Patent Application
20220290098
The present invention relates to a method of diagnosing and treating atherosclerosis using nanovesicles targeting a site with a change in blood flow.
Atherosclerosis is a leading cause of death, but current diagnosis methods cannot yet detect early etiological signals of this disease associated with an irreversible cascade. Although there remains a substantial risk of disease progression, conventional preventive and therapeutic options targeting a low cholesterol level, blood pressure or plaque formation have been widely used. The occurrence of disturbed blood flow, which is an early atherosclerotic event arising at the branch points, curved regions or distal to stenosis, results in dysfunction of endothelial cells (ECs). Under normal blood flow, ECs are aligned to a blood flow direction, and maintain anti-inflammatory and anti-thrombotic functions. In contrast, in the case of atherosclerosis, disturbed blood flow activates EC dysfunction as well as increased inflammation and thrombotic events, ultimately causing atherosclerosis. Disturbed blood flow-targeting theragnostics that can present a promising solution for this fatal chronic disease must be thoroughly demonstrated.
It has been reported that mesenchymal stem cells (MSCs) maintain therapeutic promise for anti-atherosclerosis treatments by regulating the immune response and attenuating the proliferation of vascular smooth muscle cells (VSMCs). However, their low survival rate and insufficient target efficiency must be overcome for clinical application. MSC-derived nanovesicles have emerged as therapeutic nanocarriers with prolonged circulation time in the body, which may not only have cell-originating anti-inflammatory and pre-regenerative characteristics but also promote cellular interactions. Despite attempts to develop nanovesicle-based therapeutics for several fatal diseases and injuries including a cardiovascular disease, renal injury, a liver disease and a neurological disease, maintaining size uniformity and the consistency of an active ingredient from a high yield in a long and difficult production process is still a challenge. A recent alternative engineering approach can produce cell-derived nanovesicles in 100-fold or higher yield by physically disrupting cells through micropore filtration and inducing self-assembly of fragments of the resulting cell membrane and internal contents.
An irreversible cascade leads to the critical pathogenesis of atherosclerosis, which presents an unmet need for early diagnosis and prevention. Disturbed blood flow formation is one of the fastest atherosclerotic events that increases endothelial permeability and subsequent monocyte recruitment.
The present invention is directed to providing stem cell-derived nanovesicles displaying a peptide capable of targeting a disturbed blood flow site causing atherosclerosis on their surfaces and a method of producing the same.
The present invention is also directed to providing a use of the nanovesicles for preventing, diagnosing, and treating atherosclerosis.
However, technical problems to be solved in the present invention are not limited to the above-described problems, and other problems which are not described herein will be fully understood by those of ordinary skill in the art from the following description.
To achieve the above-described purposes, the present invention provides stem cell-derived nanovesicles displaying a peptide capable of targeting a disturbed blood flow site causing atherosclerosis on their surfaces.
The present invention also provides a method of producing the nanovesicles, which includes obtaining nanovesicles displaying a peptide targeting disturbed blood flow sites on their surfaces from stem cells transfected with a vector into which coding sequences of a signal peptide, a disturbed blood flow site-targeting peptide and a transmembrane protein are sequentially inserted.
The present invention also provides a composition for diagnosing atherosclerosis, which includes the nanovesicles.
The present invention also provides a composition for preventing or treating atherosclerosis, which includes the nanovesicles.
The present invention also provides a method of treating atherosclerosis, which includes administering a therapeutically effective amount of the nanovesicles into a subject.
The present invention provides stem cell-derived nanovesicles, which are an anti-atherosclerosis theragnostic platform using a peptide capable of targeting a disturbed blood flow site, and the nanovesicles can be used as a novel theragnostic agent which can prevent the onset of atherosclerosis by providing potent anti-inflammatory and pre-endothelial repair effects similar to those of MSCs.
Hereinafter, the configuration of the present invention will be described in detail.
The present invention relates to stem cell-derived nanovesicles displaying a disturbed blood flow site-targeting peptide on their surface.
In addition, the present invention provides a method of preparing the nanovesicles, which includes obtaining nanovesicles displaying a disturbed blood flow site-targeting peptide on their surface from stem cells transfected with a vector into which coding sequences of signal peptide-disturbed blood flow site-targeting peptide-transmembrane protein-green fluorescent protein (GFP) are sequentially inserted.
The “disturbed blood flow” used herein refers to abnormal and irregular blood flow due to the structural characteristics of a blood vessel, and it is an event of early atherosclerosis causing vascular endothelial cell dysfunction.
