Reducing cholesterol level in membrane of live macrophages improves their delivery performance by enhancing adaptation to blood shear stress

doi:10.21203/rs.3.rs-3218834/v1

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Reducing cholesterol level in membrane of live macrophages improves their delivery performance by enhancing adaptation to blood shear stress

https://doi.org/10.21203/rs.3.rs-3218834/v1

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In recent years, the live cells-based drug delivery systems have attracted much interests. However, shear stress in the blood flow may cause cell death and waken their delivery performances. In this study, we found that reducing cholesterol in macrophages enhanced its tumor targeting ability by more than 2-fold. Mechanism study indicates that the reduced cholesterol in macrophages deactivated the mammalian target of rapamycin (mTOR) and consequent promoted transcription factor EB (TFEB) nucleus translocation, which enhances the expression of superoxide dismutase (SOD) in cells to reduce reactive oxygen species (ROS) induced by the flow shear stress. A proof-of-concept system using low cholesterol macrophages attached MXene (l-RX) is thus fabricated. On melanoma mice model, tumors are eliminated with no recurrence in all mice after treated with l-RX and laser irradiation. Therefore, we develop a simple and effective way to enhance the targeting performance of macrophage-based drug delivery systems.

live cell delivery system

shear stress

cholesterol

mTORC1

reactive oxygen species

Currently, insufficient targeting to diseased tissues or cells is still an unmet challenge in drug delivery research^{1, 2}. Besides, problems such as poor biocompatibility, hard-to-control degradation in vivo, unclear and possible cytotoxicity, and immunogenicity have troubled the mass application of many delivery systems^3–5.

In recent years, living cells-based delivery systems have provided new opportunities to address these issues^{6, 7}. Living cells have already been used as personalized medicine in clinic and have enjoyed significant successes in some occasions, such as the chimeric antigen receptor T cells (CAR-T) engineered for leukemia treatment⁸. The CAR natural killer (CAR-NK) cells currently being tested are less difficult to obtain and have less rejection than CAR-T cells⁹. Beyond such applications, living cells could also be utilized as drug carriers for delivery purposes. As living cells are inherently present within the body, and their own mobility and chemotaxis endow them with targeting and biological barrier penetration abilities, they may significantly improve the therapeutic efficiency of loaded-drugs without biocompatibility issues^10–12. One of the favored type of living cell vehicles for cancer treatment is macrophage.

Compared with T cells and NK cells, macrophages, as antigen presenting cells, have a wider distribution in the deep tissue of the tumor. This is attributed to cytokines such as C-C Motif Chemokine Ligand 2 (CCL2), C-X-C Motif Chemokine Ligand 12 (CXCL12), Colony Stimulating Factor 1 (CSF-1) and vascular endothelial growth factor (VEGF) released by tumor cells under hypoxia^{13, 14}. In turn, the responsive enrichment of macrophages down-regulates the expression of these cytokines. In addition, the low pH in tumor tissue also attracts macrophage infiltration¹⁵. With these synergistic effects, macrophages efficiently accumulate in tumor tissue. On the other hand, tumor microenvironment (TME) can make T cells and NK cells "dumb fire" in tumor tissue, but macrophages are less affected. Although proinflammatory M1 phenotype macrophages will be transformed into anti-inflammatory M2 phenotype macrophages in TME, the phenotypic change won’t impair their phagocytosis^{9, 16}. More importantly, compared to the irreversibility of the exhaustion phenotypes transformation of T cells and NK cells, macrophages have greater phenotypic plasticity, which provides them with more durable adaptability.

However, in contrast to the common nano-targeted delivery systems such as liposomes, PLGA nanoparticles and extracellular vesicles, living cells are generally more sensitive to the in vivo environment^17–19. They naturally response to external chemical and physical signals which may diminish its targeting function²⁰. Intravenous injection is the major route of administration of living cell carriers^10–12, and cells in the blood are exposed to a complex blood ecosystem, including massive amounts of blood cells and fluid shear stress (FSS) induced by the blood flow^21–23. Living cells will experience several different levels of FSS when they stay in blood circulation²². FSS fluctuates between 0.4 and 6 Pa in veins, arteries, lymphatics and capillaries^{24, 25}. Although the detailed mechanism is not very clear yet, it was found that FSS in the blood could increase the level of reactive oxygen species (ROS) in cells, thus damaging their mitochondria and inducing cell death^{26, 27}. Thus, enhancing the adaptability of cells to FSS can improve their survival rate in the blood²⁸. In this way, reducing cellular ROS may promote cell survival, while improve resistance to ROS is also expected to enhance its tumor targeting.

