Platelet and Erythrocyte Membranes Coassembled Biomimetic Nanoparticles for Heart Failure Treatment

doi:10.21203/rs.3.rs-3326363/v1

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Platelet and Erythrocyte Membranes Coassembled Biomimetic Nanoparticles for Heart Failure Treatment

https://doi.org/10.21203/rs.3.rs-3326363/v1

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Cardiac fibrosis is a prevalent pathological process observed in the progression of numerous cardiovascular diseases and is associated with an increased risk of sudden cardiac death. The BRD4 inhibitor JQ1 has powerful anti-fibrosis properties, its clinical application is extremely limited due to its side effects. There is still an unmet need for effective, safe and low-cost treatment. Here, a multifunctional biomimetic nanoparticle drug delivery system (PM&EM nanoparticles) is presented, which is assembled by platelet membranes and red blood cell membranes to deliver JQ1 for treating cardiac fibrosis. The platelet membrane endows PM&EM nanoparticles with the ability to target cardiac myofibroblasts and collagen, while the participation of erythrocyte membrane can increase the long-term circulation ability of the nano drug-loaded system and can be further engineered to increase fluidity. In addition, PM&EM nanoparticles can deliver sufficient JQ1 with controllable release to achieve excellent anti-fibrosis effects. Based on these advantages, it is demonstrated in both pressures overloaded induced mouse cardiac fibrosis model and MI-induced mouse cardiac fibrosis that injection of novel fusion membrane biomimetic nanodrug carrier system can effectively reduce fibroblast activation, reduce collagen secretion, and improve cardiac fibrosis. In addition, it can greatly reduce the toxic and side effects of long-term JQ1 treatment on the liver, kidney and intestinal tract. These results suggest that the integration of native platelet and erythrocyte membranes as a multifunctional biomimetic drug delivery system has great potential value in the treatment of cardiac fibrosis and the prevention of drug side effects.

Health sciences/Cardiology/Cardiac device therapy

Health sciences/Diseases/Cardiovascular diseases/Heart failure

Hybrid membrane

nanotherapy

cardiac fibrosis

Cardiac fibrosis is a characteristic feature of numerous chronic cardiovascular diseases, which ultimately result in heart failure (HF).^{1, 2} Although heart failure resulting from severe cardiac fibrosis can be treated with heart transplantation, the lack of donors and the extremely low success rate of transplantation greatly limit the treatment of cardiac fibrosis.³ Therefore, there is an urgent need for development of safe, effective and low-cost targeted therapies that reduce the burden of heart disease on both patients and health care systems.

Previous studies have identified the epigenetic reader protein bromodomain-containing protein 4 (BRD4) to be a novel target in the treatment of cancer and the BET bromine domain inhibitor JQ1 to have a therapeutic effect in mice with HF caused by long-term stress overload or extensive anterior myocardial infarction (MI).⁴ JQ1 has strong therapeutic potential in treating and reversing fibrotic diseases.⁵ However, the therapeutic effect of JQ1 is hampered by its comparative narrow therapeutic window (i.e., 50% effective dose/50% lethal dose, ED50/LD50). Oral or intravenous application of JQ1 can cause a broad effect of systemic BET inhibition, so that while it is anti-fibrotic, but cause systemic multi-system damag.^6–9 Therefore, there is an urgent need for a targeted drug delivery system that can increase accumulation of JQ1 in fibrosed cardiac tissue while mitigating its toxicity.

Recently, a new class of drug nanocarriers, fabricated by combining synthetic nanoparticle cores with biologically derived membrane coatings, has been reported.¹⁰ These biomimetic cell membrane-coated nanoparticles can exploit the versatility and complexity of cell membranes, which are produced naturally and have specific functions, especially in terms of biological interfaces. By transferring the entire membrane of the cell to the surface of the nanoparticle, all biologically relevant surface fractions are preserved, including those that may be used for immune evasion and targeting, which are highly desirable features.¹¹ Furthermore, cell-specific functions that are known to exist but have not been adequately characterized at a basic biological level can be exploited. These nanocarriers pioneered the use of erythrocytes (red blood cells) to prolong blood residency time, and these cell membrane-coated nanoparticles are immunomodulatory markers that have the same density as the original erythrocytes, such as CD47.¹² In addition to erythrocytes, other types of cells, including platelets, white blood cells, cancer cells, stem cells, and even bacteria, each of which have their own unique properties, can be used to obtain membrane materials.^{13, 14} Notably, expansion of the cell membrane-coating platform to other cells enables fabrication of naturally targeted drug carriers.

