Ultrasound-Targeting Transfection of Nano Tissue-Type Plasminogen Activator Gene to Prevent

Ultrasound-targeting transfection of nano tissue-type plasminogen activator gene to prevent in-stent thrombosis and neointimal hyperplasia after stenting intervention in injured rabbit iliac artery

Jun Ji1†,Cai-ping Ren2†, Hong Tu3*, Xia He1, Qiang Liu2, Xiao-ling Chen1, Wen-ping Ling1, Yi-feng Pan4, Jing-feng Zhao4, Yu-xiang Chen4*

1 Department of Pathology1 Shenzhen Sun Yat-Sen Cardiovascular Hospital, Shenzhen 518020, Guangdong, China

2Cancer Research Institute, Collaborative Innovation Center for Cancer Medicine,Key Laboratory for Carcinogenesis of Chinese Ministry of Health, Central South University, Xiangya Road 110, Changsha410078, Hunan, P. R. China.

3 Cardiovascular Internal Medicine2, Shenzhen Sun Yat-Sen Cardiovascular Hospital, Shenzhen 518020, Guangdong, China

4 Hepatobiliary & Enteric Surgery ResearchCenter, Xiangya Hospital,Central South University, Changsha 410008, Hunan, P. R. China

†Equal contributors

Correspondence should be addressed to: Dr.Hong Tu,; Dr.Yuxiang Chen,

Abstracts Thrombolytic therapy of systemically administered recombinant t-PAhas become a standard clinical treatment for acute myocardial infarction. However, its significant complications remain. We established the model of in-stent thrombosis with intimal hyperplasiaby deendothelialization of rabbit iliac artery followed by the bare-mental stenting (BMS) implantation and constructed a highly expressive t-PA gene plasmid packaged with albumin nanoparticlescrosslinked to albumin ultrasound microbubbles, and then this gene vector was transferred to implanted arteries under the aid of therapeutic ultrasound(1 MHz, 1.5 w/cm2, 6mins, intravenously) after stenting intervention. The expression of t-PA in the implanted arteries and the tissues around them was detected withmulticlonal antibodies to t-PA by indirect immunohistochemical method. Venous blood t-PA and D-dimercontents were tested before and weeks 1, 2, 4 and 8 after the operation. The effects of the constructed t-PA gene plasmid on in-stent thrombosis and vascular intimal hyperplasia were observed by routine pathological examination,morphometry for intimal thickness andarea, and the immuno-histochemical stains with a monoclonal antibody to PCNA for estimating theintimal SMC proliferation. The effective expression of t-PA in the implanted arteries and the tissues around them in the ultrasonic field wasobtained, followed by the persistent raises of blood t-PA and D-dimer weeks 1, 2, 4 and 8 after the targeting transfection. In-stentthrombosis and intimal hyperplasiawas successfully restrained. The transfection of the albumin nano-t-PA gene to implanted arteries under the helpof ultrasound microbubbles was successfully performed, which could prevent in-stent thrombosisand reduce intimal hyperplasiaunder implanted stents.

[Keywords] stenting intervention; thrombosis; tissue-type plasminogen activator; albumin