Unfortunately, FSS is often overlooked in live cell delivery system design, and conventional cell engineering approaches are basically unsatisfactory to reduce the negative effects of FSS^{10–12, 29, 30}. In the past decade, researchers have made various attempts to strengthen cellular resistance to ROS, such as using small molecule ROS inhibitors and nanozymes^31–33. However, adding these agents increases the complexity of the system and the difficulty of clinical transformation, and may bring unwanted side effects^34–36. Adding a layer of hydrophilic materials such as PEG on the surface of cells may also reduce the impact of FSS on cellular ROS, but the surface modification process may affect cell viability and the added layer could disturb the motility of cells³⁷. Therefore, there is an urgent need to find a new strategy to enhance the resistance of delivery cells to ROS induced by blood shear stress.

It is known that mammalian target of rapamycin complex 1 (mTORC1) could regulate cellular ROS level³⁸. Deactivation of mTORC1 promotes dephosphorylation of transcription factor EB (TFEB), which can pass through the nuclear pore and enter the nucleus, resulting in overexpression of superoxide dismutase 1 (SOD1) to reduce cellular ROS ^38–40. It was found that low cellular cholesterol level could inhibit the activity of mTORC1, which in turn activated downstream cholesterol synthesis signaling pathways^{41, 42}. Thus, reducing cholesterol level in cells may enhance their tolerance to ROS induced by FSS, at least in short term⁴¹. Hence, it appears worth checking whether reducing cholesterol content in living cell-based delivery systems could improve their performance. Noticeably, cholesterol is abundant in cell membrane and is constantly being cycled in the cell⁴³, which makes removal of cholesterol from cell membrane a viable way to reduce overall cellular cholesterol level. Methyl-β-cyclodextrin (β-CD), a highly biocompatible compound widely used as a drug solubilizer in the clinic and in various drug delivery systems, has been reported to be able to scavenge cholesterol through host-guest interaction⁴⁴. Thus, simple β-CD treatment on living cells will efficiently decrease cellular cholesterol.

To verify the above conjecture, low-cholesterol macrophages attached with MXene were used as a model system (l-RX). MXene is a 2D material generated from carbides and nitrides of transition metals like titanium, which displays high photothermal conversion efficiency and good biocompatibility^45–48. Macrophages attached MXene was used to induce photothermal therapy (PTT) in melanoma in mice. We found that l-RX accumulated more than 2-fold in tumors compared with normal macrophages, and it conferred a strong resistance to ROS induced by FSS in a TFEB-dependent way (Scheme 1).

Preparation and characterization of l-RX

MXene (Ti₃C₂T_x) nanosheets were prepared using a mild selective etching and delamination method according to previous report (Fig. 1a) ⁴⁹. Transmission electron microscope (TEM) images and scanning electron microscope (SEM) images showed the size of MXene nanosheets were around 300 nm (Fig. 1b, c). Atomic force microscopy (AFM) images of MXene nanosheets as well as AFM height profile showed that the MXene nanosheets were single-layered, the average planar size was around 300 nm and the thickness was about 1.5 nm (Fig. 1d and Fig. S1).

