In vivo quantitative characterization of nano adjuvant transport in the tracheal layer by photoacoustic imaging

Adjuvants are indispensable ingredients in vaccine formulations. Evaluating the in vivo transport processes of adjuvants, particularly for inhalation formulations, presents substantial challenges. In this study, a nanosized adjuvant aluminum hydroxide (AlOOH) was synthesized and labeled with indocyanine green (ICG) and bovine serum albumin (BSA) to achieve strong optical absorption ability and high biocompatibility. The adjuvant nanomaterials (BSA@ICG@AlOOH, BIA) were delivered as an aerosol into the airways of mice, its distribution was monitored using photoacoustic imaging (PAI) in vivo. PAI results illustrated the gradual cross-layer transmission process of BIA in the tracheal layer, traversing approximately 250 µm from the inner layer of the trachea to the outer layer. The results were consistent with pathology. While the intensity of the BIA reduced by approximately 46.8% throughout the transport process. The ability of PAI for quantitatively characterized the dynamic transport process of adjuvant within the tracheal layer may be widely used in new vaccine development.


Introduction
Public interest in inhaled vaccines has increased significantly since the Corona Virus Disease 2019 (COVID-19) outbreak [1].This is mainly because the route of infection and disease progression is mainly through the lungs, so direct inhalation of drugs into the respiratory tract is the most appropriate route of administration for the treatment of COVID-19 [2].Inhaled vaccines trigger immune defense against specific pathogens [3], including viruses and bacteria, by activating immune defense mechanisms in the respiratory mucosa.In this process, the trachea plays a vital role as it is the key airway connecting the larynx and bronchi.The tracheal structure consists of four main layers: adventitia, cartilage layer, mucosa/submucosa, and tracheal muscle [4], each of which plays an important role in the delivery of inhaled vaccines.To enhance the body's immune response to inhaled vaccines and prolong the duration of the response, adjuvants are often added during the vaccine production process [5][6][7][8].The aluminum hydroxide adjuvant is approved by the Food and Drug Administration (FDA) for use in human vaccines [9] and has been used for more than seventy years [10] with a commendable safety profile [11].However, despite the widespread use of vaccine adjuvants in billions of doses of human vaccines [12], their transport processes in the tracheal layer, visual and quantitative characterization are still insufficient, especially the specific mechanisms by which adjuvants cross the tracheal layer, time and process remain to be understood in depth.This study focuses specifically on the aluminum hydroxide adjuvant, aiming to deeply explore its dynamic transport process in the respiratory tract and achieve its quantitative characterization in the tracheal layer.
In the field of biological imaging, researchers use various medical imaging technologies to obtain biological information [13].These techniques include computed tomography (CT), magnetic resonance imaging (MRI), fluorescence imaging (FI), and positron emission tomography (PET) [14].Although current imaging techniques have made progress in the visualization of adjuvants, there are still limitations in resolution and sensitivity, particularly in the ability to accurately track and visualize the distribution of tiny drug particles or adjuvants in small tissues and organs.To address limitations, photoacoustic imaging (PAI) provides a new observation window into the transport process of adjuvants in the tracheal layer.As an emerging imaging technology in biomedical imaging, PAI combines the high contrast of optical imaging with the deep tissue penetration capability of ultrasound imaging [15][16][17][18], showing great potential in observing the vaccine adjuvant transport process in the tracheal layer.This technology achieves high-resolution imaging of tissue structure and function by detecting tissue absorption of light pulses and generating acoustic signals [19][20][21].Cause its multimodal and cross-scale advantages, PAI enables in vivo characterization of the distribution of nanomaterials of different sizes in vivo [22,23].It has shown great potential applications especially in the fields of biological deep tissue imaging and tumor theranostics [24,25].Unlike PET and CT imaging techniques, PAI avoids the use of harmful ionizing radiation and therefore has obvious advantages in terms of safety [26,27].Furthermore, compared with MRI, PAI is not only cheaper but also easier to perform [28,29].
Although vaccine adjuvants themselves are not directly trackable by PAI technology, the surface labeling of these adjuvants with a contrast agent can significantly enhance the imaging signal and improve the contrast of the adjuvant to make the transport process of the adjuvant in the tracheal layer more precise.Indocyanine green (ICG) is an FDA-approved vital dye [30,31] and is widely used as a developer in medical imaging due to its unique fluorescence properties and biocompatibility.The molecular structure of ICG gives it strong absorption and fluorescence emission properties in the near-infrared region.Its peak absorption is located at approximately 780 nm, while its fluorescence emission peak is located between approximately 800 nm and 810 nm [32,33].This makes ICG particularly suitable for imaging biological tissues, which have lower absorption in this spectral range and allow deeper light penetration.ICG has an amphiphilic molecular structure, exhibiting hydrophilicity and lipophilicity [34,35], which enhances its application versatility in various environments [36][37][38].Therefore, the use of ICG in research can significantly sharpen the imaging resolution and contrast, enabling precise visualization of the adjuvants' transport process.
In this investigation, bovine serum albumin (BSA) and ICG were amalgamated via noncovalent bonding to create BSA@ICG [39,40].Further, through electrostatic adsorption, this complex was combined with nanoscale aluminum hydroxide adjuvant (AlOOH) to formulate the nanocomposite BSA@ICG@AlOOH (abbreviated as BIA).Aluminum hydroxide adjuvant acquired PA properties and was used to track the vaccine adjuvant in the tracheal layer using the PAI technique.BIA was delivered into the respiratory tract of mice in the form of aerosol, depositing on respiratory mucosal surfaces.As time goes by, BIA is continuously transported from the inner layer of the airway to the outer layer.PAI technology was used to monitor the distribution of BIA in the tracheal layer of mice at different time points, and detailed PAI results were obtained.The detailed process of the experiment is shown in Fig. 1.