While cell membrane coatings are a powerful way of enhancing the therapeutic value of nanoparticles, additional functionality may be required depending on the intended application.¹⁵ For example, although erythrocyte membrane-coated nanoparticles can cycle for long periods of time, addition of targeted ligands can help to improve localization of these nanoparticles to desired targets, such as sites of fibrosis. This article describes a method that can be used to increase the number of functions that nanocarriers can perform by deriving functions simultaneously from multiple cell types.¹⁶ This novel approach, which involves fusion of native cell membranes with different origins, is a simple but effective method that can be used to create nanoparticles capable of performing increasingly complex tasks in biologically relevant environments. Specifically, the functions of platelets and erythrocytes of human origin were combined and the resulting platelet membrane- erythrocytes membrane complex (PM&EM) has been confirmed to retain the functions unique to each type of cell when attached to nanoparticles. A novel drug-loading system for encapsulation of JQ1 as an anti-fibrosis agent was prepared and found to have good targeting ability and an anti-fibrotic effect in mouse models of pressure overload-induced and MI-induced cardiac fibrosis. Investigation of the toxicity of JQ1 in other organs shows that this novel drug-loading system can effectively reduce accumulation of JQ1 in tissues such as the liver and kidney, and attenuate the adverse effects of this agent. This drug-loading system could be developed as an effective and safe strategy for treating cardiac fibrosis in the clinical setting.

2.1 Preparation and characterization of PM&EM/JQ1 NPs

Platelets membranes were selected as ideal candidates for coating membranes because they contain a variety of functional proteins (e.g., glycoproteins IIb/IIIa and VI) that can effectively target collagen and collagen-secreting activated fibroblasts.¹⁷ Moreover, as previously reported, erythrocyte membrane-coated nanoparticles can circulate for long periods of time.¹⁸ The functions of human erythrocytes and platelets were combined in platelet-erythrocyte hybrid membrane-coated JQ1 loaded nanoparticles (PM&EM/JQ1 NPs). This research demonstrates that PM&EM/JQ1 NPs retain functions unique to both platelets and erythrocytes.

To prepare PM&EM/JQ1 NPs, JQ1-loaded PLGA nanoparticles (JQ1 NPs) were first prepared, after which the membranes of platelets and erythrocytes are extracted using hypotonic lysis. In this study, platelet and erythrocyte membranes were fused at a concentration ratio of 4:1, which has been shown to maintain efficient membrane function after fusion of these membranes. The extracted platelet and erythrocyte membranes are then coated with JQ1 nanoparticles using ultrasound.

To determine the fusion process, a dye pair with Förster resonance energy transfer (FRET) property, DiO and Dil, was used to label the hybrid membranes¹⁶, followed by fusion with unlabeled erythrocyte membranes. After addition of the erythrocyte membrane, the fluorescence intensity at 555 nm (Dil) decreases (Fig. 2E), indicating that the platelet membrane had fused with the erythrocyte membrane, weakening the fluorescence signal of Dil (i.e., the acceptor dye). Furthermore, the platelet membranes and erythrocyte membranes were labeled, respectively, with DiO and DiD fluorescent dyes, and encapsulated DiI fluorescent dyes with PLGA. Under confocal laser scanning microscopy (CLSM), these three raw materials showed almost no overlay when they were only mixed physically but show effective overlap when assembled by ultrasonic mixing (Fig. 2A). Dynamic light scattering (DLS) analysis showed that following the hybrid membrane coating step, the diameter of the measured nanoparticles increased from ≈ 182.6 to ≈ 195.0 nm, which corresponds to the thickness of the coated biological bilayer film (Fig. 2B). The zeta potentials of the platelet membrane vessels, erythrocyte membrane vessels, JQ1 nanoparticles, and PM&EM/JQ1 NPs were − 25.73 ± 1.54 mV, − 35.1 ± 1.96 mV, − 16.83 ± 0.544 mV, and 27.8 ± 1.45 mV, respectively, and the zeta potential of the PM&EM/JQ1 NPs was between that of the platelet membrane vessels and the erythrocyte membrane vessels (Fig. 2C), indicating successful fusion of the platelet membrane with the erythrocyte membrane. Transmission electron microscopy (TEM) images further confirmed the “core-shell” structure of the PM&EM/JQ1 NPs (Fig. 2D). Together, these results demonstrated successful coating of the PLGA cores with the extracted platelet and erythrocyte membranes.