Then we prepared the low-cholesterol macrophages attached MXene (l-RX) (Fig. 1e). First, macrophages (RAW264.7) cells were trained with β-CD medium solution for 30 min to remove partial cholesterol in cells, and incubated in medium for another 1 h. Then cells were mixed with MXene to prepare l-RX. For comparation, normal RAW264.7 cells were mixed with MXene to prepare n-RX. Results show that the level of cholesterol in l-RX was about 30% of n-RX (Fig. 1f). Photothermal conversion effect of MXene was utilized to induce PTT in tumors in this study ⁵⁰. Results indicated that free MXene, n-RX and l-RX showed similar photothermal effect with 808 nm laser irradiation (Fig. 1g). SEM images show that MXene sticked to cell membrane like a patch (Fig. 1h). These results indicate that l-RX was successfully prepared.

l-RX had better survival faced with FSS in a TFEB-dependent manner

The proliferation and migration ability of cells is crucial for living cell-based drug delivery systems^{10–12, 29}. Results show that n-RX and l-RX had similar proliferation after incubating in culture medium (Fig. 2a), indicating that the cholesterol remove strategy did not affect the proliferation rate of cells. This treatment also had no effect on migration ability of cells as tested by the transwell study (Fig. 2b). Then the survival rate of l-RX and n-RX was examined in vitro using methods in previous report²⁸ (Fig. 2c). Results show that about 90% l-RX cells were alive in a constant flow of medium, while only about 20% n-RX cells were alive (Fig. 2d, e). Further study indicated that the FSS increased ROS level in cells, which was consistent with previous reports^{26, 27} (Fig. 2f). ROS level in l-RX was lower than n-RX (Fig. 2f), indicating that ROS may play an important role in the better performance of l-RX. Additionally, the ferroptosis inducer RSL3 was used to increase ROS in cells in this study⁵¹. Results showed that RSL3 increased ROS level in n-RX and killed more than 70% n-RX, while almost 100% l-RX were still alive after incubated with RSL3 (Fig. 2g-i). This cholesterol removal strategy performed better than well-known inhibitor of ROS deferoxamine (DFO) in decreasing ROS and protecting cells against ROS (Fig. 2g-i)⁵². These results indicate that the cholesterol-removal strategy can help cells resist FSS or RSL-induced ROS and improve their survival ability in these conditions.

Then we investigated the mechanism of the cholesterol-removal strategy. As mentioned above, the deficiency of cellular cholesterol inhibits the activity of mTORC1, which can promote the dephosphorylation of TFEB⁴³, and we speculated it may result in the overexpression of SOD1 to reduce cellular ROS ^38–40. Results show that the cholesterol-removal strategy could really promote the nuclear transfer of TFEB (Fig. 3a, b), and western blot analysis of TFEB indicate that more TFEB could be detected in l-RX than n-RX (Fig. S2). Moreover, SOD1 in l-RX was significantly higher than n-RX (Fig. 3c-e), indicating that TFEB-SOD1 may worked in this study.

The activated mTOCR1 could phosphorylate its downstream protein 4EBP1^{5, 41, 53}. Results show that the phosphorylation level of 4EBP1 in l-RX was significantly decreased compared with n-RX both in scramble RAW264.7 cells and RAW264.7 cells with TFEB knockout (RAW264.7^ΔTFEB) (Fig. 3c, d and Fig. S3)⁵. These results indicate that the decreased cholesterol in l-RX inhibit the activity of mTORC1. Although the inhibition of mTORC1 was also detected in l-RX made from RAW264.7^ΔTFEB, SOD1 level in l-RX and n-RX were almost the same when TFEB was knocked out (Fig. 3c-e). These results indicate that the cholesterol removal strategy could increase cellular SOD1 level through mTORC1-TFEB-SOD1 axis.

The level of ROS in l-RX was lower than n-RX and n-RX treated with DFO in a simulated blood circulation system, while this difference was disappeared when TFEB was knocked out (Fig. 3f). The survival rate of cells treated with DFO was about 60%, indicating that the increased ROS played an important role in FSS-induced cell death, which was consistent with previous reports ^{26, 27, 54} (Fig. 3g). In comparation, more l-RX cells were alive faced with FSS, but the cholesterol removal strategy failed to work when TFEB was knockout (Fig. 3g). Taken together, these results indicate that mTORC1-TFEB-SOD1 axis is a key factor in protecting l-RX against ROS induced by FSS, and l-RX has better survival rate in FSS in a TFEB-dependent manner (Fig. 3h).