The “nanovesicles (NVs)” used herein are prepared through disruption of cells by artificially passing through a filter and formed by their self-assembly mechanism, and they are understood as being different from exosomes extracted from cells.
The present invention provides stem cell-derived nanovesicles functionalized with a peptide targeting a disturbed blood flow site (see
The disturbed blood flow site-targeting peptide may be selected from the group consisting of SEQ ID NOs: 1 to 5. More specifically. SEQ ID NO: 1 is a PREY peptide having an amino acid sequence of GSPREYTSYMPH, SEQ ID NO: 2 is a myoferlin peptide having an amino acid sequence of SPREYTSYMPH, SEQ ID NO: 3 is an Eyes absent homolog 1 isoform 2 peptide having an amino acid sequence of SLSSYNGSALAS, SEQ ID NO: 4 is a partial peptide of a zinc finger protein having an amino acid sequence of ACNTGSPYEC, and SEQ ID NO: 5 is a Calsyntenin 1, isoform CRA_b peptide having an amino acid sequence of ACTPSFSKIC.
In the nanovesicles of the present invention, it is easy to confirm the expression of a disturbed blood flow site-targeting peptide, compared to the conventional disturbed blood flow site-targeting peptide-liposome, and a rate of the disturbed blood flow site-targeting peptide expression may be increased by GFP/v5tag FACS sorting in a cell stage prior to nanovesicle extraction. In addition, compared to liposomes, nanovesicles contain a stem cell-derived therapeutic substance for suppressing atherosclerosis. The nanovesicles have advantages over other chemical drugs in terms of stability or the risk of side effects. In addition, due to the use of patient-derived autologous stem cells, the nanovesicles are free from immune responses, and particularly, mesenchymal stem cell-derived nanovesicles are manufactured from cells free from host-immune rejection. Since PMSC-NVs possessing a surface marker of the mesenchymal stem cells also have the above-described characteristic, there also is a possibility for allotransplantation, and in the present invention, through a pre-clinical trial using mice and pigs, the possibility is partially confirmed. In addition, due to the host-immune rejection avoidance mechanism of mesenchymal stem cells, the mesenchymal stem cell-derive nanovesicles are expected to be free from macrophages compared to liposomes, which is expected to lead to an increase in targeting efficiency.
The nanovesicles may be separated through the size-controlled extrusion of the stem cells transfected with a vector into which coding sequences of signal peptide-disturbed blood flow site-targeting peptide-transmembrane protein (TMP)-green fluorescent protein (GFP) are sequentially inserted using a conventional transformation technology.
According to one embodiment of the present invention. plasmid DNA designed to functionalize a PREY peptide targeting a disturbed blood flow site to display it on the outside of an MSC membrane and produce NVs through physical cell disruption and subsequent self-assembly is used. To this end, a plasmid composed of external N-terminus-promoter-signal peptide-PREY-v5 tag-TMP-GFP-internal C-terminus structure is constructed (see
In addition, to improve the expression level of the disturbed blood flow site-targeting peptide, the ability of a peptide searching for and targeting the disturbed blood flow site may be maximized by using a transmembrane protein (TMP). Accordingly, the transmembrane protein may be i) a protein expressed in stem cells or nanovesicles. For example, as the transmembrane protein, an exosome marker such as CD86 and mesenchymal stem cell markers such as CD105 or CD271 may be used.
ii) The N-terminus and the C-terminus of the protein have to face in opposite directions with the cell membrane interposed therebetween.
Preferably, CD271 with high transfection efficiency may be used.
As the signal peptide, signal peptide F (BKU002587, Korea Human Gene Bank, Republic of Korea) and signal peptide R (BKU008396, Korea Human Gene Bank, Republic of Korea) may be used, but the present invention is not limited thereto.
The coding sequence of signal peptide-disturbed blood flow site-targeting peptide-transmembrane protein-green fluorescent protein (GFP) is a nucleic acid sequence, and the nucleic acid is used in the broadest sense, which includes single stranded (ss) DNA, double stranded (ds) DNA, cDNA, (−)-RNA, (+)-RNA, and dsRNA. Preferably, the nucleic acid is ds DNA.
Preferably, when DNA is selected as the coding sequence of signal peptide-disturbed blood flow site-targeting peptide-transmembrane protein, it may be used while being inserted into an expression vector.
