Engineered macrophage membrane‐enveloped nanomedicine for ameliorating myocardial infarction in a mouse model

Abstract Myocardial infarction (MI) is the serious condition causing lots of death over the world. Myocytes apoptosis, inflammation, and fibrosis are three important factors implicated in pathogenesis of MI. Targeting these three factors has been shown to ameliorate MI and rescue cardiac function. Previous studies have demonstrated that microRNA (miR) 199a‐3p protect against MI. In this study, we prepare macrophage membrane coated nanoparticles (MMNPs) containing miR199a‐3p. We evaluate the effects of these NPs on apoptosis and cell proliferation in vitro and the effects on inflammation cytokine production, expression of fibrosis related proteins, cardiac injuries, and functions in MI mice. We find that the MMNPs have receptors of interleukin‐1β (IL‐1β), interleukin‐6 (IL‐6), and tumor necrosis factor alpha (TNF‐α) and can bind to these cytokines. MMNPs prevent hypoxia‐induced apoptosis and promote cell proliferation, suppress the inflammation, and inhibit the cardiac fibrosis in MI mice. These results demonstrate that MMNPs ameliorate left ventricular remodeling and cardiac functions, and protect against MI, suggesting MMNPs containing miR199a‐3p is a potential therapeutic approach to treat MI.


| INTRODUCTION
Myocardial infarction (MI), also known as heart attack, is caused by decreased or stopped blood flow to the heart. 1 MI causes injuries to the heart muscle. Because of cardiomyocytes loss, MI damages myocardial functions and finally results in heart failure, causing major death and disability worldwide.
Apoptosis and fibrosis play essential roles in MI-induced tissue injury. 2 Following a MI, numerous cells die in response to ischemia.
Apoptosis greatly contributes to myocytes death in MI and mostly occurs in the peri-infarcted region. Apoptosis plays a critical role in determining infarct size and early symptoms of heart failure. 3 Inhibition of apoptosis has been shown to improve cardiac functions and decrease infarct size in MI mice model. 4 Due to the limited capacity of heart regeneration, fibrotic scars replace the lost cells. 5 Fibrosis is an essential process for damage repair but the accumulation of fibrosis in tissues will lead to organ dysfunction and organ failure.
Although inhibiting cardiac fibrosis from progressing has been recognized as important to prevent heart failure, there is still no efficient therapy available.
Inflammation is another hallmark of MI. 6 MI triggers inflammatory responses, which result in cytokine production and inflammatory leukocytes infiltration into myocardial region. After MI, elaborated cytokines have been identified in infarcted area. Elevated tumor necrosis factor alpha (TNF-α), interleukin (IL)-1, and IL-6 have been shown to contribute to myocytes death, myocardial injury, and healing process. 6,7 Blockage of TNF-α, IL-1, and IL-6 has been shown to preserve the cardiac function, indicating targeting these inflammatory cytokines should be a potential therapeutic approach to treat MI. [8][9][10][11] MicroRNAs (miRNAs) are short non-coding RNAs, which regulate gene expression. MiRNAs are shown to regulate various aspects of cardiomyocyte biology. 12 MiR-199a-3p has been shown to reduce the infarct size and improve cardiac function in MI mice. 13,14 Nanoparticles (NPs) have been utilized as novel approach to deliver drugs for multiple diseases treatments with higher specificity and fewer side effects. Cationic lipid-assisted PEG-b-PLGA nanoparticles (CLAN), which form a clinically translatable nucleic acid delivery system, has been also widely utilized to deliver miRNA, and these NPs containing miRNA have been shown to prevent cell apoptosis and improve myocardial remodeling after MI. 15 Recently, cell-membraneenveloped NPs have been recognized as an encouraging therapeutic platform. 16 These NPs are fused with natural cell membranes and can absorb and neutralize molecules. 17,18 The neutrophil membranecoated NPs have been described to neutralize pro-inflammatory cytokines, suppress inflammation, and against joint damage. 19 Taken together, the membrane coated NPs containing miRNA could be an efficient treatment for MI.
In the present study, we prepared NPs enveloped with membranes from engineered macrophages, which overexpressed TNF-αR, IL-1βR, and IL-6R. The NPs contained miR199a-3p, which has been shown to induce cardiomyocyte proliferation. 20 We investigated the potential effects of these membrane envelope NPs on cell proliferation, inflammatory response, and cardiac function in MI.
After removal of NPs, the remaining cytokines in the supernatant were detected. As shown in Figure 2b, the level of remaining IL-1β in the supernatant of MMNPmiR 199a-3p /IL-1β mixture decreased with increased concentration of MMNPmiR 199a-3p, indicating MMNPmiR 199a-3p bound to IL-1β. In contrast, the level of remaining IL-1β in the supernatant of NPmiR 199a-3p /IL-1β mixture did not change with increased concentration of NPmiR 199a-3p , indicating NPmiR 199a-3p did not bind to IL-1β. Similarly, we also found only MMNPmiR 199a-3p bound to IL-6 ( Figure 2c) and TNF-α ( Figure 2d).

