Sequential Targeted Delivery of Liposomes to Ischemic Tissues by

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Sequential Targeted Delivery of Liposomes to Ischemic Tissues by Controlling Blood Vessel Permeability Myung Joo Nam, Jangwook Lee, Kuen Yong Lee, and Jaeyun Kim ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00815 • Publication Date (Web): 08 Jan 2018 Downloaded from http://pubs.acs.org on January 14, 2018

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ACS Biomaterials Science & Engineering

Sequential Targeted Delivery of Liposomes to Ischemic Tissues by Controlling Blood Vessel Permeability Myungjoo Nam,1,† Jangwook Lee,2,† Kuen Yong Lee2,* and Jaeyun Kim1,3,4,* 1

School of Chemical Engineering, Sungkyunkwan University (SKKU), Suwon 16419, Republic

of Korea 2

Department of Bioengineering, Hanyang University, Seoul 04763, Republic of Korea

3

Department of Health Sciences and Technology, Samsung Advanced Institute for Health

Science & Technology (SAIHST), Sungkyunkwan University (SKKU), Suwon 16419, Republic of Korea 4

Biomedical Institute for Convergence at SKKU (BICS), Sungkyunkwan University (SKKU),

Suwon 16419, Republic of Korea †

These authors contributed equally to this work.

*E-mail: [email protected] *E-mail: [email protected]

KEYWORDS: sequential delivery, ischemia, angiogenesis, VEGF, liposomes

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ABSTRACT: Delivery systems for therapeutic angiogenesis that deliver angiogenic factors to ischemic tissues have recently been fabricated. However, these systems are designed for surgical implantation or multiple local injections which can cause pain and potential physical burden in patients. Here, we propose a minimally invasive sequential nanoparticle-mediated delivery strategy for ischemic tissue using murine hindlimb ischemic model. Intravenously injected liposomes that encapsulate VEGF, an angiogenic factor, first target the ischemic sites via the enhanced permeability and retention (EPR) effect in early stage of ischemia. VEGF released from the targeted liposomes maintains the blood vessel permeability for longer period of time compared to the delivery of empty liposome. This first nanoparticle-mediated delivery of VEGF to ischemic site enables extending the temporal window of leaky blood vessel up to 7 days so that the second liposomes could be targeted to the ischemic sites via EPR effect. This strategy will provide opportunities for the targeted delivery of other vessel maturation agents loaded in nanoparticles to ischemic tissue.

Introduction Ischemic disease is a fatal phenomenon associated with the obstruction of the blood stream, resulting in the deficient supply of nutrients and oxygen to each cell at ischemic sites.1 A potential approach to overcome this disease is promoting angiogenesis in ischemic tissues via the delivery of angiogenic factors.2,3 Angiogenic factors promote the generation of new blood vessels at ischemic sites and increase blood flow in damaged areas. For example, vascular endothelial growth factor (VEGF) promotes the proliferation of endothelial cells at ischemic sites during the initial phase of angiogenesis and results in new blood vessels.4,5 Some angiogenic factors act in the late phase of angiogenesis. For example, platelet-derived growth factor (PDGF) influences


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the maturation and stabilization of endothelial cell channels.6 Angiopoietin-1 (Ang-1) promotes vascular maturation and inhibits the leakage of blood vessels.3-5 Therefore, to enhance the efficacy of therapeutic angiogenesis, the timing of the application of various angiogenic factors is important, and the sequential delivery of angiogenic factors to target sites is an effective method. To achieve the sequential delivery of various angiogenic factors at the appropriate times, diverse drug delivery systems have been studied.6-8 A macroporous scaffold that releases dual angiogenic factors, i.e., VEGF and PDGF, has been fabricated based on poly(lactide-coglycolide) (PLG).6 As VEGF was mixed with preformed PLG particles encapsulating PDGF to form a PLGA macroporous scaffold, VEGF was released more rapidly from the polymer scaffold than PDGF. To control time-dependent sequential delivery and protect angiogenic factors, a fibrin gel and biocompatible heparin-based coacervate were developed.7 The fibrin gel incorporated VEGF and heparin-based coacervate in which PDGF was encapsulated simultaneously, resulting in the rapid release of VEGF and the slow release of PDGF. For treatment after stroke, a new sequential delivery system that can directly release epidermal growth factor (EGF) and erythropoietin (EPO) has been fabricated.8 EGF and EPO were pegylated in poly(lactic-co-glycolic acid) (PLGA) nanoparticles, which were coated with poly(sebacic acid) containing EPO. Finally, two particle types were embedded in an injectable gel of hyaluronan and methylcellulose for delivery to brain tissues. Despite great improvements in the fabrication of hydrogels and scaffolds that sequentially release different angiogenic factors, these materials need to be locally delivered to ischemic tissues via surgical implantation or local injection.4,

6, 7, 9

To achieve the minimally invasive

delivery of angiogenic factors to ischemic tissues, the targeting of nanoparticles loaded with