The term “vector” used herein refers to a nucleic acid molecule capable of delivering another nucleic acid linked thereto. As one type of vector, “plasmid” refers to a circular double-stranded DNA loop into which an additional DNA segment may be ligated. Another type of vector is a viral vector that can ligate an additional DNA segment into a viral genome. Some vectors may be self-replicated in host cells when introduced into host cells (e.g., bacterial vectors having a bacterial replication origin and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) may be integrated into the genome of a host cell when introduced into the host cell and replicated with the host genome. In addition, some vectors may direct the expression of a gene to which they are operably linked. Such a vector used herein is called a “recombinant expression vector” (or simply, called “expression vector”). Generally, the expression vector useful for a recombinant DNA method is typically a plasmid form, and since the plasmid is the most common vector type, the “plasmid” and the “vector” may be used interchangeably. However, the present invention includes different types of expression vectors such as viral vectors providing equivalent functions (e.g., an adenovirus vector, an adeno-associated virus (AAV) vector, a herpes virus vector, a retrovirus vector, a lentivirus vector, a baculovirus vector). Preferably, a lentivirus vector can be used. Transformation includes any method to introduce a nucleic acid into an organism, a cell, a tissue or organ, and it may be performed by selecting appropriate standard techniques depending on host cells as known in the art. These methods include electroporation, protoplast fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, stirring using silicon carbide fibers, agrobacteria-mediated transformation, PEG, dextran sulfate, and Lipofectamine, but the present invention is not limited thereto.
The stem cells may be stem cells derived from one or more types of tissue selected from the group consisting of bone marrow, the umbilical cord, umbilical cord blood, the placenta, blood, skin, adipose tissue, nervous tissue, the liver, the pancreatic duct, muscle and the amniotic membrane: mesenchymal stem cells; embryonic stem cells; or induced pluripotent stem cells. Preferably, stem cells containing miRNAs such as miR-21, miR-132, miR-10, miR-146, miR-143 and let 7 having an anti-atherosclerotic characteristic may be used. For example, adipose-derived stem cells with high transfection efficiency and a low apoptosis rate may be used.
According to one embodiment of the present invention, the nanovesicles of the present invention may be obtained through extrusion while porous membranes with sizes of 10 μm, 5 μm and 400 nm are sequentially changed for stem cells transfected with a vector expressing a PREY peptide. According to one embodiment of the present invention, as a result of TEM and DLS analyses, a uniform distribution of nanovesicles having an average diameter of about 47.2±12.1 nm and about 83.7±20.6 nm, respectively, may be obtained, and the diameters are smaller than 14.9±2.0 μm of stem cells measured by DLS.
The nanovesicles of the present invention contain intracellular components even after extrusion, in comparison with intracellular components of stem cells, and the levels of anti-atherosclerotic miRNA, for example, miR-21, miR-132, miR-10, miR-146, miR-143 and let 7, are increased upon extrusion.
When macrophages activated by LPS treatments are treated with the nanovesicles of the present invention, the expression of an anti-inflammatory cytokine gene increases, exhibiting an anti-inflammatory effect. In addition, the foam cell formation of macrophages caused by the uptake of oxidized LDL results in the induction of phenotypic changes and the internal growth of VSMCs, therefore, the region in which such results are obtained is important for the development of atherosclerosis. These results show that the nanovesicles can prevent an atherosclerosis process.
In addition, as a result of testing an endothelial cell repair effect by nanovesicle treatments by activating the pro-inflammatory dysfunction of iMAECs, since endothelial cells dominantly express E-selectin, ICAM-1, and VCAM-1to recruit inflammatory cells in dysfunction activation, all of them are upregulated by LPS treatments when gene expression of these markers are measured. When treated with the nanovesicles, however, they are significantly downregulated, and therefore, the nanovesicles exhibit an endothelial cell repair effect. Moreover, by the CyA treatments in HUVECs, the nanovesicles can improve the pre-endothelial cell recovery and pre-angiogenic effects.
In addition, as a result of testing a disturbed blood flow site-targeting effect of nanovesicles in murine and porcine PCL models, it can be seen that a PREY peptide, which is the disturbed blood flow site-targeting peptide, targets filamin A overexpressed in the disturbed blood flow site, which may confirm a synergistic theragnostic effect of preventing the early progression of atherosclerosis. Moreover, in an in vitro microfluidic model, the nanovesicles co-exist with filamin A, and increase under a disturbed blood flow condition, which demonstrates the theragnostic potential of the nanovesicles.
In addition, the nanovesicles of the present invention reduce the expression level of the control without noticeable toxic effects in the heart, lungs, liver and spleen after systemic circulation.
Accordingly, the present invention also provides a composition for preventing, diagnosing and treating atherosclerosis, which includes the nanovesicles.
The composition for preventing or treating atherosclerosis of the present invention may include an active ingredient and an active or inactive pharmaceutically acceptable carrier, which are used for a composition suitable for a preventive, diagnostic, or therapeutic use in vitro, in vivo. or ex vivo.
