Modification of PEG reduces the immunogenicity of biosynthetic gas vesicles

Nanobubbles have received great attention in ultrasound molecular imaging due to their capability to pass through the vasculature and reach extravascular tissues. Recently, gas vesicles (GVs) from archaea have been reported as acoustic contrast agents, showing great potential for ultrasound molecular imaging. However, the immunogenicity and biosafety of GVs has not yet been investigated. In this study, we examined the immune responses and biosafety of biosynthetic GVs and polyethylene glycol (PEG)-modified GVs (PEG-GVs) in vivo and in vitro. Our findings suggest that the plain GVs showed significantly stronger immunogenic response than PEG-GVs. Less macrophage clearance rate of the RES and longer circulation time were also found for PEG-GVs, thereby producing the better contrast imaging effect in vivo. Thus, our study demonstrated the PEG modification of biosynthetic GVs from Halobacterium NRC-1 is helpful for the future application of GVs in molecular imaging and treatment.

. More and more biomarkers which exhibit great diagnostic values on the surface of tumor cells are identified, but they are difficult to be detected by ultrasound molecular imaging because MBs-based acoustic probes cannot penetrate through blood vessels and contact extravascular tumor cells (Kose et al., 2020). Studies have shown that tumor blood vessels have some 380-780 nm gaps between endothelial cells, however, these gaps are still too small for MBs to get through. Therefore, it is desirable to develop a kind of nanobubbles which have nanoscale particle size and can penetrate through tumor blood vessels (Guvener et al., 2017;Cai et al., 2018).
In recent years, Shapiro et al. have identified gene-encoded nanoscale gas vesicles (GVs) in bacteria and archaea (Lakshmanan et al., 2017;Bourdeau et al., 2018). Physiologically, these GVs can help the buoyancy of microbes to better obtain sunlight and nutrients, with a particle diameter of 45-250 nm width and a length of 100-600 nm (Shapiro et al., 2014). GVs have protein shells, being mainly composed of GvpA and GvpC proteins (Pfeifer, 2015). Hydrophobic GvpA forms the spindle-shaped skeleton, and the hydrophilic GvpC is arranged in the outer structure of the protein shells (Distribution, 2012;Hill and Salmond, 2020;Volkner et al., 2020). Our previous studies demonstrated GVs from Halobacterium NRC-1 bacteria have about 200 nm particle size and exhibited excellent contrast imaging performance by using of clinical diagnostic ultrasound equipment at the optimized parameters (Wei et al., 2022). However, owing to the shells composed of proteins, GVs are easily removed by macrophages of the reticuloendothelial system (RES) and probably bring with some immunogenicity or side effects, limiting their use in the future clinical practice (Ling et al., 2020;Yan et al., 2020).
Evidences demonstrated that surface modification by polyethylene glycol (PEGs) greatly reduces the uptake of nanoparticles by RES and prolongs the in vivo survival time of nanoparticles in the circulation (Wang et al., 2020;Yan et al., 2020). Meanwhile, surface modification by PEGs also can shield the surface antigen of nanoparticles, reducing the occurrence of immune response. In this study, examined the immune responses and biosafety of biosynthetic GVs and polyethylene glycol (PEG)-modified GVs (PEG-GVs) in vivo and in vitro. Especially, their contrast imaging performance were also evaluated in vivo.