Preparation of AlOOH nanomaterials
In this study, the hydrothermal method was employed to synthesize aluminum hydroxide nanorods [41].Initially, aluminum nitrate nonahydrate [Al (NO 3 ) 3 •9H 2 O] (1.3933 g) was added into the beaker, and then add ultrapure water (20 mL).During stirring, EDA (0.238 mL) was gradually and carefully added to the solution, followed by continuous stirring for 15 min.The reaction mixture was then transferred to a Teflon-lined stainless-steel autoclave.The autoclave was placed in an electric oven with the temperature maintained between 160 to 200°C, and the reaction was allowed to proceed for 16 hours at this temperature.Finally, AlOOH nanomaterials were obtained.

Preparation of BIA nanomaterials
To synthesize BIA.Initially, BSA (47 mg) was added to the beaker, followed by the addition of ultrapure water (15 mL) to dissolve the BSA.For the ICG (8 mg), it was dissolved in dimethyl sulfoxide, the ICG solution was added to the BSA solution.This mixture was stirred for 12 hours in a dark environment.After stirring, the mixture underwent dialysis for 24 hours, yielding the preliminary BSA@ICG product.The dialyzed BSA@ICG solution (5 mL) was added to the beaker.Additionally, dilute the AlOOH solution to 20 mg/mL The AlOOH solution (4 mL) was then added to the BSA@ICG solution.The mixture was stirred for 12 hours in a dark environment, forming the final BIA nanomaterials.

Characterization
First, the morphology of the BIA material was imaged using transmission electron microscopy (TEM) (Talos F200S, Thermo Fisher Scientific, America).Subsequently, the analysis involved the assessment of elemental distribution within the BIA material via Energy-Dispersive Xray Spectroscopy (EDS).Additionally, dynamic light scattering (DLS) and Zeta potential experiments were conducted using a Malvern particle size analyzer (ZETASIZER NANO ZS, Malvern, England).The UV spectrophotometer (UV-2600, SHIMADZU, Japan) was employed to determine the ultraviolet-visible absorbance of various concentrations of BIA, as well as to measure the ultraviolet-visible absorbance of different samples.Finally, a Fourier transform infrared spectrometer (FT-IR) (NICOLET iS50 FT-IR, Thermo Fisher Scientific, America) was used to measure the chemical bonds and functional groups of AlOOH and BIA.