The pharmaceutically acceptable carrier includes any pharmaceutically carrier which can be mixed with nanovesicles, like protein excipients including a phosphate-buffered saline (PBS) solution, serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin and casein. Examples of carniers, stabilizers and adjuvants can be found in Martin REMINGTON'S PHARM. SCI, 18th Ed. (Mack Publ. Co., Easton (1995)) and the “PHYSICIAN'S DESK REFERENCE”, 58nd Ed., Medical Economics, Montvale, N.J. (2004). The term “carrier” may include a buffer solution or a pH adjuster, and the typical buffer solution is a salt prepared from an organic acid or base. Representative buffer solutions include organic acid salts such as a citric acid salt, an ascorbic acid salt, a gluconic acid salt, a carbonic acid salt, a tartaric acid salt, a succinic acid salt, an acetic acid salt, and a phthalic acid salt; Tris, tromethamine hydrochloride and a phosphate buffer.
Additional carriers include polymeric excipients/additives, for example, polyvinylpyrrolidone, Ficoll (polymer sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl-quadrature, 2-hydroxypropyl-cyclodextrin), polyethylene glycol, antioxidants, anti-static agents, surfactants (e.g., polysorbates such as “TWEEN 20” and “TWEEN 80”), lipids (e.g., phospholipids and fatty acids), steroids (e.g., cholesterol) and chelating agents (e.g., EDTA). An anti-icing agent or freezing point depressing agent may also be included.
The composition for preventing, diagnosing, or treating atherosclerosis may be prepared in various appropriate formulations. For example, formulations and carriers suitable for administration via parenteral routes such as intra-arterial (at a joint), intravenous, intramuscular, intradermal, intraperitoneal, intranodal and subcutaneous routes, can include antioxidants, buffers, bacteriostats, solutes that allow a formulation to have the same osmotic pressure as the blood of a target recipient, and aqueous and non-aqueous sterile suspensions including a suspending agent, a solubilizing agent, a thickening agent, a stabilizing agent and a preservative. Intravenous or intraperitoneal administration are preferable methods. The dosage of cells administered to a subject is an amount effective to achieve a desired beneficial therapeutic response in the subject over time. For example, before injection, a blood sample is obtained from the subject and then stored, and then used in subsequent analysis and comparison. Generally, at least 104 to 106, and typically 1×1087 to 1×1010 cells may be intravenously or intraperitoneally injected into a 70-kg patient for approximately 60 to 120 minutes. In consideration of overall health conditions and the weight of a subject, the nanovesicles of the present invention are administered in a proportion determined by LD-50 (or other toxicity measurement methods) according to cell type and a side effect according to cell type at various concentrations. The cells may be administered at one time or in several divided portions. The nanovesicles of the present invention may supplement treatments for other specific symptoms using some known conventional therapeutic methods including a cytotoxic agent, a nucleotide analogue, or a biological response modifier. Similarly, the biological response modifier may be selectively added to the treatments with the nanovesicles of the present invention.
The present invention also provides a method of treating atherosclerosis, which includes administering a therapeutically effective amount of the nanovesicles into a subject.
As the nanovesicles and the administration method used in the atherosclerosis treatments are described above, in order to avoid excessive complexity of the specification. descriptions of the common contents between these are omitted.
The subject may be a mammal such as a dog, a cat, a rat, a mouse, or a human, but the present invention is not limited thereto.
Hereinafter, the advantages and features of the present invention and the methods of accomplishing the same will become apparent with reference to the detailed description of exemplary embodiments and the accompanying drawings.
However, the present invention is not limited to the exemplary embodiments disclosed below, and it may be embodied in many different forms. These exemplary embodiments are merely provided to complete the disclosure of the present invention and fully convey the scope of the present invention to those of ordinary skill in the art, and the present invention should be defined by only the accompanying claims.
Hereinafter, the present invention will be described in more detail with reference to examples. The examples are merely provided to more fully describe the present invention, and it will be obvious to those of ordinary skill in the art that the scope of the present invention is not limited to the following examples.
