Boron clusters as broadband membrane carriers

We have identified a family of halogenated boron clusters capable of transporting a broad range of cargos across biological membranes. In contrast to traditional carriers, the delivery does not rely on amphiphilicity, but on the superchaotropic nature of these compounds.
Boron clusters as broadband membrane carriers

Getting water-soluble molecules inside cells is a standing challenge for drug development, as water-loving substances cannot easily cross the hydrophobic lipid membrane1,2. Up to date, the transport of hydrophilic bioactive substances inside cells has exploited the amphiphilic character of cargos and carriers. The objective is to transiently mask the water affinity of the cargo by using more hydrophobic (or amphiphilic) carriers, which complex or encapsulate the hydrophilic drug. To date, most of the synthetic carriers for drug delivery are designed by the rules dictated by this amphiphilic paradigm3. Therefore, current prototypical carriers, such as ionizable lipids, include differentiated hydrophilic and hydrophobic domains in their molecular structure to interact with the water-soluble cargo and with the lipid membrane, respectively. However, such synthetic transporters can suffer from certain limitations due to intrinsic features of amphiphilic molecules, particularly their detergent-like behaviour that can damage cell membranes, or their aggregation tendency resulting in poor water solubility, which limits their applicable concentration range.

In this article4, we present a novel class of membrane carriers that leaves behind this amphiphilic dogma. These new carriers are halogenated closo-dodecaborate anions, globular structures of barely 24 atoms that contain a 12-atom boron core, with doubly negative charge and excellent, almost sugar-like, water solubility5 (Figure 1). Despite their charge and the absence of differentiated amphiphilic domains, these boron clusters also present a high affinity for lipid membranes due to their superchaotropic nature, that by far exceeds that of smaller ions6.

Structure and space-filling model of the four closo-dodecaborate anions

Figure 1. Structures of the investigated boron clusters.

Chaotropicity has originally been associated with the ability of dissolved ions to create disorder in the water structure, experimentally reflected, among others, in a decreased viscosity. Over the years, it has matured into an overarching concept that describes the interplay between a special hydration pattern of the ions and their affinity to hydrophobic matter, including lipid membranes, peptides, and organic drug molecules. Superchaotropic ions unite a high water solubility and hydrophilicity with a significant lipophilicity, but they by-pass the classical head&tail design of amphiphiles5-8. Ultimately, superchaotropic ions can form complexes with hydrophilic cargo molecules that can interact and cross hydrophobic barriers such as the lipid membrane. In brief, the globular boron clusters allow molecules to seamlessly shift between both media: water and oil. They can transport peptides even through the macroscopic confines of a U-tube from one aqueous phase, through a chloroform layer, to an aqueous phase on the other side – acting as real carriers.

The conception of this collaborative paper can be traced back four years ago when Prof. Werner Nau hosted Dr. Javier Montenegro for a lecture at Jacobs University in the beautiful city of Bremen at the north of Germany. We are both chemists with a common shared interest in the development of conceptually new membrane carriers and transport phenomena. However, we have a slightly different perspective that is more focused on the physical and the biological projection of the chemistry behind these carriers. In the Nau group (Jacobs University, Bremen, Germany), we study the potential of superchaotropic substances that actuate and transport hydrophilic substances across lipid bilayers and we are striving to develop applications of lipid vesicles as sensors and for transport assays from the physicochemical perspective, together with the then scientific fellow co-authors and now professors Andreas Hennig and Khaleel I. Assaf. In the Montenegro group we are engaged in the design of functional supramolecular systems and synthetic membrane transporters from the more biological standpoint. Therefore, after a brief but very intense scientific discussion during this spring in 2018, a great collaborative research program was sparked that has now crystalized in a first joint publication.