nanoparticles; ultrasound microbubbles

Introduction

Heart disease is one of the leading causes of death worldwide. The acute coronary syndromes (ACS) include a spectrum of unstable coronary artery disease from unstable angina to transmural myocardial infarction (MI). Their common etiology is the formation of thrombi on an inflamed and complicated atheromatous plaque1. Despite the effort to reduce coronary heart disease mortality over the past 40 years, hospital admissions for ACS continue to increase2. One of the most common medical interventions performedin the treatment of coronary artery diseasetoday is the percutaneous coronary intervention (PCI), which opens clogged or damaged coronary arteries and has been widely used3. PCI procedures include percutaneous transluminal coronary balloon angioplasty and coronary vascular stents (or scaffolds) such as bare metal stents (BMSs), and drug eluting stents (DESs).BMSs have been identified to reduce procedural complications and late restenosis compared with balloon angioplasty in randomized trials4.Compared with BMSs, DESs have dramatically reduced restenosis, but in-stent thrombosis has emerged as a major safety concern after DES implantation5. A DES is comprised of three components: a bare metal backbone, the durable polymer, and anti-proliferative agents such as everolimus, biolimus, or sirolimus6.The first-generation of DES, containing sirolimus or paclitaxel, was shown to reduce in-stent neointimal hyperplasia and target vessel revascularizationwith reduced rates of clinical restenosis7. However,pathological studies have shown that the delayed arterial healing characterized by poor endothelialization and the accelerated neoatherosclerosis inducedby polymer hypersensitivity reaction and chronic inflammation increase the risk of stent thrombosisearly after their adoption in clinical practice, requiring prolonged dual antiplatelet therapy, especiallylate(31 days to one year) or very late(beyond one year)DES thrombosis (early thrombosis within 30 days)8.The second-generation DES was thus developed using a thinnerstent struts, permanent but biocompatible and bioabsorbable polymersto minimize inflammation or hypersensitivity reactions, and novel anti-proliferative agents9. A thin-strut, fluoropolymer-coated cobalt-chromium everolimus-eluting stent (CoCr-EES) may be associated with lower rates of definite stent thrombosis than other DES. The relative safety and efficacy of CoCr-EES, DES with bioabsorbable polymers, and fully bioresorbable scaffolds is the subject of numerous ongoing large-scale trials10.

Local drug delivery for the treatment of coronary artery disease such asDESs has been studied for many years and routinely deployed11. However, with concerns regarding late thrombosis, the pivotal step in these prevention processes is a long-term anticoagulative therapyfor these patients, which may induce complications such as hemorrhage or rebound thrombosis when the therapy ceases12. Several anticoagulant agents are commonly used clinically, including warfarin, heparin, aspirin13-16,especially in prevention of acute thrombotic events such as acute myocardial infarction, acute ischemic strokeand thrombosis after coronary bypass, transluminal balloon angioplastyand stenting and prosthetic heart valve replacement17-21.T-PA, another kind of thrombolytic agent, is a 527-aa residue serine protease and performs the primary role in fibrinolysis by preferentially catalyzing the conversion of the proenzyme plasminogen to plasmin in the presence of fibrin22,23. T-PA functions relatively clot-specific because of its high affinity for fibrin–plasminogen complexes via a fibronectin-like finger domain of lysine binding sites near the amino terminus and its relatively poor activity in the absence of fibrin. Hepatic metabolism represents the primary clearance mechanism for t-PA, and the enzyme has a half-life of 5–10 min in circulation24,25. Although thrombolytic therapy of systemically administered recombinant t-PAhas become a standard clinical treatment for acute myocardial infarction and has also been proven effective in improving the neurologic outcome in patients with acute ischemic stroke, significant complications remain. Central problem among themis hemorrhage, particularly pulmonary or intracerebral hemorrhage, each of which can be life threatening26,27.Since the cDNA of t-PA was successfully cloned, t-PA reverse-transcript virus vector could be constructed and transfected in vitro to the epithelial cells with a high expression of t-PA protein. It was demonstrated experimentally that thrombosis and restenosis after coronary stenting or bypass could be prevented with a long-term outcome when the transfected epithelial cells were spread on the surface of stent or covered on vascular anastomosis28,29.Gene therapy offers a method of local high expression of t-PA over a prolonged time period to avoid both systemic hemorrhage and local rebound thrombosis.Some problems should be addressed: On the one hand, how for t-PA gene to get to the heart or other targeting tissues in vivo is still a handicap up to date. On the other hand, gene vector for t-PA is also an issue to be tackled. In our early study, we constructed a t-PA gene plasmid and transfected it to pig myocardium, using surgery dacron suture as gene vector, and successfully prevented thrombosis after valve replacementand anastomostic restenosis after coronary bypass30,31.Although the surgery dacron suture carring gene had been proved to be safe, effective and convenient, it was alien and traumatic and unfavourable for patients as drug carrier.