Preparation of GVs and PEG-GVs
Halobacterium NRC-1 bacteria were cultured at 37 C on a shaking incubator at 220 rpm/min for 2 weeks. These bacteria were placed in a separatory funnel for 1-2 weeks to collect the upper floated bacteria. These bacteria were lysed by using of TMC lysis buffer and GVs were isolated through centrifugation at 300 g for 4 h. The isolated GVs were washed with phosphate-buffered saline (PBS) and further purified by centrifugation for 3 to 4 times at 250 g for 4 h. Finally, the GVs were stored in PBS at 4 C. The concentration of GVs was estimated using a microplate reader (Multiscan GO, Thermo Scientific, Waltham, MA, United States) at a wavelength of 500 nm.
PEG was conjugated to the surface of GVs through amidation reaction in the presence of EDC and NHS. Briefly, the GVs were dispersed in PBS (pH = 7.4) containing with EDC (5 mg) and NHS (3 mg), followed by incubation at room temperature for 2 h. After that, the mixture was slowly added to the PEG-amine (260 mg) solution and further incubated overnight. The resulting solution was centrifuged to remove excess EDC, NHS and PEG-amine and rinsed for 4 times with PBS. ICG-labeled GVs or ICG-labeled PEG-GVs were obtained by adding ICG NHS ester solution (10 mg/mL) to pure GVs solution (OD 500 = 3.0) in PBS (pH = 7.4) for 2 h incubation at room temperature. Similar rinse steps were used for removing these free ICG.

Characterization of GVs
GVs orbacterium solution was diluted in PBS at room temperature for determine the particle size. The morphologies of GVs were observed by transmission electron microscopy (TEM) (Hitachi H-7650, Japan). The particle size and zeta potential of GVs was measured using a Zetasizer analyzer (Malvern, United Kingdom). The size and zeta potential of each sample were measured three times.

Phagocytosis of CVs and PEG-GVs by macrophages
MurineRAW264.7macrophageswereseededin24-wellplateswith DMEM high glucose medium supplemented with 10% FBS and 1% antibiotic solution at 37 C and 5% CO 2 . 24 h later, cells were washed by PBS and incubated with ICG-labeled GVs or ICG-labeled PEG-GVs at 37 C and 5% CO 2 for 4 h. The cells were washed with cold PBS and fixed with paraformaldehyde for 30 min, followed by staining by DAPI for 10 min in the dark. Finally, these cells were observed under a confocal microscope (A1R, Nikon, Japan). The excitation and emission wavelengths were set at 780 nm and 800 nm. Flow cytometry was used to determine the cell number of macrophages which uptake ICG-labeled GVs. The data were analyzed using FlowJo. GVs (OD 500 = 3.0, 100 μL/well) for 24, 36 and 48 h. After that, these cells were spun at 300 g for 10 min, and the supernatant medium was collected for cytokine TGF-β and IL-6 analysis by cytokine Dakewe ELISA kits. Also, the cells were harvested and washed twice with icecold sterile PBS. Cells were labeled with anti-CD86-PC5.5 and anti-CD80-APC antibodies, followed by flow cytometry analysis of the expression of CD86 and CD80 levels. The data were analyzed using FlowJo.

In vivo immunogenicity assay of GVs and PEG-GVs
Animal experiments were conducted under protocols approved by the Ethics Committee of Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences. The male C57BL/ 6 mice (four to 6 weeks old, 18-20 g) were maintained with isoflurane anesthesia on a heating pad. The plain GVs or PEG-GVs at OD 500 3.0 were intravenously injected into the mice, once a day for three injections. 7 days later, the spleens of mice were collected and cut into small pieces, followed by treatment with collagenase V (2 mg/mL, YEASEN) in HBSS solution at 37 C for 4 h. These cells were then filtered through a 70 μm cell strainer to obtain a single cell suspension and washed with FACS buffer. The singlecell suspension was pre-blocked with CD16/32 antibody (Biolegend, Cat

Biological distribution of GVs and PEG-GVs in vivo
Ten C57BL/6 mice were randomly allocated into two groups, (a) ICG-labeled GVs were injected into the tail vein of the mice. (b) ICG labeled PEG-GVs at OD 500 3.0 were injected into the tail vein of the mice. Hearts, livers, spleens, kidneys, lungs were acquired at 1 h, 4h, 6 h, 12 h or 24 h after injection and fluorescent signal intensities of these organs were detected by IVIS Spectrum (Caliper Life Sciences, United States; Excitation Filter: 745 nm, Emission Filter: 800 nm). Also, the major organs of mice at varying time points (i.e. 1 h, 4 h, 6 h, 12 h or 24 h) post intravenous injection of GVs or PEG-GVs were collected, weighted, homogenated and centrifuged at 1,200 rpm for 5 min and the supernatant was subjected to absorbance measurement.