The protein composition of the platelet and erythrocyte membrane vessels was determined by 3D- Data Independent Acquisition (DIA). Principal component analysis (PCA) is a mathematical procedure used to reduce the dimensionality of data. It achieves this by transforming the data into principal components using an orthogonal transformation, while still preserving the majority of the variance in the dataset.¹⁹ PCA indicated that PM&EM hybrid vessels were distinguishable between PM vessels and EM vessels groups. (Fig. 2F) By searching the UniProt database, total of 4872 membrane proteins were identified in PM&EM hybrid vessels. A functional classification of the identified proteins based on the biological process (Fig. 2G) involved in localization (613), cellular component organization biogenesis (98), single-organism process (60), positive regulation of biological process (36), multicellular organismal process (23), cellular process (8), response to stimulus (8), metabolic process (5), biological regulation (3), muti-organism process (2), negative regulation of biological process (2) and others (71). The membrane proteins were also classified according to cellular component (Fig. 2H): membrane-enclosed lumen (1000), organelle (549), extracellular region (255), cell junction (248), synapse (133), supramolecular complex (99), macromolecular complex (76), cell part (60), organelle part (27), other organism part (23), extracellular region part (18), membrane (15), nucleid (8), supramolecular fiber (2) and others cell component (2238) and others (61). Heatmap analysis of the levels of 523 proteins revealed notable differences between PM vessels and PM&EM vessels and 2771 between EM and PM&EM vessels. The hybrid membrane inherits most of the proteins on both the platelet membrane and the erythrocyte membrane (Fig. 2I). Ponceau S staining further confirmed that the PM&EM/JQ1 NPs contained most of the cell membrane proteins, similar to those in platelet and erythrocyte membrane vesicles, and that the PLGA-encapsulated JQ1 did not show the expected band of protein (Fig. 2J). To further investigate whether PM&EM/JQ1 NPs can retain the key physiological characteristics of platelets and erythrocytes, western blotting was used to analyze membrane proteins targeting collagen (glycoproteins IIb/IIIa and VI) and membrane proteins that enable immune evasion (CD47). As expected, after derivation and fusion of the membranes, the PM&EM/JQ1 NPs contained both platelets and potent membrane proteins (Fig. 2K). Together, these findings demonstrated successful fusion of platelet and erythrocyte membranes, preserving the key physiological characteristics of the source platelets and erythrocytes.

The drug loading rate (LE) and encapsulation rate (EE) of PM&EM/JQ1 NPs were 3.58 ± 0.27% and 37.15 ± 2.9%, respectively. In order to study the release kinetics of JQ1 from nanoparticles, we placed the JQ1 NPs and PM&EM/JQ1 NPs in 20% FBS. After 62 h, the measured JQ1 release rates were76.31 ± 4.94% and 67.19 ± 0.65%, respectively, suggesting that most of JQ1 can be released at the site of fibrosis after injection (Figures S1B).

2.2 Targeting and immune evasion in vitro

The ability of PM&EM nanoparticles to target collagen in vitro was examined to determine whether this novel drug-carry system can retain the advantages of both erythrocyte and platelet membranes. After 4 hours of co-incubation with collagen in vitro, CLSM showed that platelet-erythrocyte hybrid membrane-coated DiI loaded nanoparticles (PM&EM/DiI NPs) adhered to collagen more efficiently than DiI NPs (Fig. 3A).

Mouse embryonic fibroblasts stimulated with angiotensin II simulated cardiac fibroblasts activated after HF. Then, the targeting effect of PM&EM/DiI NPs on activated fibroblasts (myofibroblast) was evaluated. In detail, after stimulation of fibroblasts with angiotensin II, there was a clear increase in expression of collagen I (Fig. 3B). After incubating DiI NPs and PM&EM/DiI NPs with activated fibroblasts for 4 hours, the nanoparticles became aggregated within cells. The number of nanoparticles in myofibroblast were much higher in the PM&EM/DiI NPs group (Fig. 3C). Further flow cytometry (FC) analysis confirmed that myofibroblast had approximately 30% higher uptake efficiency for PM&EM/DiI NPs than for DiI NPs (Fig. 3D). These results suggest that our modified macrophage membrane-encapsulated nanoparticles have higher adhesion and affinity for myofibroblast.

Next, we tested the ability of PM&EM/DiI NPs to evade phagocytosis by macrophages in vitro. After 4 hours of incubation with RAW 264.7 cells, cellular uptake of PM&EM/DiI NPs were monitored by CLSM. As shown in Fig. 3E, fewer PM&EM/DiI NPs were observed within macrophages than in PM&EM/DiI nanoparticle-treated and PLGA nanoparticle-treated cells. Furthermore, LysoTracker staining showed that most of the internalized DiI nanoparticles were in lysosomes. These results suggest that CD47 proteins derived from erythrocyte membranes were effective in preventing PM&EM/DiI NPs from being phagocytosed by macrophages (Fig. 3F).