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VEGF to ischemic sites via intravenous injection has been studied.4, 10, 11 The targeting strategy is similar to the passive targeting of nanoparticles to tumors via the enhanced permeability and retention (EPR) effect12; nanoparticles extravasate through leaky blood vessels around the tumor region and then accumulate in tumor tissues.13 In hypoxic conditions induced in ischemic regions, various angiogenic factors are secreted in ischemic tissues, leading to an increase in blood vessel permeability.14 It has been reported that intravenously injected fluorescent nanoparticles could be targeted to ischemic muscles in a murine hindlimb ischemic model due to the increased permeability of blood vessels in hypoxic tissues.11 Interestingly, there is a temporal window for the efficient targeting of nanoparticles to ischemic sites after the induction of ischemia. The targeting efficiency of nanoparticles to ischemic muscle upon the injection of nanoparticles decreases as time after the induction of ischemia increases, and was enhanced during the early stages of ischemia within a few days. Similarly, passive targeting of adenosine-loaded silica nanoparticles to the ischemic-reperfused myocardium has also been explored.15 These examples indicate that the early administration of nanoparticles after the occurrence of ischemia is necessary for efficient targeting. However, considering the appropriate timing of different types of exogenous angiogenic factors (e.g., VEGF for early phase vs. PDGF or angiopoietin-1 for late phase) in ischemic regions to enhance therapeutic angiogenesis, the sequential delivery of nanoparticles carrying different angiogenic factors is desired. To date, no reports have examined the sequential delivery of nanoparticles to ischemic tissues. In this context, a strategy for the efficient sequential targeting of nanoparticles to ischemic tissues is necessary and has the potential to open a new route for the delivery of diverse angiogenic factors after ischemia in a minimally invasive manner.


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Here, we propose a new strategy for the sequential delivery of different nanoparticles to ischemic tissues using a murine hindlimb ischemic model (Figure 1). We first delivered liposomes loaded with VEGF to the ischemic site in a murine hindlimb model based on the EPR effect in the early stage of ischemia. The delivered VEGF had dual roles; it initiated angiogenesis and also further extended the duration of blood vessel permeability in ischemic tissues. Owing to the extended vessel permeability, the targeting of the second liposomes to the ischemic site was more efficient (Figure 1b). However, if there is no VEGF loaded on first liposome, the targeting ability of the second liposome is reduced due to naturally decreased permeability (Figure 1c). If any therapeutic agents, such as PDGF or Ang-1, are loaded on the second liposomes, these agents will target ischemic sites where pre-delivered VEGF initiated angiogenesis and will have an increased potential to contribute to the late state of angiogenesis, i.e., blood vessel maturation. Accordingly, the effects of proper therapeutics delivered to ischemic tissues may be maintained for a sufficient period of time, depending on the timing of delivery, to treat ischemic diseases.16 Experimental Reagents and Materials: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt) (DSPEPEG 2000), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and cholesterol were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Alexa Fluor 680 NHS ester (succinimidyl ester) and Alexa Fluor 750 NHS ester (succinimidyl ester), which are infrared dyes (IRDye), were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Recombinant human VEGF was purchased at R&D Systems (Minneapolis, MN, USA). Triethylamine (TEA) and chloroform were purchased from Sigma Aldrich (St. Louis, MO, USA). Liposome extruder (LiposoFast Basic) was purchased from Avestin (Ottawa, Canada).


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Synthesis of Alexa Fluor-labeled DSPE: Alexa Fluor-labeled DSPE was synthesized by conjugating Alexa Fluor dye (NHS ester) to DSPE.20 Alexa Fluor powder (1 mg) was dissolved in chloroform (1 mL) in a glass vial. DSPE (25 mg/mL) was dispersed in 3% TEA/CHCl3 (1:2 v/v). The solution was sonicated for 5 minutes. Alexa Fluor dye/CHCl3 (0.1 mL) and the DSPE solution (0.05 mL) were mixed in a glass vial and wrapped with foil. The solution was reacted for 20 hours at 37°C in the dark with shaking. Preparation of Alexa Fluor-labeled liposomes: Each lipid and cholesterol were dispersed in chloroform; 0.22 mL of DSPC solution (25 mg/mL), 0.24 mL of cholesterol (10 mg/mL), and 0.1 mL of DSPE-PEG (25 mg/mL) were mixed in a glass vial (molar ratio, 16:13:4). Then, 0.2 mL of the Alexa Fluor-labeled DSPE solution was added to the lipid solution. The solvent was evaporated using a rotary evaporator and a vacuum evaporator for 1 hour. The lipid film was rehydrated with 1 mL of 60°C phosphate-buffered saline. The solution was vortexed and sonicated and then the liposomes were reduced using a 200-nm polycarbonate membrane in a liposome extruder. The final liposome solution was filtered using a 200-nm filter for the animal experiment. Preparation of VEGF-loaded Alexa Fluor-labeled liposomes: The procedure was the same as that used for the preparation of Alexa Fluor-labeled Liposomes, but VEGF powder (0.1 mg) in phosphate-buffered saline (60 oC) was used for the rehydration procedure. The liposome solution was then vortexed and extruded. In vitro release experiment of encapsulated BSA-FITC: 0.1 mg of BSA-FITC (65.5 kDa) was encapsulated into the DSPC-based liposomes in rehydration procedure on preparation of liposome and the released protein was determined by measuring the fluorescence intensity of the suspension. Fluorescence intensity was monitored at 490 nm excitation and at 530 nm emission.


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Murine hindlimb ischemic model: C57BL/6J mice (J.Kim Laboratory) were used to create a model of unilateral hindlimb ischemia. The iliac and femoral artery and vein were tightly ligated under anesthesia. Incisions were sutured using Ethilon sutures (Johnson & Johnson). Liposome injection: Liposome solutions were intravenously injected into mice (n = 4) at a dose of 100 µl/mouse after inducing ischemia. The injected mice were imaged using the IVIS imaging system after 1 day and the measured fluorescence signals were analyzed to reveal targeting of fluorescent liposome in ischemic tissue using fluorescence signal in normal healthy limb. Statistics: All data are shown as mean ± standard deviation. All statistical comparisons were done by using Student t-test (two-tail comparisons). Significance levels are indicated as p

Sequential Targeted Delivery of Liposomes to Ischemic Tissues by

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