Using polymer-constructed nanoparticles as the carrier for gene transfection has become a new method for the past few years32. Albumin has been used as the nano material because of its high biocompatibility, biodegradability, no immunogenicity and no cytotoxicity33.Albumin is positively charged in tart medium. The albumin nanoparticlescan absorb the gene DNAs with negative charges by static electricity. Directly injecting a gene of interest loaded with a plasmid DNA or an adenovirus vector to the targeting tissues for gene expression represents the major method in modern experimental study. This method is hard to be received clinically because of traumatic conclusion or immunogenicity. In vivo distribution of a drug or a gene carried by nanoparticles can be changed by modification on nanoparticle surface, which display the targeting of nanoparticles. The nano-targetingconsists of two patterns, active and passive. The former has real significance and includes physical-chemical (such as PH-sensitive, temperature-sensitive and magnetic targeting) and biological targeting (such as antigen-antibody, receptor-ligand targeting), which have been commonly used in nano-targeting study. The drug or gene can be precisely sent to the targeting organ or diseased region for treatment or transfection34.

Ultrasound contrast agents have been used in diagnostic ultrasound imaging in the past few years35 and can also carry drug or gene to the selected targeting tissues and cells for treatment or transfection36.These methods have been experimentally confirmed to be effective, safe and non-traumatogenic37.The fundamentalprinciples of ultrasound for targeting treatment are: ⑴ Cavitations and machinery effects produced by the therapeutic ultrasound make the cell membrane reversible injury and increasein permeability.⑵ The capillary vessels(≤7mm) in the ultrasound field are injured and interspaces between endothelial cells become wide, through which the drug or specific gene can get to targeting tissue38. And ⑶ when the ultrasound microbubbles in blood circulation pass through the tissue or organ treated with the ultrasound, they are quickly destroyed and the carried drug or gene is very easily released to the targeting tissue or organ. Systemic toxicity and side effect produced by the drug and gene significantly decrease because of targeting localization39.

In this study, deendothelialization of rabbit iliac artery was performed followed by the bare-mental stenting (BMS) implantation. The model of in-stent thrombosis with intimal hyperplasia was made. A highly expressive t-PA gene plasmid packaged with bovine serum albumin (BSA) nanoparticlewas constructed and further crosslinked to ultrasound microbubble prepared with sucrose and BSA for t-PA gene targeting transfection. This agent was given intravenously followed by a therapeutic ultrasound treatment of the local tissue around the stenting artery. The expression of t-PA by implanted arteries and local tissues including skeletal muscle was detected. Venous blood t-PA and D-dimer contents were tested before and 1,2, 4 and 8 weeks after operation. This targeting gene therapy provides a new experimental method forpreventing in-stent thrombosis after stenting intervention.

Materials and methods

Animals

Thirty six healthy New Zealand rabbits, male, 2.3-2.5kg in weight, were provided by Southern Medical University Animal Center, Guangzhou, Guangdongprovince, China. All animal experiments were performed in compliance with animal protection policy of Chinese government (license SYXK Guangdong) and standards of bioethics and bio-security of our institute committee.

Main reagents and instrument

Chinese Hamster Ovary (CHO) cell line, pSecTag2B plasmid, E.Coli JM109, rabbit anti-human t-PA multiclonal antibody, mouse anti-α-actin and anti-proliferative cell nuclear antigen(PCNA) monoclonal antibodies, fluoresceine isothiocyanate（FITC）coupled sheep anti-rabbit IgG antibody and rabbit anti-sheep multiclonal antibody, immunohistochemical reagents and bovine serum albumin(BSA) were purchased from JingMei Biotech, Shenzhen, Guangdong province, China. Restriction enzymes HindIII, KpnI, BamHI and XhoI, Vent DNA polymerase, T4 DNA ligase, polymerase chain reaction（PCR）product purification kit and DNA marker DL2000 were purchased from New England Biolabs, Hong Kong, China. Perfluoropropane (Halocarbon-218) was supplied by JieRui Co.Ltd, Fushan, Guangdong province, China. Zeta potential analyzer is a product of Brookhaven Instruments Corporation. Diagnostic ultrasonic generator(PHILIPS-iE33) is a product of PHILIPS, Co Ltd, Japan. Therapeutic ultrasound Unit (US-700) was made by ITO Co. Ltd, Japan. Balloon catheters with bare-metal stents (3.0mm ballon catheter in diameter) were kindly presented by Kinhely Bio-tech Co. Ltd, Shenzhen, China.