In vivo ultrasound contrast imaging
Five C57BL/6 mice (6-8 weeks old, male, 20-24 g) were subcutaneously injected with a suspension of 1 × 10 6 MB49 cells in PBS (100 μL) to established the tumor model. When the tumor volume reached 100-200 mm 3 , mice were randomly divided into two groups, including GVs group and PEG-GVs group. Ultrasound image was performed in these tumors by an ultrasound diagnostic system (Resona 7, Mindray, China). All imaging parameters remain the same during all imaging procedure (acoustic power = 5.13%, gain = 70 dB). Images were acquired continuously for 10 min after injection of GVs or PEG-GVs. When the acoustic signals enter in plateau after a single injection, a burst pulse is applied to collapse the PEG-GVs. Images were acquired for another 6 min again. One frame of image per second was extracted in these videos and the perfusion area of tumors was defined as the region of interest (ROI) when the contrast signal achieved strongest. The average acoustic signal intensity of the ROI per frame was measured using ImageJ to quantify the contrast signal.

Cytotoxicity detection of GVs
The MB49 cells and bEnd.3 cells were seeded in 96-well plates at a density of 1 × 10 4 cells per well for overnight at 37 C and 5% CO 2 . The next day, cells were washed 3 times with PBS and incubated with GVs at a given concentrations (OD 500 = 2.0, 2.4, 2.8, 3.2 or 3.6). After 24 h, cell viability was evaluated by CCK-8 assay kit.

In vivo biosafety assay of GVs
Ten mice were systemic administration of PBS, GVs or PEG-GVs (100 μL, OD 500 = 3.0). The blood samples were collected from the ophthalmic arteries after 1 day, 7 days and 14 days to detect the blood biochemical indicators, including liver function markers (alanine aminotransferase, ALT; aspartate aminotransferase, AST) and kidney function makers (blood urea nitrogen, BUN; creatinine, CREA). The main organs (hearts, livers, spleens, lungs and kidneys) were acquired at 7 days after three consecutive injections of GVs a week, fixed with paraformaldehyde (4%, W/V) for H&E staining.

Statistical analysis
The data were expressed as the mean ± standard deviation. Comparisons among groups were analyzed by independent-samples one-way ANOVA test using Graphpad Prism 8.0.1 software unless otherwise specified. The asterisks (*p < 0.05, **p < 0.01, ***p < 0.001) were considered significant and n. s represented no significance. center). After modified with PEGs, the resulting PEG modification did not significantly change their appearance ( Figure 1B, right). However, PEG-GVs became slightly larger than that of plain GVs in the hydrodynamic particle size, with 342 nm average particle size ( Figure 1D). The Zeta potential of plain GVs was −35.53 ± 0.61 mV while the potential of PEG-GVs was nearly neutral, with −0.78 ± 0.31 mV Zeta potential. These data shown PEGs were successfully coated to the surface of GVs, which are consistent with the previous report that nanoparticles (NPs) typically become slightly larger due to the surface coating of PEGs [22].

Immune escape ability of PEG-GVs in vitro
To examine whether of PEG modification can help GVs to escape from RES uptake, we labeled the GVs and PEG-GVs with indocyanine green (ICG), a near-infrared (NIR) fluorescent dye. The ICG-labeled GVs or ICG-labeled PEG-GVs were then co-incubated for 6 h with RAW264.7 murine macrophage and then the cell uptake of GVs was detected by fluorescence microscopy and flow cytometry (Figure 2A). From Figures 2B,C, we can see that numerous ICGlabeled GVs entered the macrophages by the process of phagocytosis, emitting a strong red fluorescence signal. By contrast, there was almost no detectable signals for ICG-labeled PEG-GVs incubated macrophages, revealing that the PEG-GVs can escape from RES uptake after PEG modification.