Animal and ethics statement
All procedures involving animals were conducted by the Guidelines for Institutional Animal Care and Use Committee, and the ethical approval for this study was provided by the Guangzhou Medical University Institutional Animal Care and Use Committee (GY2023-218).This research utilized Kunming mice (8-10 weeks, weighing 35-45 g) as the study subjects.Twelve mice were randomly allocated into four groups, with three mice per group.One group inhaled saline, serving as the normal control group (NC group).The remaining three groups inhaled AlOOH, ICG, and BIA, respectively, and were designated as the ICG group, BSA@ICG group, and BIA group.

PA performance of BIA
Different concentrations of BIA (0, 0.5, 1, 2 and 4 mg/mL) were injected into capillary glass tubes and then PA images and intensities were obtained.To better observe the effect on the stability of BIA under multiple laser pulses, PA images at different time points (0, 15, 30, 60, 90, and 120 min) were obtained to assess the PA stability of the nanomaterials by monitoring the changes in PA intensity.

Cytotoxicity of BIA
The effect of BIA on cell viability was determined using cell counting.Initially, a suspension of human normal lung epithelial Beas-2B cells (100 µL, 5 × 10ˆ4 cells per well) was seeded into a 96-well plate.The 96-well plate was incubated in a 37°C cell culture incubator for 12 to 24 hours.Subsequently, the cells in each well were incubated with varying concentrations of BIA solution (0, 0.2, 0.4, 0.6, 0.8, 1, 2, 4 mg/mL) for 24 hours.After the 24-hour incubation, CCK-8 solution (10 µL) was added to each well.The plate was then returned to the 37°C incubator for 3 hours.Finally, the absorbance of each well at a wavelength of 450 nm was measured using a spectrophotometer.

Histological analysis
To assess the impact of nebulized inhaled materials on the major organs of mice, histological analysis is required.On the first day, mice from the respective groups inhaled materials through a nebulizer.On the seventh day, the mice were euthanized.Following euthanasia, major organs, including the heart, liver, spleen, lung, and kidney were extracted for histological analysis.The extracted organ samples were fixed in paraformaldehyde solution.Subsequently, the tissues were dehydrated; the embedded tissue blocks were subjected to sectioning.Next, these tissue sections were stained with H&E.Finally, the stained tissue sections were scanned with a slide scanner (Aperio CS2, Leica Biosystems, Germany) to obtain histological images of mouse organs.

In vivo PAI experiment
In this study, we used a series of refined experimental procedures to explore the transport of different materials in the tracheal layer of mice.Initially, mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital.After anesthesia, use electric clippers and depilatory cream to remove fur from the mouse's neck and upper chest.Subsequently, a nebulizer was used to atomize the materials to be tested into the respiratory tract of the mice.The nebulizer has a frequency of 110 K Hz and an aperture of 5 µm.This process lasted for 15 min to ensure that these materials effectively adhered to the respiratory tract.After the nebulization process is completed, use surgical scissors to expose the mouse's tracheal area in preparation for PAI.
Mouse tracheal PAI was performed under 770 nm laser excitation.Using a photoacoustic multi-modal small animal imaging system (PASONO-ANI, Photoacoustic Technology, China), which has a center frequency: 30 MHz; -6 dB bandwidth: 100%; the imaging speed is 10 × 10 mm 2 per 6 min.A pulsed laser with a wavelength of 770 nm was used as the optical excitation source with a repetition frequency of 50 kHz.The spatial resolution of the system was measured to be 7.7 µm, system imaging depth up to 2.4 mm, as shown by Fig. S1.PA signal profiles on the sharp edge of the scalpel blade was measured to fit the edge spread functions.Deriving the edge spread function gives the line spread function and calculates its full width at half-maximum (FWHM).Insert the blade into the agar block to obtain the system imaging depth.This system provides detailed images of the distribution of adjuvants in the tracheal layers.Finally, the PAI results were quantitatively analyzed using Matlab.By quantifying the intensity of the PA signal and its propagation distance within the trachea, this study was able to accurately assess the distribution of the material within the trachea of the mouse.

