The characterization of complex mixtures is a difficult issue in analytical chemistry. Taking the analysis of active ingredients in plant medicine Scutellariae Radixas (SR) an example, the pharmacopoeia of many countries stipulates that baicalin, the main effective ingredient of SR, shall be quantitatively analyzed by HPLC, and the quality control content shall not be less than 9%, and it is concluded that its primordial form is baicalin. However, the carbonyl peak (about 1725cm-1) of glucuronic acid in baicalin did not appear when the SR was analyzed by infrared spectrum, but its spectrum was similar to that of baicalin magnesium, indicating that the primordial form of baicalin in SR was probably baicalin magnesium. In this study, In order to confirm the primordial form of baicalin in SR, Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS), combined with Plasma Emission Spectrometry (ICP-OES) and Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-TOF) and other analytical technology, was used to detect and analyze SR samples, baicalin standard samples and baicalin magnesium standard samples, and accordingly characterized the primordial form of baicalin in SR. The results of this study support the viewpoint that the primordial form of baicalin, the main effective component of SR, is baicalin magnesium.

Supramolecular hydrogel formulations have the potential to increase topical delivery of active agents and are well suited being biocompatible, with facile gel formation from cationic surfactant bis-imidazolium salts and combination with anionic, cationic or neutral drugs [Limón et. al., Eur J Pharm Biopharm, 2015]. Although the potential of hydrogels for improved topical skin permeation analysis has been demonstrated using time of flight secondary ion mass spectrometry (ToF-SIMS) [Starr et. al., Int. J. Pharm, 2019], the chemistry of the systems themselves have not been chemically characterised in their native state. This is primarily due to ion beam induced fragmentation and limitations of mass resolving power, as well as the obscuring of the spectra of frozen hydrated samples with water fragment ions.

In this work we investigate the application of cryo-OrbiSIMS in the molecular characterisation of supramolecular hydrogels loaded with two different porphyrins (0.1% w/v). Skin permeation studies were performed to evaluate the delivery of 5,10,15,20-Tetrakis(4-hydroxyphenyl)porphyrin (TPPOH) and 5,10,15,20-Tetrakis(4-carboxylatephenyl)porphyrin (TCPP). It was observed that in ex vivo porcine skin permeation studies the TPPOH appeared to have permeated the skin whereas the TCPP had not. Gel monomer skin permeation was below detectable levels in all cases. In order to understand this difference in delivery, cryo-OrbiSIMS and SEM were performed to determine if there were any variations in the physicochemical properties of the gels.

In native state gels as well as those loaded with porphyrin, the cryo-OrbiSIMS spectra show the detection of a range of secondary ions attributable to the gel, [M-H]+ (m/z 901), TPPOH, [M-4H]+ at m/z 677, and TCPP [M-4Na]^- at m/z 788. Ions detected include molecular and fragments ions. The data suggests that the chemistry of the supramolecular gel is confirmed and that the porphyrins have been successfully loaded into the gels and are uniformly distributed. Using a controlled sample sublimation approach to expose the fibrous microstructure of the frozen hydrated gels, cryo-SEM images indicate structural differences between gels with and without porphyrins, with longer, more interconnected fibres present in gels systems without porphyrins. However, the two porphyrin containing systems are comparable, as such the release behaviour is proposed to relate to a difference in their affinity to the gel fibres.

References:

D. Limón, E. Amirthalingam, M. Rodrigues, L. Halbaut, B. Andrade, M. L. Garduño-Ramírez, D. B. Amabilino, L. Pérez-García, A. C. Calpena, European Journal of Pharmaceutics and Biopharmaceutics, (2015), 96, 421-436.

N J. Starr, K. Abdul Hamid, J. Wibawa, I. Marlow, M. Bell, L. Pérez-García, D. A. Barrett, D. J. Scurr, International Journal of Pharmaceutics, (2019), 536, 21-29.

Gathering a substantial understanding of the molecular composition of a complex biological system is often a tedious task. Achieving this goal frequently requires the use of multiple characterization techniques and experiments, each collecting a small portion of the global information. When following such workflows, researchers have to tackle the inherent complexity of puzzling together this fragmented information in order to extract its biological meaning.