| DISCUSSION
In the present study, we investigated the potential protective effects In addition, MMNP miR199a-3p prevented fibrosis, improved left ventricular remodeling, and promoted cardiac function. Therefore, these results suggested MMNP miR199a-3p could be utilized as a potential effective approach to treat MI.
Myocardial infarction is the leading cause of death. Cell apoptosis and cardiac fibrosis are two important events occur after MI. Apoptosis plays an significant role in myocardial loss after MI and is involved in the process of subsequent left ventricular remodeling and development of heart failure. 3 In MI, dominant apoptosis is present and correlated with left ventricular remodeling. 25 Using animal model, Bialik et al described that MI resulted in apoptosis, which only emerged in hypoxic regions during acute infarction. The hypoxiainduced apoptosis was independent of p53. 26 Inhibition of apoptosis has been shown to protect left ventricular function and attenuate remodeling in MI rats. 27 Therefore, targeting apoptosis could be a useful approach to treat MI. miRNAs are small, non-coding RNAs, which can silence gene by inhibiting mRNA translation. 28 Increasing evidences have shown that miRNA is involved in the pathogenesis of MI. 29 For example, he and colleagues reported that inhibition of miR-124 inhibited cardiomyocytes apoptosis and protected against MI. 30 Overexpressing miR-325-3p attenuated the cardiac tissue injury and decreased the infarct size. 31 MiR199a-3p has been described to regulate the cardiac cell proliferation and inducing cardiac regeneration after MI, either when expressed by adeno-associated virus (AAV) vector 32 or directly intra-cardiac injection. 13 These results suggested miR199a-3p was a potential candidate for MI treatment. In the present study, we utilized NPs containing miR199a-3p as the approach for treatment. We demonstrated that NPs containing miR199a-3p (NP miR199a-3p and MM NP miR199a-3p ) prevented hypoxia-induced apoptosis of cardiovascular cells, promoted the expression of cell cycle related proteins, and induced cell proliferation. Interestingly, the free miR199a-3p, which was directly added to the cell culture had no effect on these parameters. We found that the cardiovascular cells HL-1 cells only up-took NPs containing Cy5-labeled miR199a-3p but not the free Cy5-labled miR199a-3p. These results strongly suggested that the correct and efficient delivery approach is one key factor in designing disease treatment. The CLAN NPs is an efficient delivery system to deliver the miRNA into target cells and enable the active function of miRNA in cells.
MI also induces inflammatory response and cytokine production.
Robust production of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6 has been detected after MI induction in rodents. 33 The excessive inflammatory responses promote fibrosis and results in pathological remodeling and the progression of myocardial disease.
Therefore, it has been suggested that inflammation could be an important target for disease preventing. 34 Li and colleagues produced microparticles with anti-IL-1β antibodies and they found that these microparticles neutralized IL-1β after MI, prevented cardiac remodeling, and induced cardiac repair. 8 In present study, we utilized the membrane from macrophages overexpressing the receptors of TNF-α, IL-1β, and IL-6 and coated to the NP miR199a-3p . We found that the macrophage membrane coated NP miR199a-3p (MMNP miR199a-3p ) can bind to and absorb TNF-α, IL-1β, and IL-6 while NP miR199a-3p cannot.
In MI mice, the MMNP miR199a-3p had significantly enhanced antiinflammatory activities compared to NP miR199a-3p , suggesting the membranes containing TNF-αR, IL-1βR, and IL-6R contribute to the antiinflammatory activities of MMNP miR199a-3p . Our findings are similar to previous study, which described that the neutrophil membrane-coated NPs neutralized pro-inflammatory cytokines and protected against joint damage. 19 In the present study, delivery of NP miR199a-3p also resulted in decreased inflammation in peri-infarct tissues, indicating miR199a-3p itself had the anti-inflammatory activities. Previous study by Bardin et al described that miR-199a-3p negatively regulated IκB Kinase β (IKKβ) expression and inhibited the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway. 35 This result supported our finding that miR199a-3p itself suppressed inflammation. However, the underlying mechanism of how miR199a-3p inhibits inflammation need to be further investigated. It should be interesting to detect the NF-κB activation in MI heart after administration of NP miR199a-3p .
Several questions of current study need to be further addressed.
We found the differences of anti-inflammatory activity between NP miR199a-3p and MMNP miR199a-3p . The cytokines absorption ability of MMNP miR199a-3p should contribute to the differences. It has been described that the cell membrane coated NPs is with the biological properties of the source cell from which their membrane is derived, and have prolonged circulation and disease-relevant targeting. 36 It is well-known that the macrophages accumulate into heart after MI and play significantly role. 37 It is possible that the MMNP miR199a-3p should better target to heart tissue when compared to NP miR199a-3p in MI mice while direct evidence and careful comparison are needed. In addition, to maximize the effects of NPs in MI heart, it is desired to enhance the biodistribution of NPs in MI heart. The factors involved in NPs distribution should be evaluated.
Macrophage membrane enveloped NPs were prepared as previously described. 39