2.3 Targeting Research In Vivo

Next, we investigated the in vivo targetability of PM&EM-coated nanoparticles to the site of cardiac fibrosis in a pressure overload induced HF mouse model. The adult mice were subjected to transverse aortic constriction (TAC) surgery. The same numbers of DiI NPs and PM&EM/DiI NPs were injected through the tail vein in the pressure overload mouse models. 24 hours later, heart tissue was collected from the mice for fluorescence imaging. Some faint fluorescence signals were demonstrated in the hearts of the DiI NPs group, and the amount of DiI dye was significantly higher in PM&EM/DiI NPs than in DiI NPs (Fig. 4A). To explore the distribution of drug-loaded systems in various other murine organs, fluorescence signals of DiI were detected in other major organs 24 hours after intravenous injection, mainly in the liver and kidneys. This may reflect the clearance of mononuclear phagocyte system (MPS) in the liver and kidneys when nanoparticles were injected directly into the bloodstream. At the same time, we also found that, unlike the enrichment of the DiI fluorescence signal in the heart, the DiI NPs group had a large amount of fluorescence signal while the amount of DiI dye in PM&EM/DiI NPs was significantly reduced (Fig. 4C). This finding indicates that PM&EM/DiI NPs are better able to target fibrotic hearts without accumulating in other tissues, such as the liver and kidney, which may be related to glycoproteins IIb/IIIa and VI acquired from the platelet membranes on PM&EM/DiI NPs and CD47 inherited from the erythrocyte membranes.

2.4 Evaluation of Treatment Effects in the TAC-induced HF Mouse Model

Given that PM&EM nanoparticles were observed to accumulate in fibrotic lesions in mice with TAC-induced HF, we then explored the role of PM&EM/PLGA nanoparticle drug delivery systems in treating cardiac fibrosis and improvement in HF after JQ1 loading. To evaluate the therapeutic efficacy of each preparation (free JQ1, JQ1 NPs, and PM&EM/JQ1 NPs), we first established a model of pressures overload in mice (Fig. 5A) based on previous treatment regimens with appropriate improvements. On postoperative day 18, the time point when severe cardiac hypertrophy and left ventricular dysfunction is established in this model, mice were randomized to daily tail vein injections of 50 mg/kg of bare JQ1 or the various types of nanoparticle systems, which were continued until postoperative day 45.⁴

Cardiac ultrasound was performed on day 45 of TAC to assess heart function and contractile capacity. As shown in Fig. 5B, C and D, cardiac function and left ventricular contractility improved in mice treated with bare JQ1, JQ1 NPs, and PM&EM/JQ1 NPs but not in untreated mice. PM&EM/JQ1 NPs significantly improved the ejection fraction and left ventricular short axis shortening rate in mice with TAC-induced cardiac fibrosis, and cardiac function increased from 48.9 ± 5.16% and 49 ± 5.16% to 58.49 ± 5.5% compared with Free JQ1 and JQ1 NPs. The rate of shortening of the left ventricular minor axis also increased from 24.25 ± 3.09% and 24.25 ± 3.09% to 30.37 ± 3.44%. These findings suggest that the prognosis of TAC-induced HF was significantly better in mice that received PM&EM/JQ1 NPs than in those that received free JQ1 or JQ1 NPs.

Collagen in cardiac tissue was then stained with Sirius red to assess the degree of cardiac fibrosis further. The results showed that the free JQ1 and JQ1 NPs were able to improve the degree of fibrosis in mice to some extent. However, injection of PM&EM/JQ1NPs significantly reduced the collagen content by 6.23 ± 1.18% (Fig. 5E and G). PM&EM/JQ1 NPs were more effective in reducing the degree of fibrosis in the heart than free JQ1 or JQ1 nanoparticles.

The process of cardiac fibrosis is often accompanied by hypertrophy of cardiomyocytes, which typically means a worse prognosis.²⁰ Assessment of the size of cardiomyocytes by wheat germ agglutinin staining showed that, compared with the sham surgery group, cardiomyocyte size increased by an average of 1.84 ± 0.23 times in the mice with TAC-induced fibrosis in the free JQ1 or JQ1 NPs groups, but increased by only 1.15 ± 0.02 times in the mice treated with PM&EM/JQ1 NPs (Fig. 5F and H). Therefore, PM&EM/JQ1 NPs were more effective in reducing hypertrophy of cardiomyocytes than free JQ1 or JQ1 NPs.

2.5 Evaluation of Treatment Effects in the MI-induced HF Mouse Model

The TAC model is commonly used to study HF and cardiac fibrosis. However, it is important to note that the acute onset of severe hemodynamic overload in this model does not accurately represent the clinical situation typically encountered in human patients.²¹ On the other hand, myocardial infarction (MI) is a frequent cause of pathological cardiac remodeling and HF in humans.²² Furthermore, animal models of HF after MI have become the main avenue for exploration of the role of PM&EM/JQ1 NPs in the treatment of cardiac fibrosis and their ability to improve HF. Referencing a previous regimen and making appropriate improvements, we established an MI-induced mouse model of HF by administering 25 mg/kg of free JQ1 or JQ1 encapsulated in the various nanoparticle systems via the tail vein on postoperative day 6 for 6 days (i.e., the early postoperative period), then gradually increased to 50 mg/kg per day until postoperative day 28 (Fig. 6A).