Although an ideal investigation technique yielding the totality of the information about a given system in one experiment is yet to be invented, the field of materials science has been consistent over the last decades in providing biological and medical research with useful investigation techniques. Among these, Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) has proved particularly successful in characterizing the molecular composition of a wide range of biological systems. Indeed, one single ToF-SIMS acquisition yields detailed molecular maps of the sample composition, with a lateral resolution reaching the 50 nm range for state-of-the-art instruments. When applied to the study of a biological system like a tissue cross section, this methodology allows the simultaneous detection of molecular fragments of a size up to that of lipids with very minimal sample alteration. Because the maximum size of molecular fragments generated in ToF-SIMS only reaches a few thousand atomic mass units, the technique is however less adapted to the specific identification of proteins in such systems.

In this work, we applied ToF-SIMS to the investigation of in vitro epidermal model cross sections¹ and were able to successfully map the distribution of numerous endogenous molecules involved in the epidermal barrier function². Moreover, we report the design of metallic nanoparticle-conjugated antibody probes destined for the specific labelling and detection of proteins in tissue cross sections using ToF-SIMS. Finally, we discuss the process of indirect immunolabelling of epidermal proteins using these metallic probes and their detection in ToF-SIMS thanks to low energy sample etching.

References

1. De Vuyst, E., Charlier, C., Giltaire, S., De Glas, V., de Rouvroit, C.L., Poumay, Y. (2013). Reconstruction of Normal and Pathological Human Epidermis on Polycarbonate Filter. In: Turksen, K. (eds) Epidermal Cells. Methods in Molecular Biology, vol 1195. Springer, New York, NY. https://doi.org/10.1007/7651_2013_40
2. Delvaux X., Noël C., Poumay Y., Houssiau L. (2023). Probing the human epidermis by combining ToF-SIMS and multivariate analysis. Biointerphases 18, 011002. https://doi.org/10.1116/6.0002289

The proof-of-concept of the transfer of fragile biomolecules from a target reservoir to a collector substrate using large Ar gas cluster ion beams (Ar_n⁺-GCIB) was recently demonstrated in our laboratory [1]. For instance, biomolecules as large as 14 kDa (lysozymes) could be transferred intact and with retention of their bioactivity.

Currently, properties such as deposition thickness and bioactivity can be controlled very precisely because they are linearly proportional to the total Ar_n⁺ ion dose used to bombard the reservoir [2]. Moreover, because no solvent is used, separate layers can be built on any collector substrate (e.g. paper, PET, silicon, gold, ect.). Therefore, the construction of new biomaterials such as “thick” multilayers has become possible.

With these advances, the work can now move towards applications. For example, molecular transfer can be used to increase ToF-SIMS sensitivity for molecules in tissues. In this approach, Ar_n⁺-GCIB is used to expand a microvolume from the sample to a collector, which is a material ideally enhancing the ionization yield. The collector is then analyzed using a liquid metal ion gun (LMIG). By doing so, the ion signal is increased by 4 orders of magnitude when pure phosphatidylcholine (PC) is expanded using 10 keV Ar₃₀₀₀⁺ and Ar₅₀₀₀⁺ on a sublimated layer of α-cyano-4-hydroxycinnamic (CHCA). This matrix-assisted laser desorption/ionization (MALDI) matrix was selected as the best candidate amongst a pattern of different matrices. The transfer of PC from the grey matter of a mouse brain was also achieved on different materials (e.g. MALDI matrices, Polyethylene, Si and Au) with varying degrees of success.

References

[1] V. Delmez, H. Degand, C. Poleunis, K. Moshkunov, M. Chundak, C. Dupont-Gillain, A. Delcorte, J. Phys. Chem. Lett. 12 (2021) 952–957.

[2] V. Delmez, B. Tomasetti, T. Daphnis, C. Poleunis, C. Lauzin, C. Dupont-Gillain, A. Delcorte, ACS Appl. Bio Mater. 5 (2022) 3180–3192