(Plasmid Design and Cloning for PREY Expression)
A plasmid displaying and localizing PREY on a cell membrane consists of external N-terminus-signal peptide-PREY-V5-TMP-GFP-internal C-terminus. As the signal peptide, Signal peptide F (BKU002587, Korea Human Gene Bank, Republic of Korea) or Signal peptide R (BKU008396, Korea Human Gene Bank, Republic of Korea) are used. In the plasmid sequence, i) the signal peptide induces localization of the PREY peptide to the outside of the cell membrane: and ii) the V5 tag and GFP monitor the location and the expression level of PREY. An expression vector was cloned using a NEB Gibson Assembly kit (New England Biolabs, MA) according to the manufacturer's instructions. Signal peptides and each type of transmembrane protein were amplified y PCR using the following templates: Signal peptide F (BKU002587, Korea Human Gene Bank. Republic of Korea). Signal peptide R (BKU008390, Korea Human Gene Bank, Republic of Korea), NGFR (Addgene plasmid #27489, Addgene, MA) for CD86, CD105, and cleaved CD271 (LNGFR). The vector components were inserted into a Cas9-digested p3S-Cas9-HNa (Addgene plasmid #104171) backbone with plasmid synthesis (Macrogen Republic of Korea). According to the above procedure, PCR amplification and Gibson cloning were performed. All primers and plasmids are listed in Tables 1 and 2.
(Measurement of Transfection Efficiency)
PREY transfection efficiency was compared among the test candidates (CD86, CD105, and CD271) of transmembrane proteins with two types of MSCs (ASC and BMSC) one day after transfection by flow cytometry using FACSCanto (BD Biosciences, CA) with quantitative analysis. Therefore, cells were immunostained with an anti-v5 tag primary antibody (ab27671, Abcam, MA) and an Alexa Fluor 647-conjugated secondary antibody (Jackson Immuno Research, PA). To evaluate plasmid dose-dependent apoptosis, transfected ASCs and BMSCs were harvested 30 minutes after transfection, immunostained with Alexa Fluor 488-conjugated annexin V (Thermo Fisher Scientific, CA), and they were subjected to flow cytometry. The number of viable cells was also counted by trypan blue staining one day after transfection.
(NV Extrusion)
Three days after transfection, human ASCs (Promocell, Germany), BMSCs (Lonza, Switzerland), and PMSCs were washed twice with PBS, and detached with 0.25% trypsin/EDTA. Subsequently, to produce nanovesicles, 1×106 cells/mL of the cell suspension in PBS was extruded 6 times sequentially with polycarbonate membrane filters with pore sizes of 10 μm, 5 μm, and 400 nm (Whatman, UK) using an extrusion kit (Avanti Polar Lipids, AL). The NVs were collected by centrifugation at 15,000 g for 30 minutes. Subsequently, the pellet was re-suspended in PBS, filtered using a 0.20-μm syringe filter (Avantec, Japan), and the resulting product was stored at −70° C. until use. The size and morphology of PMSC-NVs were determined by transmission electron microscope (TEM; JEM-F200, JEOL, Japan) and dynamic light scattering (DLS; ELS-1000ZS, Otsuka Electronics, Japan).
(Measurement of In Vitro Anti-Inflammatory Effects)
RAW264.7 cells were seeded on a24-well plate (5×105 cells/well). The pro-inflammatory activation of the RAW264.7 cells was induced in ASC-NV or BMSC-NV (10 μg/mL) after 24-hour LPS (Sigma-Aldrich: 100 ng/mL) treatments. To visualize cellular uptake, the RAW264.7 cells and NVs were labeled with DiO and DiI (Invitrogen, CA), respectively, and imaged using a confocal microscope (LSM780; Zeiss, Germany). For qRT-PCR analysis, the cells were collected 24 hours after NV treatments. Primer sequences of IL-10, IL-10, IL-6, and TNF-α are listed in Table 3. For cytokine analysis using a mouse inflammation antibody array (ab133999, Abcam), a cell supernatant was collected according to the manufacturer's instructions. The anti-phagocytic effects of MSC-NVs were measured according to the manufacturer's instructions using a Vybrant™ Phagocytosis Assay Kit (V6694, Molecular Probes, OR). Images were obtained using a confocal microscope, and a fluorescent intensity was measured using a Vanoskan™ LUX multimode microplate reader (Thermo Fisher Scientific, MA).
(Evaluation of In Vitro Pro-EC Recovery Effects)
iMAECs (ATCC, VA) were seeded on a 24-well plate (1×105 cells/well) and then treated with LPS (100 ng/mL) for 24 hours. Subsequently, the cells were additionally treated with ASC-NVs, BMSC-NVs or PMSC-NVs (10 μg/mL) for 24 hours. For visualization of cell uptake, the iMAECs and NVs were labeled with DiO and DiI, respectively, and visualized using a confocal microscope. For qRT-PCR analysis, iMAECs were collected 24 hours after NV treatments. Primer sequences of E-selectin, ICAM-1, and VCAM-1 are listed in Table 3. The iMAECs were immunostained with a VCAM-1 antibody (ab134047, Abcam) and imaged using a confocal microscope with quantitative analysis by ImageJ. HUVECs (Lonza) were treated with NVs (10 μg/mL) and CyA (25 μg/mL; Santa Cruz Biotechnology, CA), and they were cultured on Matrigel (BD Biosciences, MA) for 2 hours or 24 hours, followed by measuring the pro-EC recovery effects caused by anti-angiogenesis and angiorrhexis. Images were obtained using a confocal microscope and quantified using ImageJ.