l-RX showed enhanced tumor targeting ability in vivo

Encouraged by the above results, we first tested performance of l-RX in mice bearing melanoma. B16F10 cells (7 × 10⁵ cells/mouse) were inoculated subcutaneously in C57BL/6 mice, and l-RX were intravenously injected on day 8. Results show that the ROS level in n-RX were increased more than 3-fold after circulated in the blood for 4 hours (Fig. 4a, b). Flow-cytometric analysis results indicated that more l-RX could be detected in the blood compared with n-RX (Fig. 4d), and the fluorescent images of blood also confirmed this result (Fig. 4e). At last, l-RX accumulated more than 2-fold than n-RX in tumor (Fig. 4g, h), and no significant difference was observed in the distribution of l-RX and n-RX in major organs (Fig. S4). These results indicate that the ROS level in l-RX is lower than that of n-RX in the blood, and more l-RX remained alive in the blood to accumulate in tumors.

Then, we tested the PTT effect of l-RX in mice (Fig. 5a). The infrared thermal images show that the temperature of tumors was about 60 ℃, 49 ℃ and 45 ℃ in l-RX, n-RX and MXene group respectively (Fig. 5b). As PTT has been reported to activate the tumor immune microenvironment⁵⁵, and we found l-RX enhanced the expression of calreticulin in tumor cells under laser irradiation (Fig. S5), we then detected the immune cells in tumors. Results show that compared with n-RX, l-RX increased the level of CD4 T cells, CD8 T cells, Granzyme B⁺ CD8 T cells, and M1 macrophages in tumors under laser irradiation (Fig. 5c-f). The proportion of matured dendritic cells (DCs) in tumor draining lymph node (TdLN) was also increased in l-RX group (Fig. 5g). Finally, all tumors did not recur after PTT in l-RX group, while all mice were died from tumor recurrence within 30 days both in MXene and n-RX group (Fig. 5h, i).

Additionally, the biosafety of l-RX was assessed in mice treated by the same scheme described in Fig. 5a. The number of red blood cells, platelets and white blood cells in l-RX-treated group was similar to that of the control group (Fig. S6a-c). Biochemical assays showed that the levels of ALT, AST, UREA and creatinine (CREA) in the serum of treated mice were all within normal range, suggesting that renal and hepatic functions is normal (Fig. S6a-d). These results indicate that l-RX is relative safe in mice.

Live cells are complex self-sustained machines that actively respond to many different types of external and internal stimuli in different ways. When live cells are used as delivery vehicles, their intrinsic responses to the environment will decisively influence the delivery performances. Though it was reported that the FSS may affect cell viability, it is not yet clear how strong the effect is for various types of cells that may enter blood flow.

It has been shown that endothelial cells (ECs) can down-regulate the level of cholesterol on its cell membrane under horizontal FSS in vitro and thus trigger the release of mitochondrial adenosine triphosphate (ATP)²⁰. Cholesterol is a significant factor in mammalian cell metabolism, and the disturbance of its content will affect the regulation of many active proteins in the cell. The mTOR pathway mentioned in this paper has been shown to be one of the important pathways in intracellular lipid regulation, which is regulated by the feedback of reduced cholesterol content⁴². We artificially assisted macrophages to strip cholesterol, which may be a "mock exam" for drug carrier macrophages to improve its adaptability in the blood. In this study, the results show that the prepared macrophages have a significantly increased ability to survive in the blood, and show better targeting as a drug carrier.

The flowing blood in the body not only facilitates the targeted delivery of live cells to the target site, but also poses significant challenges to the cellular drug delivery system^{22, 28}. It is reasonable to suspect that other cells and other cellular regulation pathways such as metabolic control and migration, may also be affected by FSS. The mechanical signal may jointly regulate cell behavior with other signals in vivo, though the detailed mechanisms are still mostly unclear^{26, 27, 54}. For drug delivery purpose, these results demand us to further investigate the role of FSS on macrophage and other cells that may be used as delivery vehicles, in order to rationally engineering cells for even better delivery performance. This is also an important clue for biological researches that focus on cell activity in blood.