Consistent with the findings in the stress overload model, all three treatments (free JQ1, JQ1 NPs, and PM&EM/JQ1 NPs) improved cardiac function and left ventricular contractility in mice with MI-induced cardiac fibrosis, but the treatment effect was more pronounced in the mice that received PM&EM/JQ1NPs (Fig. 6B, C and D). These nanoparticles also achieved better results in terms of the degree of cardiac fibrosis and cardiomyocyte size (Fig. 6E–H). Those results showed that PM&EM/JQ1 NPs could inhibit and improve HF after large-scale anterior MI and had a significantly better therapy effect.

2.6 Biosafety Induced by JQ1

The novel drug-loading system investigated in this study was found to have enhanced efficacy but its effect on organs other than the heart was unclear. To determine the status of the major organs (liver, kidney, gastrointestinal tract) after each type of treatment, we stained the liver and kidneys with hematoxylin-eosin and the intestines with Alcian blue which was used to characterize goblet cells.⁶ We found that significant pathological changes occurred in the liver, kidneys, and intestines when 50 mg/kg of free JQ1 or JQ1 nanoparticles were injected via the tail vein for 30 days but that there were no obvious differences in lesions between the group treated with PM&EM/JQ1NPs and the control group. (Fig. 7A) Biochemical analysis showed that alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and serum creatinine (CRE) levels were normal in the sham, MI-Control, and PM&EM/JQ1 NPs groups but were significantly elevated in the free JQ1 and JQ1 NPs groups.(Figure S3A-D) These findings indicate that long-term use of free JQ1 and JQ1 NPs for the treatment of cardiac fibrosis would impair the biological function of the liver and kidneys, whereas toxicity would be reduced by administration of PM&EM/JQ1 NPs, which do not accumulate in liver and kidney tissue to the same extent.

A total of 981 JQ1-related genes, 745 genes related to liver toxicity, 681 genes related to renal toxicity, and 7423 genes related to intestinal toxicity were extracted from GENECARDS (https://www.genecards.org/) to explore the potential mechanism of liver, kidney, and intestinal damage caused by long-term use of JQ1 in more detail. The four gene datasets were imported into Hiplot (https://hiplot.com.cn/home/index.html). Forty-four common genes were obtained and displayed in Venn diagrams (Fig. 7B). In order to explore the potential pathways in which these common genes are involved, GO pathway enrichment analysis were performed using clusterProfiler R package. Biological Process (BP) mainly involves response to oxidative stress, cellular response to oxidative stress, cellular response to chemical stress and response to reactive oxygen species, whereas Molecular Function (MF) mainly involves DNA-binding transcription factor binding, RNA polymerase II-specific DNA-binding transcription factor binding, DNA-binding transcription activator activity and RNA polymerase II-specific which plays a role in the toxicity of JQ1 in the liver, kidney, and gastrointestinal tract. (Fig. 7C, Figure S4A and B).

To further confirm this, these genes were then introduced into Metascape (http://metascape.org/) for GO enrichment analysis (a false discovery rate of < 0.01 is considered significantly enriched). The highest levels of enrichment were also in the response to oxidative stress signaling pathway, which is mainly involved in oxidative stress-related and apoptosis-related disfunctions. Based on the false discovery rate and hit genes, the top 10 GO entries pathways were selected and showed in Fig. 4SC. The key regulatory molecules were also showed in Figure S4D.

Finally, the results of pathway enrichment showed that JQ1 can effectively alleviate oxidative stress and apoptosis in murine liver and kidney tissues, which may be the mechanism for the relatively mild hepatic and renal toxicity of these nanoparticles. For further verification, reactive oxygen species (ROS) test and Tunnel test were performed on the liver and kidney of mice in those treatment groups. Compared with sham and MI-control group, Free JQ1 and JQI NPs treatment group significantly increased ROS and apoptosis cells. However, PM&EM/JQ1 NPs inhibits these side effects indicating that PM&EM NPs enable effectively reduce JQ1 accumulation in liver and kidney tissues. (Fig. 7D and Figure S5).

2.7 Biosafety Assignment of PM&EM nanoparticles

To assess the hemocompatibility of biological materials, it is important to evaluate their blood compatibility. In this study, we used an in vitro direct contact method to detect the hemolysis of PLGA nanoparticles and PM&EM NPs. As shown in Figure S6A, the results indicated that after incubating the PLGA nanoparticles and PM&EM NPs with blood, there were no abnormal phenomena or particle sediment in the supernatant, suggesting that the red blood cells were not destroyed (Figure S6B).

To assess the biological effect of PM&EM NPs, we incubated them with fibroblasts, endothelial cells (ECs), smooth muscle cells (SMCs), and macrophages, along with PLGA nanoparticles. The cytotoxicity of these nanoparticles was then investigated using the Cell Counting Kit-8 (CCK-8) assay. The results, as shown in Figure S6C-F, indicated that there was no significant difference between the different treatment groups and the untreated group, suggesting good cytocompatibility of our drug delivery system.