Initial results were acquired in vesicles by the first author of the paper Dr. Andrea Barba-Bon by using a short peptide as the initial cargo. The smallest, and least chaotropic, cluster (B12H122–) was inactive, as there was no evidence for cargo transport, while the largest and most chaotropic one (B12I122–) interacted too strongly with the membrane lipids, breaking the vesicles. However, dodecaborates with intermediate chaotropicity (B12Cl122– and B12Br122–) carried the cargo into the lipid vesicles without leaking their contents. Most interestingly, these carriers did not behave as classical amphiphilic transporters. For example, the transport was not affected by the sequence of addition of cluster and cargo to the vesicles, nor by the vesicle’s membrane charge. Moreover, with the exception of negatively charged molecules, the brominated cluster transported a wide variety of neutral and cationic hydrophilic molecules, ranging from small molecules such as vitamins or neurotransmiters to larger peptides.

Scheme of the proposed mechanism for cargo delivery across lipid bilayers.

Figure 2. Proposed mechanism. The interaction of the cluster with the cargo facilitates the shedding of the water solvation layer that retains the molecules in the aqueous phase, allowing their insertion into the lipid membrane and their transport across the bilayer.

Following studies were developed in Santiago de Compostela, by Giulia Salluce, Ph.D. candidate in the Montenegro group, who tested different cluster carriers for the intracellular delivery of bioactive cargos. In accordance with the vesicles results, the B12Br122– cluster was the star in the series and it was indeed able to deliver different cargos into the cytosol, for instance, phalloidin – an impermeable cyclic peptide traditionally employed for labelling the cytoskeleton of fixed cells. Giulia found that thanks to the boron clusters we could use phalloidin to readily stain the actin cytoskeleton of several living cellular types with no toxicity under the assay conditions. In addition, together with Dr. Irene Lostalé-Seijo, we found that the cluster was capable of transporting other drugs, such as the low permeable PROTAC dBET1 or the antineoplastic monomethyl auristatin F, which were internalised 2-3 times more efficiently in the presence of the superchaotropic cluster.

Confocal microscopy images of cells incubated with phalloidin-TRITC in the absence or presence of the brominated cluster.

Figure 3. The actin cytoskeleton of living cells can be stained with phalloidin using the B12Br122– cluster (right).

It should be noted that part of the experimental work was performed under the restrictions imposed by the pandemic. However, the new and exciting discoveries and the great cargo scope of these new cluster carriers encouraged us to finish the work and reinforced the collaboration. After all, efficient cytosolic delivery of different hydrophilic molecules and functional peptides was being triggered by a small, inorganic, and anionic boron cluster! We believe that superchaotropic anions such as these boron clusters offer a conceptually new tool for the delivery of hydrophilic molecules into cells. So far, we have only demonstrated their potential on in vitro cell culture, and we are aware that, to reach practical applications, there is still a lot of work ahead and many open questions. Is there any limit to the size of the cargo? How are these clusters going to be processed by the cellular machinery? Which substances can be employed to control (enhance/inhibit) their activity? We couldn't be more excited to try to find the answers to these and many other questions in the near future.

The article can be found at: https://doi.org/10.1038/s41586-022-04413-w

References

  1. Lostalé-Seijo, I. & Montenegro, J. Synthetic materials at the forefront of gene delivery. Nat. Rev. Chem. 2, 258–277 (2018). https://doi.org/10.1038/s41570-018-0039-1 
  2. Chiper, M., Niederreither, K. & Zuber, G. Transduction Methods for Cytosolic Delivery of Proteins and Bioconjugates into Living Cells. Adv. Healthc. Mater. 7, 1701040 (2018). http://doi.org/10.1002/adhm.201701040
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  4. Barba-Bon, A. et al. Boron Clusters as Broadband Membrane Carriers. Nature (2022). https://doi.org/10.1038/s41586-022-04413-w
  5. Boron‐based compounds: Potential and emerging applications in medicine. (eds C. V. Teixidor & E. Hey-Hawkins, John Wiley & Sons, Inc., 2018). https://doi.org/10.1002/9781119275602
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  8. Fernandez-Alvarez, R., Ďorďovič, V., Uchman, M. & Matějíček, P. Amphiphiles without Head-and-Tail Design: Nanostructures Based on the Self-Assembly of Anionic Boron Cluster Compounds. Langmuir 34, 3541–3554 (2018). https://doi.org/10.1021/acs.langmuir.7b03306