The cell engineering method applied in this study is simple and seemingly effective. Macrophages were used as model cells, and quick β-CD pretreatment was shown to inhibit mTORC1 and activate the TFEB-SOD axis in cell, which weakened the influence of FSS on drug-loaded cells. The survival rate of drug-loaded cells was guaranteed and a longer blood half-life period was obtained, which endowed drug-loaded cells better targeting ability. It is possible that inhibitors (e.g., rapamycin) of mTORC1 may also weakened the influence of FSS on drug-loaded cells, but it will require extra cargo loading capacity and procedure, and the residual inhibitors in cells may also induce side effects on the body. Other option such as modifying polymer materials on the surface of cell membranes may affect the activity and active transport ability of cells, and also increase the complexity of the product, which is not conducive to clinical transformation. Moreover, our “subtractive” cholesterol removal is likely suitable for other carrier cells such as neutrophils, NK cells, and T cells.

Additionally, this subtractive strategy did not influence the proliferation and transfer of cells (Fig. 2a, b), and due to the inhibition of mTORC1, the intracellular cholesterol content may be repaired during the later self-renewal process of cells^{41, 42}, which may be beneficial for cells function at the target site.

In conclusion, we found that removing cholesterol in cells enhanced the adaption of cells to FSS in the blood. Mechanism study indicate that this “subtractive strategy” increased cellular SOD1 through mTORC1-TFEB-SOD1 axis, and thus it endowed cells with the ability to resist ROS induced by FSS. As a model system, the cholesterol-removed cells attached MXene (l-RX) was used to treat melanoma in mice. The tumor targeting ability of l-RX is more than 2-fold than n-RX, and it activated anti-tumor immunity to prevent tumor recurrence under laser irradiation (Scheme 1). This highly versatile “subtractive strategy” is worth further investigation in the future.

Materials

Ti₃AlC₂ powder was purchased from MACKLIN in China; Lithium fluoride (LiF, AR) was purchased from Aladdin Inc. (China); RPMI 1640 medium (include penicillin/streptomycin) was brought from KeyGEN BioTECH in China; Antibodies for flow cytometry assay were obtained from Biolegend; anti- calreticulin antibody was obtained from Abcam; anti-TFEB, anti-4E-BP1, anti-Phospho-4E-BP1 (Thr37/46) antibodies were obtained from Cell Signaling Technology; anti-SOD1 antibody was obtained from Proteintech in China; HRP-conjugated secondary antibodies were obtained from Proteintech in China; PMSF protease inhibitor was bought from Solarbio in China; RIPA cell lysate, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD) dyes was brought from Beyotime Biotechnology in China; DCFH-DA was brought from Sigma; Methyl-β-cyclodextrin was purchased from MACKLIN in China; Cholesterol Assay Kit was obtained from ApplyGEN in China.

Animals and cells

Male C57BL/6 mice (18 ± 2 g, 6–8 weeks) were obtained from GemPharmatech in China. All animal experiments were approved by the Institutional Animal Care and Ethics Committee of Sichuan University. RAW264.7 and B16F10 were brought from American Type Culture Collection and were cultured in a complete RPMI-1640 medium. RAW264.7^{TFEB KO} cells and scramble RAW264.7 cells were established in our previous work⁵, and they were cultured in a complete RPMI-1640 medium. All cells were maintained in a humidified atmosphere incubator containing 5% CO₂ at 37°C.

Preparation of MXene

Ti₃C₂T_x nanosheets were prepared from a mild selective etching and delamination according to previous study ⁴⁹. Specifically, 1 g of LiF was mixed in 20 mL of HCl solution. 1 g of Ti₃AlC₂ was slowly added to the mixed solution. Then, the mixture was stirred for 24 h at 35 ℃ to allow the etching of Ti₃AlC₂. After reaction, the acidic suspension was repeatedly washed with deionized water for 6 times. Subsequently, the clay-like Ti₃C₂T_x sediment was dispersed in deionized water, and delaminated through vigorous hand-shaken for 8 min. The suspension was repeatedly centrifuged at 3500 rpm for 5 min. The supernatant of each centrifugation was collected as few-layered Ti₃C₂T_x nanosheets suspension.