| Quantification of cytokine binding
To test the cytokine binding ability, 10 ng/ml mouse IL-1β, IL-6, or TNF-α was mixed with 0 to 1.6 mg/ml NP miR199a-3p or MMNP miR199a-3p . The mixtures were incubated at 37 C for 2 h. Then the NPs were removed by centrifugation at 13,000 rmp for 10 min. Cytokine concentration in the supernatant was measured using commercial mouse IL-1β, IL-6, or TNF-α enzyme-linked immunosorbent assay (ELISA) kit (Abcam, Beijing, China).

| ELISA
Tissues were homogenized in tissue extraction buffer (Abcam, Beijing, China) and homogenates were centrifuged for 20 min at 13,000 rpm at 4 C. The levels of IL-1β, IL-6, and TNF-α were measured using commercial ELISA kit from Abcam (Beijing, China) following manufacturer's protocols.

| Western blot
The western blot was performed as described previously. 40 Briefly, NPs, MMNPs, macrophage membrane extraction, or total protein extracted from heart tissues were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then were transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat milk for overnight at 4 C. Next day, primary antibodies were added to the membrane and incubated at room temperature for 2 h. After wash, corresponding horse radish peroxidase-conjugated secondary antibodies were incubated for 1 h at room temperature. Immuno-reactive bands were visualized using ECL sub-

| Immunofluorescence assay
HL-1 cardiac muscle cells were incubated with free or nanoparticle encapsulated Cy5-labeled miRNA for 4 h. Then the cells were washed with PBS and fixed with 4% paraformaldehyde. The DNA was stained with 4 0 ,6diamidino-2-phenylindole (DAPI) (Thermo Fisher) at room temperature for 5 min. After wash, cells were analyzed by confocal microscopy.

| Mice treatment
MI was induced in Balb/c mice by coronary artery ligation as described previously. 41 1 h post coronary artery ligation, 2.0 mg/kg free miRNAs or NPs were injected by tail vein every day for consecutive 7 days. After treatment, tissues were harvested for analysis. The study was approved by the ethics commitment of Tangdu Hospital, Air force Military Medical University.

| Masson staining
The fibrosis of heart was determined using Masson Trichrome Stain kit (Sigma, St. Louis, MO) following manufacturer's protocols. The collagen fibers were stained as blue and the viable myocardium was stained as red. The area of fibrosis and the scare size was quantitated using ImageJ software.

| Echocardiography and hemodynamic study
Mice were anesthetized by isoflurane and then M-mode images were obtained by using Visualsonics Vevo 770 echocardiography machine (Visualsonics Inc., Toronto, Canada) as described previously. 42 Hearts areas between two papillary muscles were viewed. LV internal dimensions (LVID) at diastole (LVIDd) and systole (LVIDs) were measured.
The LV ejection fraction (EF) was calculated by the software with the machine. The cardiac hemodynamic function was evaluated using a Millar tip-pressure catheter as described previously. 42 LV end-diastolic pressure (LVEDP) was measured by catheter advancement into the LV cavity. Data were analyzed using the PowerLab System.

| Statistical analysis
All data are shown as means ± standard deviations (SD). Statistical analysis was performed by one-way ANOVA followed with a Tukey's post hoc test. p < .05 was considered statistically significant.

CONFLICT OF INTEREST
No conflicts of interest, financial, or otherwise, are declared by the authors.

DATA AVAILABILITY STATEMENT
Data could be obtained upon reasonable request to the corresponding author.