Moreover, Clinical biochemistry analysis demonstrated that PLAG nanoparticles and PM&EM NPs did not affect liver and kidney functions, as evidenced by normal levels of ALT, AST, BUN, and CRE (Figure S7A-D). Furthermore, testing of mouse serum cytokines (TNF-α, IL1β, IL6, CCL2) showed no significant difference between the treatment groups, indicating that our drug delivery system does not cause obvious side effects in vivo (Figure S8A-D).

H&E staining was performed on pathological sections of various organs, and after comparing the H&E-stained sections of different main organs from MI-induced HF mice. we found no significant difference between the control group and different treatment groups (Figure S9). This suggests high biocompatibility of PLGA nanoparticles and PM&EM nanoparticles after long-term treatment in MI-induced HF mice.

We have developed a hybrid biomimetic nanodrug-loading system that co-assembles with natural platelet and erythrocyte membranes and uses the advantages of both membranes to target fibrosis in a sustained manner in vivo. We have demonstrated that PM&EM nanoparticles can adhere efficiently to collagen fibers in vitro because of the key receptor proteins on platelet membranes. This fusion membrane can also effectively reduce phagocytosis of macrophages in vitro. Moreover, when compared with the traditional single-cell membrane drug delivery systems, the fusion membrane-coated drug delivery system can combine two or more cell functions, making the new fusion membrane more functional. This hybrid system was further investigated in mouse models of TAC-induced and MI-induced HF. After long-term intravenous injection of PM&EM/JQ1 NPs, heart function was significantly improved, the degree of cardiac fibrosis was significantly reduced, and the toxicity of JQ1 to organs other than the heart was effectively attenuated in mice.

There is a need for more biofilm fusion research in the future. Rational and effective fusion of various biofilms can effectively improve the multifunctional characteristics of targeted drug delivery systems. This will provide more insights into drug delivery strategies after MI. This work describes a novel hybrid biomimetic drug delivery system that integrates native platelet and erythrocyte membranes to enhance the function of nanoparticles. This system paves the way for combining other cell membranes and even cell membrane hybrids with engineered artificial lipid membranes to form biomimetic nanoparticles with various biomedical applications.

Materials and Cells

The anti-human GP IBA antibody was obtained from ProMab Biotechnology (CA, USA), while the anti-human GP IIbIIIa antibody was acquired from ABclonal Biotechnology (Shanghai, China). The anti-human GP VI antibody was purchased from AbBox Biotechnology (Suzhou, China), and the anti-human CD47 antibody was obtained from Santa Cruz Biotechnology (USA). Additionally, we used the anti-mouse Collagen I antibody, which was purchased from ABclonal Biotechnology (Shanghai, China).

JQ1, a chemical compound, was obtained from Selleck (USA). Dimethyl sulfoxide (DMSO) and Wheat germ agglutinin (WGA) were acquired from Sigma (USA). PLGA (50:50; MW = 90,000), a type of polymer, was purchased from Dalian Meilun Biotechnology in China. The fluorescent dyes 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO), 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine,4-Chlorobenzenesulfonate Salt (DiD) and 6-diamidino-2-phenylindole (DAPI) were obtained from Beyotime (China). Masson’s Trichrome, Hematoxylin and Eosin were purchased from Servicebio (Wu han, China). Ponceau S, Cell Counting Kit 8 and BCA Protein Assay Kit were purchased from Biosharp (China). Mouse TNF-𝛼, IL-1𝛽 and IL-6 ELISA kit was purchased from LunchangshuoBiotech (Xiamen, China) and Mouse CCL2 ELISA kit was purchased from Fankew (Shanghai, China). LysoTracker Deep Red was purchased from Thermo Fisher (USA).

The Murine RAW 264.7 and NIH/3T3 cell lines were purchased from ZQXZ Biotechnology in Shanghai, China, while the Murine smooth muscle cell line was obtained from Beinart Biotech in Beijing, China. HUVECs were purchased from Science Cell in Carlsbad, CA.

Human platelet isolation and membrane extraction:

Platelet membrane vesicles (PMV) were obtained using a previously described method of repeated freeze-thaw cycles.¹³ To obtain PMV, aliquots of platelet suspension were frozen at -80°C, thawed at room temperature, and then centrifuged at 4,000 g for 3 minutes. The resulting pellet was resuspended in PBS and sonicated for 5 minutes using a Fisher Scientific FS30D water bath sonicator at a frequency of 42 kHz and 100 W power. The presence of PMV was confirmed through dimensional measurements using dynamic light scattering (DLS) and morphological examination by transmission electron microscopy (TEM).

