APS Review—The team of Dean Guo/Wanying Wu focused on the spatial distribution of heterogeneity in natural medicine R&D, and reviewed the role and research progress of mass spectrometry imaging

Figure 2: Application of mass spectrometry imaging in the discovery of medicinal plants/microbial natural products

(Source: Hou JJ, et al.
, APS, 2022).

After obtaining biologically active NPs from medicinal plants/microorganisms, MSI techniques can be applied to facilitate preclinical research based on the following three aspects [15]: First , in absorption, distribution, metabolism and excretion (ADME) and pharmacokinetic-pharmacodynamics (ADME).
Pharmacokinetic-pharmacodynamic, PK-PD) studies, MSI can provide a direct spatial distribution of compounds to facilitate the understanding of NPs and their ADMES Characteristics for more intuitive spatial heterogeneity analysis
.
At the same time, the spatial association between NPs and in situ endogenous pharmacodynamic biomarkers can be established (Figure 3).

In drug development, determining the in vivo ADME of a drug is critical
to determining its druggability.
Various molecular imaging techniques, including radiolabeling, magnetic resonance imaging, fluorescence imaging, and Raman imaging, have been used to study the tissue distribution of drugs [16].

However, MSI technology has unique advantages in spatial resolution, chemical information provision, and label-free [17].

First, mass spectrometry imaging can provide label-free visualization of spatial heterogeneity information within the drug
.
This has been applied to the study of the in vivo distribution heterogeneity of many NPs, such as adenosine YZG330/YZG331, powdered prohexine, paclitaxel, sutrazine methyl ether, sitterin, leptosine7 tetracyclic indole alkaloids, etc
.
MSI can also provide visual access to the spatial process of drug absorption, addressing processes
that are difficult to observe with traditional analytical methods.
Cutaneous absorption of drugs [18-20] and intestinal absorption [21, 22] are the main links in the study of drug absorption in vivo, and the use of mass spectrometry imaging technology can provide the depth and degree
of drug skin and intestinal absorption intuitively.
In addition, MSI can intuitively provide spatial distribution information of multiple metabolites at the same time, and provide the spatial correlation of relevant pharmacodynamic markers of drugs at the same time
.
The label-free advantage of MSI enables this technique to not only visualize the spatial distribution characteristics of NPs and their metabolites with high spatial resolution, but also to simultaneously observe the spatiotemporal changes
of endogenous pharmacodynamic markers in vivo after drug intervention.
Second, "therapeutic heterogeneity" is the characteristic of drug action [23], and MSI technology can promote the accuracy and prediction of NPs efficacy and toxicity analysis in efficacy and safety evaluation and prediction studies (Figure 4).

First, the spatial heterogeneity analysis of drug distribution in target organs can better understand the pharmacodynamic heterogeneity
of drugs.
Second, when the drug is not well distributed at the target, it will reduce the efficacy of the drug, which is difficult to observe
in traditional tissue distribution.
The organs of drug enrichment and distribution in the body must be necessarily related
to its efficacy or toxicity.
With the widespread application of MSI and its spatial resolution and sensitivity increase, it is possible
to predict the potential efficacy or toxicity of natural drugs based on the results of mass spectrometry imaging analysis.
Third, MSI can provide rationality
for NPs modification, formulation optimization, and nanomaterial selection.
NPs can effectively improve drug targeting through chemical modification, formulation optimization or selection of appropriate nanomaterials
.
MSI can use its intuitive analysis to visually display the target distribution of optimized drugs, providing visual evidence
for optimization.
In addition, as metal nanomaterials play an increasingly important role in the development of pharmaceutical formulations, more attention is paid to their distribution in vivo
.
Due to the characteristics of metal nanomaterials, under the laser-based mass spectrometry imaging platform, metal nanomaterials have characteristic mass spectrometry signals, which can be used to monitor them and analyze their spatial distribution [23, 24].

Figure 3: Application of mass spectrometry imaging in drug development ADME and PK-PD research

(Source: Hou JJ, et al.
, APS, 2022).

Figure 4: Application of mass spectrometry imaging in the accuracy, predictability, and design of chemical modifications and dosage forms in drug efficacy and toxicity analysis

(Source: Hou JJ, et al.
, APS, 2022).

MSI technology adds new spatial dimension analysis tools to the research and development of NPs, but obtaining the ideal analysis results requires the following problems or optimization
.
First, the two analysis modes of MSI determine the direction
of analysis method.
For discovery model studies, scientific hypotheses are proposed by exploring the unknown spatial distribution of components such as elements, small molecules, peptides, proteins, or N-glycans [25, 30], and further combined with other techniques to elucidate scientific questions
.
This research model requires full understanding of different sample processing methods, and the choice of different ion sources and mass spectrometry instruments can affect the observed spatial distribution characteristics and component types, that is, "what is not seen".
For the verification mode research, based on the existing research distribution law, it better visualizes and displays the unseen spatial distribution, and confirms it with other technologies
.
This research mode needs to reasonably select and optimize the sample processing and ion source parameters according to the characteristics of the target components to obtain the best imaging sensitivity and spatial resolution, that is, "unseen"
.
In addition, in situ matrix effects should be considered when performing quantitative distribution analysis [31].

Second, sample processing methods are a key part of
MSI.
The importance of sample processing has been repeatedly mentioned in several mass spectrometry imaging reviews, some of which have been specifically reviewed [32, 33].

Mass spectrometry imaging sample processing includes at least the following five aspects: 1) tissue type selection; 2) Slice acquisition method; 3) Processing method of slicing; 4) whether the slice is derivatized and derivatization method [34, 35]; 5) Selection and spraying method
for matrix in ion sources that require matrix assistance.
Again, ion source selection is at the heart of
MSI.
The core component used in MSI is an in-situ ion source
.
At present, according to the principle of in situ extraction and ionization, it can be divided into three categories of ion sources: laser-based (UV or IR) ion sources; Nanospray-based ion sources and ion beam-based ion sources
.
In addition, ion mobility adds a separation dimension to MSI, and the impact of mass spectrometry instrument matching and the balance of spatial resolution, mass spectral resolution, sensitivity, and data acquisition time on the analysis cannot be ignored
.

As a powerful visual analysis technology, MSI plays a unique role
in the study of NPs and their understanding of disease intervention by displaying the distribution of thousands of molecules in tissue space without labels.
With the development of MSI, it will have more exciting innovative applications in the following four aspects: first, with the development of mass spectrometry imaging instrument platform, the improvement of acquisition pixel resolution and acquisition speed will make single-cell and subcellular mass spectrometry imaging analysis easier to achieve, and will be better integrated with existing biological research results; Second, with the development of other spatial omics technologies, especially the improvement of spatial transcriptomics to single-cell resolution, the combination of the two will provide more powerful analytical means for the study of spatiotemporal resolution.
Third, with the deep combination of ultra-high-resolution mass spectrometry imaging and metabolic flow technology, the dynamic changes
of metabolism in the spatial dimension in vivo will be better studied.
Fourth, the establishment of public databases for mass spectrometry imaging analysis and the application of data mining will promote the wide application of
mass spectrometry imaging technology.
The development of MSI in the above four aspects will provide a powerful spatiotemporal analysis method for the in-depth development and research of NPs.

However, MSI, as a lossy analysis, must be analyzed on tissue sections, making it difficult to analyze the dynamic distribution
of molecules in living tissues in real time.
Real-time visual analysis of all molecules inside plants and animals may be one of humanity's dreams, but it's the number one driving
force leading the way in technology.

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