Characterization of BIA
Under ambient room temperature conditions, BSA was successfully employed as an intermediary to label ICG onto AlOOH, thereby synthesizing the BIA nanomaterials.The preparation process of this nanomaterial is documented in Fig. 1.
The morphology of BIA was observed using TEM.The TEM results revealed that BIA has a rod-like structure, with a diameter of approximately 40 nm and a length of approximately 320 nm. Figure 2(a) distinctly displays these structural characteristics.Furthermore, the hydrodynamic diameters of both AlOOH and BIA materials were measured using DLS.As shown in Fig. 2(b), the results indicated that the average hydrodynamic diameters of AlOOH and BIA were approximately 160 nm and 320 nm, this is similar to the results found under TEM.To assess the long-term stability of the materials, DLS measurements were conducted over seven days. Figure 2(c) shows that within seven days, the average hydrodynamic diameters of AlOOH and BIA have little difference, indicating good stability of the synthesized materials.In further analysis of BIA, the Zeta potential of the materials was measured, with results presented in Fig. 2(d).Figure 2(e) shows the absorption spectra of different materials in the near-infrared region.Among them, BIA has two characteristic peaks at 730 nm and 810 nm respectively.The peak absorption of ICG is observed around 750-780 nm, whereas the absorption peaks of BIA are around 800-830 nm and show a significant redshift due to the aggregation of ICG [42,43].In addition, the characteristic peaks of BIA did not shift at different concentrations, and the absorption value increased with increasing concentration, as shown in Fig. 2(f).
Subsequently, the lyophilized powders of AlOOH and BIA were obtained, and their functional groups and chemical bonds using FT-IR.In the FT-IR analysis of AlOOH and BIA, a series of significant absorption peaks were observed, as illustrated in Fig. 3(a).For the AlOOH sample, the peak at 3443 cm −1 is attributed to the stretching vibrations of the O-H bond, indicating the presence of hydroxyl groups.In contrast, the BIA sample exhibited a similar O-H stretching vibration peak at 3443 cm −1 .Still, a peak at 1644 cm −1 highlighted the amide vibration of the C = O in proteins, a typical hallmark of the secondary structure of BSA.Additionally, the peak at 1512 cm −1 resulted from the combined contributions of the amide II vibration in proteins and the C = C stretching vibrations in ICG.Lastly, the peak at 1033 cm −1 in the BIA sample is associated with the C-O single bond vibration in ICG, reflecting the structural characteristics of ICG.These spectral features reveal the distinctions in chemical composition and structure between the two samples.The composition and structure of BIA were further confirmed by EDS analysis, and the results are shown in Fig. 3(b).EDS analysis showed the rod-like structure of AlOOH, and the elements of aluminum, sodium and sulphury represented AlOOH, ICG and BSA, respectively, which demonstrated the successful labelling of aluminum adjuvant with ICG.

BIA in vitro PA performance
PAI was performed on five different concentrations of BIA under laser excitation and the results are shown in Fig. 4(a).It can be observed that BIA exhibits PA effect even at low concentrations.As the concentration of BIA increased, the corresponding PA signal was significantly enhanced.Figure 4(b) shows the quantitative analysis of PA signals from PA images at each concentration, and it was found that the concentration of BIA increased exponentially in relation to its PA intensity, and that concentrations above 4 mg/mL are appropriate to use when performing imaging.Figure 4(c) shows the PA images of the material acquired at different time points, and the PA signal did not undergo significant signal attenuation after multiple image acquisitions.Then, it was quantitatively analyzed, and the results are shown in Fig. 4(d), the PA intensity of BIA does not show a sharp decrease, which indicates the photostability of BIA under multiple laser pulses.Therefore, the above results show that BIA exhibited desirable photoacoustic performance in vitro.