(Murine PCL Model)
All animal studies were carried out by procedures (2018-0044) approved by the Institutional Animal Care and Use Committee (IACUC) of the College of Medicine of Yonsei University, Republic of Korea. The surgical procedure was performed with 6-week-old male Balb/c (Orient Bio Inc, Korea) or KOR-ApoE (shl) (SLC, Japan) mice. For PCL surgery, the animals were anesthetized by intraperitoneal injection of a mixture of xylazine (10 mg/kg) and zoletil (50 mg/kg). The neck of each animal was shaved and disinfected with betadine. Subsequently, a midline incision was performed (5 mm). After LCA exposure, three (ECA, ICA and OA) of the four branches of the LCA were ligated with 10-0 polyamide suture, and the STA was left unligated. Subsequently. the incision was closed with 6-0 silk suture. In addition, the mice were monitored and fed an atherosclerotic diet (Research Diets, NJ), followed by intravenously administering MSC-NVs, PMSCs, or PMSC-NVs three days after ligation.
(Measurement of Targeting Efficiency and Anti-Atherosclerotic Effects in Murine PCL Model)
The in vivo PMSC-NV targeting efficiency of disturbed blood flow sites was measured in murine PCL models through IVIS imaging (PerkinElmer, WA) and histological analysis 24 hours after injection of the test groups. The MSC and NV groups were labeled with VivoTrack 680 (PerkinElmer) for 30 minutes and injected into the PCL models. Subsequently, IVIS imaging was performed under inhalational anesthetization with isoflurane. Afterwards, the mice were sacrificed, and their LCAs, RCAs and major organs were harvested for ex-vivo IVIS imaging and histological analysis. Tissue sections of the LCAs and RCAs were immunostained with an anti-filamin A antibody (ab51217, Abcam), and the corresponding fluorescence intensity was quantified using ImageJ software. Tissue sections were also stained with H&E, or immunostained with an anti-CD68 antibody (ab125212, Abcam) and an anti-VCAM-1 antibody (ab134047, Abcam). Neointima structure factors (area ratio of neointima to neointima+ lumen, area ratio of neointima to media. and neointima area) or the corresponding fluorescence intensities were quantitatively analyzed using ImageJ.
(Measurement of Targeting Efficiency in Porcine PCL Model)
PCL surgery was performed on female Yorkshire pigs with a weight of 25 to kg (XP bio, Republic of Korea) according to a previous study. The pigs were subjected to intramuscular injection with atropine (0.04 mg/kg), xylazine (2 mg/kg), and azaperone (2 mg/kg) as premedication. The pigs were anesthetized with alfaxan (1 mg/kg) and maintained in this state by endotracheal intubation of 2% isoflurane during surgery. The neck was disinfected using betadine and then a midline skin incision was performed. A sterilized stainless-steel rod (outer diameter=0.9 mm) was placed as a spacer on the LCA and ligated together with 5-0 silk suture. The rod was sequentially removed, and the incision was closed by suturing, resulting in 80% occlusion of a carotid artery. The RCA was left unligated as a normal group. A blood flow pattern was observed using ultrasound (S22V; SonoScape Medical Corp., China). At three days after ligations, Vivotrack680-labeled MSC-NVs or PMSC-NVs were intravenously injected into an ear vein (1 mg/pig), and the pigs were sacrificed on day 1 or 21 after injection. For ex-vivo IVIS and histological analysis, RCAs, LCAs, and aortic arches were collected. These tissue sections were immunostained with an anti-filamin A antibody and an anti-CD31 antibody (sc-1506, Santa Cruz Biotechnology, CA), followed by fluorescence imaging with ImageJ analysis.