In vitro immunogenicity of GVs and PEG-GVs
To explore the immunogenicity of GVs, we used PEG modification to reduce the immunogenicity of GVs. The plain

In vivo immunogenicity of GVs and PEG-GVs
To further examine the immune response caused by GVs and PEG-GVs in mice, we intravenously injected GVs or PEG-GVs at OD 500 3.0 three times a week and collected mouse immune cells from the spleen after 7 days post the last injection. The percentage of M1 phenotype macrophages (CD80 + /MCHⅡ + ) and M2 phenotypes macrophages (CD206 + ) in spleen were assessed by flow cytometry. Figure 3A showed that there were 20.8% CD80 + /MCHⅡ + cells (M1 phenotype macrophages) in the spleen of GVs-treated mice but only 14.1% CD80 + cells in the spleen of PEG-GVs-treated mice, Frontiers in Bioengineering and Biotechnology frontiersin.org similar with PEG-treated mice. Notably, the proportion of M2 phenotype macrophages (CD206 + F4/80 + ) in spleen for PEG-GVs-treated mice was significantly recovered to 17.3%, similar with PBS-or PEG-treated mice while only 9.92% in spleen for GVstreated mice ( Figure 3B). Significantly higher CD206 + /CD86 + cells were found in the spleen of GVs-treated mice (20%) but not the spleen of PEG-GVs-treated mice (11.9%), indicating that PEG-GVs did not promote the maturation of BMDCs cells ( Figure 3C). The proportions of CD4 + and CD8 + T-cells were 14.7% and 19.0% in the spleen of PEG-GVs-treated mice, significantly lower than those of GVs-treated mice, with 17.9% CD4 + cells and 24.0% CD8 + cells, respectively ( Figure 3D).

Biodistribution and ultrasound imaging of GVs and PEG-GVs in vivo
Next, we determined the in vivo biodistribution of GVs and PEG-GVs (Bowery et al., 1978;Xu et al., 2022a;Xu et al., 2022b). ICG-labeled GVs or ICG-labeled PEG-GVs at OD 500 3.0 were intravenously injected The signal intensities of GVs in livers were 1.41 ± 0.03 a. u and 1.49 ± 0.01 a. u after 4 h and 24 h respectively, while the signal intensities of PEG-GVs in livers were 1.27 ± 0.02 a. u and 0.68 ± 0.01 a. u, respectively. It should be noted that PEG-GVs exhibited significantly lower accumulation in livers than GVs.
Also, we examined the ultrasound imaging performance of GVs and PEG-GVs through the tail vein into tumor-bearing mice. GVs(OD 500 = 2.8, 100 μL)and PEG-GVs(OD 500 = 2.8, 100 μL)were injected into the tail veins of tumor-bearing mice and then the tumors were imaged in the contrast mode at 0 s, 60 s, 120 s, 180 s and 240 s after injection. From Figure 4D, we can see that the tumors received with PEG-GVs showed stronger ultrasound contrast signals after 60 s, compared with the tumors received with GVs. The contrast signals of the tumors received with GVs began to decline and tended to Frontiers in Bioengineering and Biotechnology frontiersin.org disappear after 120 s. By contrast, the contrast signals of the tumors received with PEG-GVs could keep the enhanced contrast signals for a longer time (Figures 4D,E). The results show that PEG modification of GVs can prolong the circulation time. To confirm the contrast signals from PEG-GVs, we applied a short high-power ultrasonic pulse to collapse these PEG-GVs after 10 min post-injection. Figures 4F,G showed these contrast signals disappeared immediately after the ultrasonic burst and reappeared in the tumor, confirming these contrast signals were really from PEG-GVs.