Cytotoxicity results of BIA
In the study, cytotoxicity experiments were conducted to evaluate the biocompatibility of BIA.Through these cellular assays, the impact of the material at varying concentrations on the viability of Beas-2B cells was observed.As indicated by the data in Fig. 5, BIA exhibited negligible toxicity to the cells.Cell viability remained relatively stable at concentrations below 1 mg/mL, with a vitality value of approximately 0.98, suggesting minimal impact of BIA on the cells within this concentration range and almost no toxicity at lower concentrations, demonstrating good biocompatibility.
When the BIA concentration is equal to or greater than 1 mg/mL, it promotes cell viability, and as the concentration increases, enhanced cell viability is observed.This may be due to the nutrient-supportive environment of BSA that indirectly promotes cell growth and maintenance.This indicates that BIA not only exhibits good biocompatibility within a certain concentration range, but may also have the potential to promote cell growth.
To comprehensively assess the biosafety of BIA, further in vivo investigations were conducted on its potential harm to biological organs.The experimental results, as illustrated in Fig. 6, indicate that compared to normal organs, there were no apparent tissue damages in the organ samples from each experimental group.This implies that aerosolized inhalation of BIA does not cause significant harm to the major organs of the mice, suggesting that it does not exert significant toxic effects on the major organs when applied in vivo.

Trachea layered dynamic PAI
In this work, with the demonstration of BIA's PA capabilities, its application to transport changes in the tracheal layer of a normal mouse model was further explored.This work was accomplished   using a PA multimodal small animal imaging system.The PA signal was visualized with a "HOT" color bar, while ultrasound (US) imaging provided a background for the location of the trachea.After nebulization was completed, high-resolution and high-contrast images in the tracheal layer were acquired at different time points over the next 2 hours, as shown in Fig. 7.The results in Fig. 7 clearly show the position changes and intensity changes of the PA signals of each group.In contrast, during the two-hour monitoring period, no obvious PA signal position changes and intensity changes were observed in the NC group and AlOOH group.The ICG group and the BIA group showed obvious transport changes in the tracheal layer (specific areas are marked with green boxes).Specifically, at the beginning of the experiment, both the ICG group and the BIA group showed peak PA signal intensity and were in the inner layer of the trachea.As time went by, the intensity of these signals gradually attenuated and moved toward the outer layer of the trachea.
To conduct further quantitative analysis of these results, we used Matlab software to conduct a detailed analysis and quantified the PA intensity and movement distance of each group at different time points, as shown in Fig. 8(a) and Fig. 8(b).The analysis results show that during the entire experiment, the signal intensity of the NC group and AlOOH group was roughly stable at 4, and there was no obvious moving trend.As time went by, the PA signal intensity of the ICG group and BIA group continued to decrease, while the signal intensity continued to decrease.Transported to the outer layer of the trachea.At the beginning of the experiment (0 min), both the ICG group and the BIA group showed good adhesion to the inner layer of the trachea.At this time, they were not affected by metabolism in the body.The signal intensity of both groups was about 30.As the experiment progressed, we observed that ICG and BIA began to cross the mucosal layer.At 15 min, the signal intensity of the ICG group decreased by approximately 30% and moved approximately 100 µm; the signal intensity of the BIA group decreased by approximately 13% and moved approximately 80 µm.This is due to the smaller size of the ICG, which moves faster, but the number of ICGs being cleared simultaneously is also greater.At 30 min, the moving distance of both groups was approximately 150 µm, but there was still a difference in PA intensity.Neither the ICG group nor the BIA group showed significant changes in signal intensity and moving distance during the periods between 30 and 60 min and between 60 and 90 min respectively, which may be because greater resistance is encountered when crossing the cartilage layer and takes longer to overcome.By 120 min, the PA intensity of the ICG group and BIA group dropped by about 72.4% and 46.8% respectively, and both groups finally moved about 250 µm and reached the outer layer of the trachea.To verify the accuracy of PAI results, the trachea of mice was sampled and pathologic sections were made.The thickness of the trachea in the region imaged by PAI was about 235 µm, as shown by Fig. S2.The results of the sections were in good agreement with those of PAI.Based on the PAI results and related quantitative characterization data of all experimental groups, the transport process of the adjuvant within the tracheal layer was successfully observed.We believe that early in this transport process, the adjuvant begins its transport process after adhering to the lining of the trachea.As the first point of contact, the mucosa/submucosa have a certain ability to clear foreign substances.This is a natural defense mechanism designed to protect the respiratory tract from harmful substances and pathogens.Therefore, a portion of the adjuvant may be cleared during this process, explaining the rapid decrease in the adjuvant PA signal.Subsequently, the adjuvant reaches the cartilage layer, probably because the density and structural properties of the cartilage layer provide greater resistance to the adjuvant, resulting in an increase in the residence time of the adjuvant in this layer.Eventually, the adjuvant reaches the adventitia and completes its transport in the tracheal layer.The adventitia, as the outermost layer of the trachea, marks the completion of the adjuvant's passage through the tracheal layer.This process not only shows the physical crossing ability of the adjuvant, but also reflects the complexity of the adjuvant's behavior in the body, involving multiple factors of biophysics and biochemistry.These may include the physical and chemical properties of the adjuvant, such as particle size, morphology, and surface charge.In addition, the transport process of adjuvants in the body is also affected by the biological characteristics of the host, including the pH value of the local microenvironment, enzyme activity, and immune cell responses.These factors together determine the transport capacity of the adjuvant, which requires further study.