Isolation of red blood cells and membrane extraction

Human type O blood is treated with 1.5 mg/ml EDTA and erythrocyte isolation is performed approximately 16 h after blood collection. Collect red blood cells by centrifugation at 720 g for 10 min and wash twice using pre-chilled PBS solution (containing 1 mm EDTA, 50 µm leutin, and 1 µg/mL aprotinin). The resulting red blood cell membrane is suspended in quadruple volume of 0.2 mm EDTA 2Na deionized water to induce membrane rupture. After 60 min of low permeability solubilization, centrifuge the sample at 20,000 g for 20 min and collect the red blood cell membrane. Repeat the low permeability dissolution step described above once. Wash the erythrocyte membrane using pre-chilled PBS solution (pH 7.4 containing 50 µm leucin peptide and 1 µg/mL aprotinin) until the supernatant is colorless. All of the above operations are performed at 4°C to maintain the structural and functional stability of membrane proteins. Fresh erythrocyte membranes are stored at 4°C and used within 6 h.

Verification of membrane fusion

To determine the fusion process between platelet and red blood cell membranes, we employed the FRET method. Specifically, we added DiO and DiI FRET dye pairs (0.1%, w/w) to the platelet membrane and then introduced the red blood cell membrane to the DiO and DiI-labeled platelet membranes. The resulting mixture was sonicated and extruded, and the fluorescence spectrum of a sample was measured at 400 nm using a Tecan fluorescence spectrometer.

To visualize the fusion between platelet and erythrocyte cell membranes, we labeled the platelet membranes with DiO and the erythrocyte cell membranes with DiD, following the fusion methods described above. As a control, we also prepared a physical mixture of the two membranes. The resulting samples were imaged using laser confocal microscopy (CLSM) from Leica..

Total protein of PM&EM/JQ1 NPs was validated using SDS-PAGE. The specific proteins GP IIbIIIa, GP Ibα, GP VI, CD47 were then characterized by Western blot.

Protein identification and classification:

We used 3D-DIA based on mass spectrometry (MS) to perform proteomic analysis of the protein composition of PM&EM NPs, as previously described (reference 23). Briefly, the samples were dissolved in lysis buffer containing 8M urea, 100mM Tris-HCL (Sigma, MO, USA), pH 8.5, and 1% protease inhibitor cocktail (Biyuntian Biotechnology Co., Shanghai, CN). The resulting mixture was sonicated and centrifuged at 15,000g at 4°C for 15 minutes to remove the sediment. Protein digestion was performed using the FASP method with Trypsin (Promega, Madison, WI) in 50mM NH4HCO3 (Sigma, MO, USA). Data Independent Acquisition (DIA) analysis was conducted on a Q Exactive HF mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with an EASY-nLC 1200 system (Thermo Fisher Scientific). Forty-five variable DIA windows were set for DIA acquisition, and identification and quantification were performed using Spectronaut 17.4 (Biognosys, Schlieren, Switzerland) with the directDIA model. The DIA raw files were searched against the human fasta database (from uniprot) to generate a spectral library using BGS factory settings. All results were filtered by a Q value cutoff of 0.01 (corresponding to a FDR of 1%). P-value estimator was performed by Kermel Density Estimator.

JQ1 released

The sample solution was incubated in a dialysis bag (Sigma, MWCO 100 kDa) containing PBS at pH 7.4 and maintained at 37°C. At a specific time point, the solution inside the bag was collected and the amount of JQ1 was measured using a spectrometer (Tecan) with an absorption wavelength of 300 nm. JQ1NPs were prepared and used as a control.

Biocompatibility of PM&EM nanoparticles

In vitro biocompatibility of PM&EM NPs was determined using RAW264.7, mouse embryonic fibroblasts, ECs and SMCs. Cells are seeded separately in 96-well plates and cultured overnight. Add different concentrations of PM&EM NPs and incubate for another 24 or 48 h. Cell viability was determined using a cell counting kit (Beyotime, China).

Blood Compatibility Tests

To evaluate the hemocompatibility of PLGA nanoparticles and PM&EM NPs, 1 mL of rat blood was mixed with 1.25 mL of 0.9% sodium chloride solution. Then, 0.1 mL of the diluted whole blood was added to the PLGA nanoparticles or PM&EM NPs solution (5 mL, 1 mg/mL) and incubated at 37°C for 1 hour. The mixture was then centrifuged at 3000 rpm for 5 minutes, and the absorbance of the supernatant was measured at 540 nm to determine the hemoglobin released from the lysed red blood cells.

Methods of animal experimentation

All animal experiments were conducted in compliance with the Guidelines for the Protection and Use of Laboratory Animals of the National Institutes of Health and were approved by the Ethical Review Committee of Beijing Anzhen Hospital.