(Measurement of Human EC Targeting Efficiency in In Vitro Blood Flow Model)
As previously described (Sei, Y. J. et al. Sci Rep 7, 10019, 2017), a microfluidic device was produced with polydimethylsiloxane (PDMS, Dow Corning, MI) by soft lithography, bonded with glass coverslips (VWR, PA), and placed in a polystyrene box (Ted Pella Inc., CA). The devices were sterilized with 70% ethanol and washed with PBS. Channels were then coated with 50 μg/mL of collagen 1 (Corning, MA) at 37° C. for 1 hour. Human coronary artery endothelial cells (hCAECs; Lonza) or human aortic endothelial cells (hAECs; Lonza) were seeded into the channel for 12 hours (2×107 cells/mL). The outlet of each device was connected to a PhD Ultra syringe pump (Harvard Apparatus, MA) to create a normal medium flow or a disturbed blood flow. To create the normal laminal flow, the medium was perfused with a shear stress of 10 dyne/cm2 at 22.5 μL/min. The disturbed blood flow was generated by repeated cycles of injecting and removing the medium at 22.5 μL/min (10 dyne/cm2) and 20 μL/min (9 dyne/cm2), respectively. After the human ECs were exposed to one of the flow types, NVs (10 μg/mL) were perfused into the channels at 37° C. for 1 hour. NV uptake into human ECs in each test group was quantitatively analyzed using ImageJ.
(qRT-PCR and miRNA Array)
Total RNA was extracted from each sample using a I mL TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The RNA was dissolved in diethyl pyrocarbonate (DEPC) water, and cDNA was synthesized using AccuPower CycleScript RT Premix (Bioneer, Republic of Korea). Subsequently, PCR was performed with SYBR Green PCR mix (Thermo Fisher Scientific) in a StepOnePlus real-time PCR system (Applied Biosystems, CA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was provided as a housekeeping gene, and the gene expression of each marker was measured using the relative quantification method (2−ΔΔCt). Primer sequences are listed in Table 3. The dissolved RNA was profiled using a miRNA array (GeneChip 4.0 microRNA Microarray; Affimetrix, Japan) according to the manufacturer's instructions.
(Immunofluorescence Staining of Cells and Tissue)
Cell samples were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 10 minutes, tissue samples were fixed with 10% paraformaldehyde for 3 days, and both were performed at room temperature. The fixed samples were washed with PBS, and embedded in paraffin to make tissue sections. Subsequently, the sections were hydrated using a series of xylene and ethanol solutions (100%, 95%, 80% and 70% v/v in distilled water) and treated with a pepsin reagent (Sigma-Aldrich) for 30 minutes at 37° C. for antigen retrieval. The tissue sections were then treated with a blocking solution (5% bovine serum albumin (Millipore, MD)+0.3% Triton X-100 (Sigma-Aldrich)) at room temperature for 1 hour. The primary antibodies are an anti-v5 tag antibody (ab27671, Abcam), anti-VCAM-1 (ab134047, Abcam), anti-CD68 (ab125212, Abcam), anti-Filamin A (ab51217, Abcam), and anti-CD31 (sc-1506, Santa Cruz Biotechnology, CA, USA). These antibodies were treated in PBS with 1:100 dilutions, and subsequent secondary antibodies were treated with PBS in 1:200 dilutions. The secondary antibodies are an anti-mouse antibody conjugated to Alexa Fluor 594, an anti-rabbit antibody conjugated to Alexa Fluor 594, an anti-rabbit antibody conjugated to Alexa Fluor 488 and an anti-goat antibody conjugated to Alexa Fluor 488 (all from Jackson Laboratories). Subsequently, the samples were mounted and counterstained with a mounting solution containing 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, CA) to visualize the cell nuclei.
(Western Blotting)
The samples were lysed with RIPA buffer (Sigma-Aldrich) to obtain total proteins, and the concentrations of the proteins were measured using a Bradford assay (Sigma-Aldrich). Protein extracts were run on a 10% (w/v) SDS-PAGE gel by electrophoresis and then transferred to a nitrocellulose membrane. The membrane was blocked in TBST (20 mM Tris, 0.9% NaCl, 0.1% Tween 20, pH 7.4) with 5% (w/v) skim milk and then incubated with primary antibodies such as anti-v5 tag (ab27671, Abcam), anti-CD271 (345102, Biolegend, CA), anti-GFP (ab32146, Abcam), anti-CD9(ab92726, Abcam), and anti-β-actin (ab8227, Abcam). Subsequently, secondary antibodies such as goat anti-mouse IgG (H+L)-HRP conjugated and goat anti-rabbit IgG (H+L)-HRP conjugated antibodies (all from Vector Laboratories) were applied according to the manufacturer's instructions. The signals were visualized using a CL Plus Western Blotting Detection Kit (Amersham Biosciences, UK) according to the manufacturer's instructions and analyzed using a LAS-3000 image reader (Fujifilm, Japan).