Remove cholesterol in RAW264.7 cells

RAW264.7 cells were washed with PBS, and then cells were incubated with methyl-β-cyclodextrin (β-CD) in serum free RPMI-1640 medium (without serum) for 0.5 h at 37 ℃ (5 mM of β-CD was used in most experiments otherwise stated). The level of cholesterol in RAW264 cells as well as n-RX and l-RX was detected by Cholesterol Assay Kit.

Preparation of n-RX and l-RX

RAW264.7 cells in a petri dish (1*10⁷ cells per dish) were washed twice with PBS and then re-suspended with 1 ml PBS. 0.2ml MXene solution (MXene 0.4mg) was added to the cell suspension and the system was slowly shaken in a shaker for 10 min at 37℃. The cells were collected by centrifugation and suspended with 1ml normal saline for later use.

The injection dose of mice was based on MXENE content (MXene 2 mg/kg).

Cell migration of n-RX and l-RX

l-RX and n-RX were seeded in the transwell inserts (8 µm, 1*10⁵ cells per well) for 2 h in serum-free medium. After cells settled, the medium containing 20% serum was added as outer culture. After 24 h training, we washed the transwell inserts with PBS gently and fixed with 4% paraformaldehyde for 15 min. Then 0.1% crystal violet were dripped to the inserts gently and stained for 15 min. Images were detected by optical microscope.

Detection of ROS in vitro

In order to evaluate cellular ROS level, the treated cells were stimulated with DCFH-DA-dissolving medium (10*10^− 6 M) for 30min. After that we centrifuged the collected samples (500 xg, 3 min) and discard the supernatant. The cells were resuspended in PBS and detected by flow cytometry for quantitative analyses. ROS-induced DCFH-DA fluorescence in cells could be detected at Ex/Em of 488/525 nm.

Detected ROS level and viability of n-RX and l-RX in the blood

n-RX and l-RX were labeled by DiD perchlorate and then washed three times with PBS and resuspended in PBS. This process takes place in a sterile environment. After quantifying the fluorescence intensity of the cells with a fluorescence spectrophotometer, the cells were intravenously injected into the mice (1*10⁵ cells per mouse). The mice were kept in a dark environment for 4 h before blood was taken. Red blood cell lysate was added to the collected blood in a 1:9 ratio and left to lyse at 4°C for 10 min. Then we centrifuged the cells and discarded supernatant. After three washes with PBS, intracellular ROS labeling was performed as mentioned above and detected by flow cytometry.

Immunofluorescence staining of TFEB in cells

RAW264.7 cells was treated as described above, and washed with PBS three times. Then the cells were blocked with a PBS solution containing 0.1% triton X-100 and 5% BSA for 1.5 h. After this, the primary antibody was added and incubated at 4 ℃ overnight. Samples were washed with PBS three times again. Then Cy3-rabbit secondary antibodies (1:100) was added and incubated at room temperature for 1 h. Before detected by CLSM, the cells were washed with PBS.

In vitro fluid shear stress model

The microfluidic system mimicking blood flow was used in this study according to the published literature²⁸. The microfluidic system aimed to mimic the fluid shear stress of bloodstream by controlling the flow rate of peristaltic pump. The inner radius of circulatory tube is 0.15 mm. The flow rate of RPMI-1640 medium is 1.2 mL/min. The circulation system was sterilized with 75% ethanol, followed by sterile water, finally RPMI-1640 medium. 2.5*10⁴ l-RX/mL was used in this study, and cells were collected after 50 min circulation time. Then cell viability was detected by CCK8 kit and cellular ROS was detected by flow cytometry.

Detected protein expression by western blot analysis

n-RX and l-RX were treated with RIPA lysis buffer (1 mM PMSM) for 40 min at 4 ℃. Samples with loading buffer were boiled in water bath for 10 min. Then samples were separated by SDS-PAGE gel and transferred to PVDF membranes. PVDF membranes were blocked with 5% BSA in TBST for 1 h at room temperature and incubated with primary antibodies including SOD1 (1:1000), 4EBP1 (1:1000, Phospho-4E-BP1 (Thr37/46) (1:1000) and β-actin (1:500) overnight at 4°C. Then PVDF membranes were washed 3 times with TBST, and incubated with HRP-conjugated secondary antibodies (1:8000) for 1 h at room temperature. The PVDF membranes were incubated with an enhanced chemiluminescence (ECL) substrate kit.