For this study, male C57BL/6J mice (8 week) were obtained from Beijing Huafukang Technology Co., Ltd. The mice were housed under controlled temperature and humidity conditions in a pathogen-free environment with a 12-hour light/dark cycle. They were provided with free access to standard laboratory rodent feed and water. During surgery, the mice were placed on a temperature-controlled small animal operating table to maintain their body temperature.

TAC-induced chronic heart failure model: In the aortic arch coarctation (TAC) model, open heart surgery is performed after anesthetized mice using pentobarbital. Use a 7 − 0 silk thread and a 27-gauge needle to contract the aortic arch between the left common carotid artery and the brachiocephalic trunk artery in mice, as described in a previous article (PubMed: 23911322). On day 18 after TAC surgery, mice are randomly divided into 4 groups (n = 6). Free JQ1 group, JQ1 NPs group and PM&EM/JQ1 NPs group, dissolved in 0.9% sodium chloride solution for injection, containing JQ1 at a dose of 50mg/kg/day, lasting until the 42nd postoperative day, the TAC-Con group was injected with the same volume of normal saline, and the Sham group was set up with sham surgery. Treat until postoperative day 45 to perform an ultrasound of the mouse heart and sacrifice the mouse under anesthesia.

Model of post-MI heart failure: After inducing anesthesia in mice using isoflurane, adjust the anesthetic concentration and make the mouse continuously inhaled, permanent ligation of the proximal left anterior descending coronary artery (LAD) at the left atrial margin using a 7 − 0 silk thread. Mice are randomly divided into 4 groups (n = 6) on postoperative day 6. Free JQ1 group, JQ1 NPs group and PM&EM/JQ1 NPs group intraperitoneal injection, the dose containing JQ1 was 25mg/kg/day, and the dose of JQ1 was increased to 50mg/kg/day on the 15th postoperative day, and continued until the 27th postoperative day, the MI-Con group was injected with the same volume of normal saline, and the Sham group was set up with sham surgery. Mouse heart ultrasound is performed until day 30 postoperatively and mice are sacrificed under anesthesia.

Echocardiography

After hair removal at the heart site of the mouse, 1–2% inhaled isoflurane was anesthetized, supine on a physiological information detection platform, and imaged using the Vevo high-resolution imaging system. Images were acquired in B mode on the left ventricular minor axis, and the left ventricular central section was measured in two dimensions to obtain the thickness and inner diameter of the anteroposterior wall during diastolic and systolic periods of the left ventricle.

Organizational analysis

Cardiac tissue is collected, PBS washed, fixed with 4% paraformaldehyde fixative solution, and sliced after conventional dehydration embedding. The cross-sectional area of cardiomyocytes was determined using WGA staining, and the degree of cardiac fibrosis in the TAC heart failure model and the post-MI heart failure model was assessed using Sirius red staining and Mason staining, respectively. Quantitative analysis of cardiomyocyte cross-sectional area and cardiac fibrosis area using ImageJ.

In vitro imaging

Mice are injected with 100ul DiI-labeled NPs and PM&EM/NPTs solution through the tail vein, anesthetize mice at predetermined time points after injection, and detect fluorescence signal distribution using in vivo imaging systems. After 24 h, mouse organs were removed and IVIS spectroscopic imaging was performed to assess the distribution of fluorescence signals in different organs.

Construction of Pathways. JQ1 and main organ toxicity-related genes were retrieved from GeneCards (https://www.genecards.org/). Vene diagram which shown common genes were designed in Hiplot (https://hiplot.com.cn/home/index.html). The common gene profiles were analyzed using the bioconductor packet. R and Metascape (http://metascape.org/) were used for GO enrichment analysis. The interactions of target genes were analyzed using Metascape.

Statistical analysis

All experimental results were statistically analyzed using GraphPad Prim (version 8.02), which was performed using one-way ANOVA, and each result was expressed as mean ± standard deviation. The difference significance level is set to *p < 0.05, ** p < 0.01, *** p < 0.001.

Acknowledgements

Yuyu Li and Jiaqi Yu contributed equally to this work. The authors are very grateful to Dr. Yi Xin for scientific advices. The authors wanted to thank the stuff of PROMAB for some technique supports. This work was supported by the National Key R&D Program of China (2021YFA0805100)

Conflict of Interest

The authors declare no conflict of interest.

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Platelet and Erythrocyte Membranes Coassembled Biomimetic Nanoparticles for Heart Failure Treatment

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Abstract

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1 INTRODUCTION

2 RESULTS AND DISCUSSION

2.1 Preparation and characterization of PM&EM/JQ1 NPs

2.2 Targeting and immune evasion in vitro

2.3 Targeting Research In Vivo

2.4 Evaluation of Treatment Effects in the TAC-induced HF Mouse Model

2.5 Evaluation of Treatment Effects in the MI-induced HF Mouse Model

2.6 Biosafety Induced by JQ1

2.7 Biosafety Assignment of PM&EM nanoparticles

3 Conclusion

4 Experimental Section

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