(Statistical Analysis)
Quantitative data is expressed as mean±standard deviation (stdev). The results are statistically analyzed through by one-way ANOVA by a Tukey's significant difference post hoc test using SigmaPlot 12.0 (Systat Software, Inc., California. USA).
To enable NVs to target disturbed blood flow sites, a targeting peptide PREY was externally displayed by expressing a specifically-designed plasmid across the MSC membrane (PMSCs: PREY-expressing human MSCs). The plasmid was designed to express a PREY extracellular domain with a signal peptide at the N-terminus so that the signal peptide can induce the outward localization of the PREY-extracellular domain from the MSC membrane and deactivate the induction signal by cleavage.
In addition, the ability of three transmembrane proteins (TMPs), that is, the exosome marker (CD86) and MSC markers (CD105 and CD271), for improving PREY expression was tested. In this way, the ability of PREY to search for and target disturbed blood flow sites was maximized. PREY was linked with V5 tag and then with GFP (external N-terminus-promoter-signal peptide-PREY-V5 tag-TMP-GFP-internal C-terminus) to confirm the localization and the expression levels of PREY outside and inside the cell membrane (
Among the test groups having the three transmembrane proteins, the expression level of PREY-CD271 (nerve growth factor receptor, NGFR) was higher than those of PREY-CD86 and PREY-CD105 in both adipose-derived stem cells (ASCs) and bone marrow-derived stem cells (BMSCs), showing excellent transfection efficiency (
To verify the external display of PREY on the MSC membrane, the V5 tag was immunostained when placed next to the PREY in the sequence (external N-terminus-promoter-signal peptide-PREY-V5-TMP-GFP-internal C-terminus). Since neither of the two types worked to clearly obtain both information types, adhered (
In the adhered form, PREY was highly expressed along the cell membrane as shown by the v5 tag and GFP signal, indicating successful transfection. When suspended forms were formed by inserting cells into a gelatin hydrogel, the confocal images showed the appearance of the v5 tag (red) and GFP (green) signals outside and inside the boundary of the MSC membrane, respectively, confirming the external PREY display on the cell membrane.
To produce PMSC-NVs, PMSCs were sequentially extruded through a series of polycarbonate micropore membranes while gradually decreasing the pore diameters from 10 to 5 μm and finally to 0.4 μm (
Next, comparing the intracellular components in MSC-NVs, PMSCs and PMSC-NVs, the preservation of intracellular components was confirmed after PREY transfection and NV extrusion. miRNAs are provided as important therapeutic agents, and most of them are delivered by exosomes. Since NVs are inherently similar to exosomes, the miRNA contents of PMSC-NVs, MSC-NVs, and PMSCs were profiled by miRNA array (
It is well known that MSCs and MSC-derived intracellular contents exhibit a potent anti-inflammatory effect. Accordingly, the anti-inflammatory effects of BMSC-NVs and ASC-NVs were examined before PREY transfection (
The anti-inflammatory activity of BMSC-NVs, ASC-NVs, and PMSC-NVs was analyzed by qRT-PCR (
The EC-recovery effects of MSC-NV treatment was examined by activating the pro-inflammatory dysfunction of immortalized murine aortic endothelial cells (iMAEC) by LPS treatments (
Cyclosporin A (CyA) treatments are known to disturb angiogenesis by inhibiting angiogenic EC activity. Therefore, the pro-angiogenic (
The theragnostic efficiency of PMSC-NVs was determined in a murine PCL model. As three of four left carotid artery (LCA) branches, i.e., the external carotid artery (ECA), internal carotid artery (ICA), and occipital artery (OA) were ligated (
Since the anti-inflammatory and pro-EC recovery effects of MSC-NVs (
As a pre-clinical large animal model, a porcine PCL model was used to confirm the targeting efficiency of PMSC-NVs for disturbed blood flow sites according to a previously established method. Briefly, a stainless-steel rod (diameter=0.9 mm) was tightly ligated to the LCA (diameter=4.5 mm) and then removed (top,
Finally, under a disturbed blood flow, the efficiency of PMSC-NV targeting hCAECs (
As above, as specific parts of the present invention have been described in detail, although it is clear to those skilled in the art that this specific technique is merely a preferred embodiment, the scope of the specification is not limited thereto.
Since stem cell-derived nanovesicles using a peptide capable of targeting a disturbed blood flow site according to the present invention provide potent anti-inflammatory and pre-endothelial recovery effects similar to mesenchymal stem cells as an anti-atherosclerosis theragnostic platform, they are expected to be effectively used in related medical industry as a novel theragnostic agent for preventing and treating atherosclerosis.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2019-0107218 | Aug 2019 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2020/011581 | 8/28/2020 | WO |