In vivo imaging of biodistribution

Subcutaneously inoculated B16F10 cells (0.7*10⁶ cells/mouse) in male C57BL/6 mice. n-RX and l-RX were labeled by DiD perchlorate and then washed three times with PBS and resuspended in PBS. After modeling for 8 days, injections were given at 1*10⁶ cells per mouse. After 12 hours in the dark, the mice were used for in vivo fluorescence photography.

H&E staining

For H&E assay, the main organs (including heart, liver, spleen, lung and kidney) of the mice were isolated at the end of the treatment and fixed in 4% paraformaldehyde solution. The slices were prepared routinely and stained with hematoxylin and eosin, and then detected by optical microscope.

In vivo anticancer treatment

Subcutaneously inoculated B16F10 cells (0.7*10⁶ cells/mouse) in male C57BL/6 mice. l-RX and n-RX were intravenously injected on the day 8. The tumor was irradiated with 808nm laser for 5 minutes (1.0 W/cm²) at 12 h post the first injection. Size of tumors was recorded every two days. Mice were sacrificed when the tumor volume⁵⁶ is greater than 2000 mm³.

Statistical analysis

Statistical analysis was performed using GraphPad prism software. All quantitative parameters were presented as mean with Standard Error of Mean. For two-group comparison, Student’s two-sided t test was performed for the statistical analysis. For multiple comparisons, the data were analyzed using a two-way analysis of variance (ANOVA). P < 0.05 was considered statistically significant. ns, no significant difference. *p < 0.05, **p < 0.01 and ***p < 0.001, ns, no significant difference.

ACKNOWLEDGMENTS

We acknowledge the support from the National Science Fund for Excellent Young Scholars (No. 82022070), the Regional Innovation and Development Joint Fund (No. U20A20411), and the Innovation Program of Med-X Center for Materials from Sichuan University (MCM202103).

AUTHOR CONTRIBUTIONS

Ling Zhang and Lin Li designed this research. Lin Li and Mengxing Zhang performed and analyzed most experiments. Jing Li, Na Ji, Qixue Bao, Ningyun Sun, Hongding Rong, Xu Peng, Qin Lin, Zhirong Zhang assisted in analyzing the results and doing experiments. Lin Li and Ling Zhang prepared the manuscript. All authors have given approval to the final version of the manuscript.

COMPETING INTERESTS

The authors declare no competing financial interest.

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Scheme 1 is available in the Supplementary Files section.

Supportinginformation.docx
Scheme1.png
Scheme 1. Schematic illustration showing the preparation of l-RX and the proposed mechanism of l-RX to target to tumors. n-RAW264.7, normal RAW264.7 cells; l-RAW264.7, low-cholesterol RAW264.7 cells; n-RX, normal RAW264.7 cells with MXene; l-RX, low-cholesterol RAW264.7 cells with MXene.

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Reducing cholesterol level in membrane of live macrophages improves their delivery performance by enhancing adaptation to blood shear stress

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Abstract

Figures

INTRODUCTION

RESULT AND DISSCUSION

Preparation and characterization of l-RX

l-RX had better survival faced with FSS in a TFEB-dependent manner

l-RX showed enhanced tumor targeting ability in vivo

CONCLUSION

MATERIALS AND METHODS

Materials

Animals and cells

Preparation of MXene

Remove cholesterol in RAW264.7 cells

Preparation of n-RX and l-RX

Cell migration of n-RX and l-RX

Detection of ROS in vitro

Detected ROS level and viability of n-RX and l-RX in the blood

Immunofluorescence staining of TFEB in cells

In vitro fluid shear stress model

Detected protein expression by western blot analysis

In vivo imaging of biodistribution

Statistical analysis

Declarations

References

Scheme

Supplementary Files

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