In vitro and in vivo biosafety of GVs and PEG-GVs
The results of the in vitro cytotoxicity test showed that GVs and PEG-GVs had no significant cytotoxicity to the mouse brain microvascular endothelial cells (bEnd.3 cell line) and mouse bladder cancer (MB49 cell line) at the all tested concentrations (OD 500 = 2.0, 2.4, 2.8, 3.2 and 3.6) ( Figures 5A,B). H&E staining analysis showed systemic administration of GVs or PEG-GVs did not produce significantly pathological damage to the hearts, livers, spleens, lungs, and kidneys, similar the PBS control ( Figure 5C). In addition, an analysis of blood samples from mice administered systemically to 1-, 7-, and 14-day mice showed all of blood biochemical markers, including alanine aminotransferase (ALT), aspartate aminotransferase (AST) for liver function, and creatinine (CREA) and urea nitrogen (BUN) for renal function, were within the normal arrange ( Figures  5D-G). All these results indicated that GVs and PEG-GVs have good safety in vitro and in vivo.

Discussion
Compared to conventional ultrasonic contrast agents, nanoscale GVs extracted from Halobacterium NRC-1 can Frontiers in Bioengineering and Biotechnology frontiersin.org perfuse tumor ischemic regions and generate contrast signals due to their small particle size (Wei et al., 2022). However, similar to conventional nanoparticles, most of GVs after tail vein injection were usually uptake by liver macrophages, resulting in a weak signal in the tumor (Kreuter, 1996;Le Floc'h et al., 2018).
Although studies have shown that systemic administration of GVs does not cause acute toxicity in mice, it is necessary to study the immune response caused by GVs composed of protein shells (DasSarma and DasSarma, 2015;Hill and Salmond, 2020;Adamiak et al., 2021;Kim et al., 2022). In this study, we used PEG to modify GVs, aiming to reduce the phagocytosis of liver macrophages and to reduce the immunogenicity of GVs (Cruz et al., 2011;Cui et al., 2018). Our findings suggest that the plain GVs have some immunogenicity. But the immunogenicity of the GVs can be greatly decreased through PEG surface modification.
In this study, we found that PEG modifications can reduce the clearance of GVs, significantly prolong blood circulation time, and increase accumulation of GVs within tumors. The ultrasound contrast signals of PEG-GVs immediately disappear when using a short, high-power ultrasonic pulse to collapse these PEG-GVs after 10 min of injection, confirming these signals from PEG-GVs. In addition, the ultrasonic signal of PEG-GVs can be observed again due to the reperfusion of PEG-GVs. Although there were not obvious cytotoxicity to the in vitro cultured bEnd.3 and MB49 cells and damage to liver and kidney functions for systemic administration of GVs and PEG-GV S . It is noteworthy that GVs have a certain immunogenicity to induce the immune responses in the mice. The possible reason for this may attribute to the fact that GVs are consisted of protein shells synthesized by bacteria. Fortunately, the protein shells provide lots of carboxyl and amino groups, making it possible modify these GVs with PEGs. Just as our data shown in Figures 4D,E, significantly longer circulation time can also be observed when using of PEG-modified GVs.

Conclusion
In this study, we successfully synthesized PEG-GVs by coating GVs with PEG, greatly reducing the immunogenicity of GVs and RES uptake, and increasing blood circulation time. We demonstrated that PEG-modified GVs can escape liver uptake and prolong tumor imaging performance. Importantly, our findings suggest that these GVs can induce the macrophage polarization from M2 to M1 phenotype and promote the mature of BMDCs. But these effects can be greatly attenuated by using of PEG surface modification. In conclusion, our study provides new ideas for the future clinical translation of GVs in molecular imaging and disease diagnosis and treatment.

Data availability statement
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.

Ethics statement
The animal study was reviewed and approved by The animal study protocol was approved by the Institute's Animal Care and Use Committee (IACUC) of Shenzhen Institutes of Advanced Technology, Shenzhen Academy of Sciences. Author contributions YW, MF, and YY: data collection, data analysis, and manuscript writing. JZ, ZZ, and JX: data collection. YZ and FY: project development. All authors contributed to the article and approved the submitted version.