Conclusion
In this work, the aluminum hydroxide adjuvant was labeled by ICG, and PAI technology was used to achieve quantitative characterization of the transport process of the adjuvant in the tracheal layer.The study meticulously records the distribution and transport process of BIA in the trachea over time, demonstrating a progressive translocation from the mucosa/submucosa to the outer adventitia layer, indicative of the adjuvant's dynamic movement and its interaction with the biological barriers within the tracheal structure.BIA has potential applications in respiratory diseases such as respiratory syncytial virus infections and whooping cough, and viral pneumonia such as COVID-19.Quantitative characterization of the adjuvant transport process in the tracheal layer not only provides an important scientific basis for the development of inhaled vaccines, but also broadens the scope of PAI technology in biomedical applications.With the further development and optimization of PAI technology, its role in biomedical research and clinical applications will become more significant, especially in non-invasive, real-time monitoring of biological processes and therapeutic effects.

Fig. 1 .
Fig. 1.AlOOH labeled with ICG and BSA, and its transport process within the mouse trachea layers was monitored by PAI.

Fig. 2 .
Fig. 2. Characterization of BIA.(a) Morphology under TEM.(b) DLS measurement of hydrodynamic size.(c) Measurement of hydrodynamic size for seven consecutive days.(d) Measurement of Zeta potential.(e) UV absorption intensities of different materials.(f) UV absorption intensity of BIA at different concentrations.

Fig. 4 .
Fig. 4. In vitro PA property of BIA.(a) PA images at 770 nm excitation for different concentrations of BIA.(b) Quantitative analysis of the variation of PA intensity with BIA concentration at 770 nm.(c) PA images of BIA at different time points.(d) Quantitative analysis of the photostability of BIA at different time points.

Fig. 6 .
Fig. 6.H&E staining images of heart, liver, spleen, lung, and kidney sections of mice in each group.The scale bar is 200 µm.

Fig. 8 .
Fig. 8. (a) PA intensity values of the four groups.(b) PA signal movement distance of the four groups.