing or chitin/chitosan modifying enzymes have the potential to deliver defined partially acetylated chito-oligosaccharides with a much higher specific biological activity, allowing dramatic reductions in dosages required to obtain reliable effects. The use of defined, biotechnologically produced chitosans may, thus, even allow the use of these precious biopolymers/biooligomers in application fields such as agriculture which compared to medical applications, would require much larger amounts of materials, lessening the environmental burden of agriculture and relieving the stress on natural resources.

A serious ethical issue with modern agricultural practices relying on plant protection is its global ac-cessibility, even to resource-poor subsistence farmers, e.g. in Africa or parts of South/South East Asia and South America. Today’s chemical plant protectants are too often not affordable to these farmers. In addition, globalization and increased transcontinental travels have led to a situation where diseases easily spread across natural barriers such as oceans or mountain ranges. As a consequence, agricultural production is massively threatened by the emergence of diseases new to a given location, putting additional stress on farmers who cannot afford chemical plant protection. Once the best performing chitosans are known e.g. for plant strengthening, and reliable bio-technological tools and processes for their mass production have been developed, these can easi-ly be transferred to almost any country in the world. Of course, access to biotechnological infrastructure will be a prerequisite, but examples for low cost, robust agro-biotechnological processes are available, and NGOs have already successfully introduced such techniques even to rural areas of e.g. India. Such a reduction in complexity of the technology required is not within reach of the Nano3Bio project proper, but can clearly be envisaged for follow-up research projects.

As a conclusion, we can state that while nanotechnology and synthetic biology do raise a number of ethical issues, these are not relevant in the framework of the Nano3Bio project. In contrast, the goals pursued by this project, when realised, will have a number of clear advantages for human health, consumer safety, and environment protection. Even the most threatening scenario of mili-tary or criminal misuse of nanotechnology or synthetic biology is not a serious issue with the Nano-3Bio project as there is no toxic potential of the materials and organisms used.

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