Construction and expression of the pSecTag2B -t-PA gene

Three expressed tag(EST) sequences were obtained from Internet Blast according to t-PA gene sequence. The ID numbers were 6251209, 4861268 and 5190656, respectively. The primers were synthesized as follows: t-PA-1F: 5’-CCC AAG CTT ATG GAT GCA ATG AAG AGA GGG- 3’, t-PA-1R: 5’-GGG GTA CCA CGG TAG GCT GAC CCA TTC-3’, t-PA-2F: 5’-GGG GTA CCC ACA GCC TCA CCG AGT CG-3’, t-PA-2R: 5’-CGG GAT CCA GCA GGA GCT GAT GAG TAT GCC-3’, t-PA-3F: 5’-CGG GAT CCT CTC TGC CGC CCA CTG CT’-3’, t-PA-3R: 5’-CCC TCG AGG CGG TCG CAT GTT GTC AC-3’. As the PCR amplification template, three EST clone strains were abstracted and the three t-PA fragments were amplified. The pSecTag 2B and three t-PA fragments t-PA-1, t-PA-2 and t-PA-3 were digested by HindIII and XhoI, HindIII and KpnI, KpnI and BamHI, BamHI and XhoI, respectively. These enzymatic products were purified with PCR product purification kit and were linked by T4 DNA ligase at 14 ℃ overnight. The linked products were transfected to E.Coli JM109 and the resistance colony in the aminobenzypenicillin LB plate culture was chosen. This t-PA plasmid was sequenced and was transfected to CHO cells by calcium phosphate coprecipitation. The expression of t-PA was detected using a rabbit anti-human t-PA multiclonal antibody by indirect immuno-fluorescence method. For in vitrot-PA activity, human umbilical-vein endothelial cells were harvested and grown on gelatin-coated plates in M199, supplemented with 20% fetal bovine serum, 0.1 mg/mLporcine heparin, 5 mg/mL extracellular matrix, 100 mg/mL penicillin, and 100 mg/mL streptomycin at 37°C. These cells were infected with albumin nano-tPA(0.5μg/mL in final concentration) or PBS for 10 min under the treatment of ultrasound (1MHz, 1.5w/cm2) on the surface of medium and then incubated in dye-free DMEM for 48 h. Aliquots were withdrawn for detection of thet-PA content by ELISA.

Preparation of BSA nanoparticles loaded with t-PA gene plasmid

The preparation of BSA nanoparticles loaded with t-PA gene plasmid was according to the methods published by Arnedo32and Zhang40 with some improvement. Briefly, 2mg t-PA plasmid DNA was incubated with 10mL albumin aqueous solution (1% w/v; pH 5.5) for 30 min. Then, this aqueous phase was dissolved with ethanol dropwise (ethanol: water=2:1). The coacervates were hardened with 30μl glutaraldehyde (concentration: 0.5%, w/v) for 2 h. After ethanol was eliminated by evaporation, the nanoparticles were purified by centrifugation at 17000 r.p.m. for 30 min to eliminate free albumin and excess cross-linking agent. The purified nanoparticles by centrifugation were resuspended in pure water and dispersed with ultrasound generator (180W, 20 kHz, for 30 sec) and stored at 4 ℃ for further use. The amount of albumin transformed into nanoparticles was determined by a standard Bicinchoninic acid(BCA) protein assay. The pellet of nanoparticles obtained after centrifugation was digested with NaOH 0.1M for 2 h at room temperature. The resulting solution was analyzed with a spectrophotometer at 562 nm. The resulting absorbance was compared with the data obtained after same digestion of a control albumin solution. The stability of nanoparticles was investigated in RPMI 1640 cell culture medium supplemented with fetus bovine serum at 37 ℃ over 48 h by turbidity and particle size measurements. Some of nanoparticles were taken for particle size, morphological observation, surface zeta potential, envelopment rate and the electrophoresis.

In Vitrot-PA activity Assay

Human umbilical-vein endothelial cells were harvested by the method of Yang41 and grown on gelatin-coated plates in RPMI-1640, supplemented with 20% fetal bovine serum, 0.1 mg/mL porcine heparin, 100 mg/mL penicillin, and 100 mg/mL streptomycin at 37°C. The medium was changed 48 hour later.These cells were digested by trypsin and counted before transfection, and then were inoculated in six-well plate with each well containing 5.0×106 cells(2.5 mL). Human umbilical-vein endothelial cells were infected with the constructed nano-pSecTag2B -t-PA diluted with DMEM free of serum. Serial dilutions(containing DNA 1,2,4 and 8μg, respectively) were performed for determining the relation of added DNA content with t-PA activity. PBS was employed for substitution of nano-t-PA in control experiment. For better effect of transfection, an ultrasound treatment(1MHz, 0.5w/cm2, 6min) was given on the surface of culture medium which was replaced 4 hour later. Supernatant was obtained for testing t-PA activity after 48 hour further cultivation. T-PA activity detection(choromogenic substrate assay) was performed according to the manufacturers instruction. The calibration sample was diluted to different concentrations (2.5×102, 2.0×102, 1.5×102, 1.0×102, 0.5×102, 0 IU/mL), which were added into the wells of an enzyme-linked plate. The supernatant samples were diluted 1000 times with buffer. 0.1mL of diluted sample and 0.01mL choromogenic substrate were added into the same well and incubated at 37℃ for 150 minutes. After the reaction was ended by the stop solution, the absorbency value of each well was measured at 405 nm with a microplate reader.

Preparation of ultrasound microbubbles

The preparation of albumin ultrasound microbubbles was according to the method reported by Du42 and Li43 with some improvement.10mL BSA solution (5%, w/v) with sucrose (final concentration: 10%, w/v) was prepared in 50mL plastic centrifuge tube and saturated with oxygen and perfluoropropane（flow rate: 6mL/min）by turns for 10 min and dispersed with ultrasound generator (180W, 20kHz, for 1 min). All procedures were operated under sterility. The prepared microbubble solution was stored at 4 ℃ for further use.

Linkage of nanoparticles to microbubbles

Nanoparticles loaded with t-PA gene plasmid (containing 1 mg plasmid DNA) were mixed with 5mL microbubbles (containing 1.0×109/mL) at the room temperature. 10μLof 50% glutaraldehyde (final concentration: 0.1%, w/v) was added to the prepared mixture for 2 hours at 4 ℃. The linked product was centrifugated at 200 r.p.m. for 1 min and the floatage was taken and washed with 0.9% sodium chloride under the centrifugation at 200 r.p.m. for 3 times and the supernatant was stored at 4 ℃ for ultrasound targeting transfection.

Vascular intervention and targeting gene transfection

Thirty rabbits were randomly divided into two groups: control (n=15) and experimental (n=15). Residuary six animals were used for experiment-related controls. All surgical procedures were carried out on the animals under general anesthesia (25 mg/kg body weight sodium pentobarbital). Rabbit iliac arteries were denuded with a 2.5-mm diameter balloon catheter as previously described44 and vascular stenting interventions with 3.0 mm balloon catheters with bare-metal stents were performed in injures arteries. In the experimental group, liver was chosen as a ultrasonic control organ and was observed with 2Ddiagnostic ultrasonic generator soon after intravenously injecting 5mL linked ultrasound microbubbles(containing 1 mg plasmid DNA)and a strong resonance of the ultrasound signal from liver was observed. After that, a therapeutic ultrasound treatment (1MHz, 1.5w/cm2, 6 min) was given on the tissue around the stenting artery, followed by a normal liver resonance seen. In control group, physiologic saline injection with the ultrasound treatment was given after the surgical operation. In addition, each of 5mLmicrobubbles and nanoparticles withblank plasmid (n=2), nanoparticles and t-PA plasmid without microbubbles (n=2) and microbubbles without nanoparticles t-PAplasmid (n=2) were givenfor experiment-related controls. The all animals were observed for 8 weeks. The venous blood was taken before operation, and 1,2,4 and 8 weeks after operationfor t-PA and D-dimer contents and prothrombin time. Any anticoagulant agent was not given for all animals before and after operation.