Endogenous Polysialic Acid Based Micelles for Calmodulin Antagonist

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Endogenous polysialic acid based micelles for calmodulin antagonist delivery against vascular dementia Xiao-Juan Wang, Yin-Ping Gao, Nan-Nan Lu, Wei-Shuo Li, Ji-Fang Xu, Xiao-Ying Ying, Gang Wu, Mei-Hua Liao, Chao Tan, Ling-Xiao Shao, Ying-Mei Lu, Chen Zhang, Kohji Fukunaga, Feng Han, and Yong-Zhong Du ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b13052 • Publication Date (Web): 17 Oct 2016 Downloaded from http://pubs.acs.org on October 18, 2016

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ACS Applied Materials & Interfaces

Endogenous polysialic acid based micelles for calmodulin antagonist delivery against vascular dementia Xiao-Juan Wanga1, Yin-Ping Gaob1, Nan-Nan Luc1, Wei-Shuo Lia, Ji-Fang Xua, Xiao-Ying Yinga, Gang Wuc, Mei-Hua Liaoc, Chao Tanc, Ling-Xiao Shaoc, Ying-Mei Lub, Chen Zhangd, Kohji Fukunagae, Feng Hanc*, Yong-Zhong Dua* a

Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University,

Hangzhou 310058, China; b

c

School of Medicine, Zhejiang University City College, Hangzhou 310058, China;

Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences,

Zhejiang University, Hangzhou 310058, China; d

Institute of Materia Medica, College of Pharmaceutical Sciences, Zhejiang

University, Hangzhou 310058, China; e

Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku

University, Sendai 980-8574, Japan; (1) These authors contributed equally to this work (2) Correspondence should be addressed to the following: Dr. Y. Z. Du (Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University), 866 Yu-Hang-Tang Road, Hangzhou, 310058, China. Tel: +86-571-8820-8435; Fax: +86-571-8820-8435. E-mail: [email protected] Dr. F. Han (Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University). Tel: +86-571-8820-8402; Fax: +86-571-8820-8402. E-mail: [email protected] 1

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ABSTRACT Clinical treatment for vascular dementia still remains a challenge mainly due to Blood-Brain Barrier (BBB). Here, a micelle based on polysialic acid (PSA) which is a hydrophilic and endogenous carbohydrate polymer, was designed to deliver calmodulin antagonist for therapy of vascular dementia. PSA was firstly chemically conjugated with octadecylamine (ODA), and the obtained PSA-ODA copolymer could self-assemble into micelle in aqueous solution with a 120.0 µg/mL critical micelle concentration. The calmodulin antagonist loaded PSA-ODA micelle, featuring sustained drug release behavior over a period of 72 h with 3.6 % (w/w) drug content and 107.0 ± 4.0 nm size was then fabricated. The PSA-ODA micelle could cross BBB mainly via active endocytosis by brain endothelial cells followed by transcytosis. In water maze test for spatial learning, calmodulin antagonist loaded PSA-ODA micelle significantly reduced escape latencies of right unilateral common carotid arteries occlusion (rUCCAO) mice with dosage significantly reduced versus free drug. The decrease of hippocampal phospho-CaMKII (Thr286/287) and phospho-synapsin I (Ser603) was partially restored in rUCCAO mice following calmodulin antagonist loaded PSA-ODA micelle treatment. Consistent with the restored CaMKII phosphorylation, the elevation of BrdU/NeuN double-positive cells in same context was also observed. Overall, the PSA-ODA micelle developed from the endogenous material might promote the development of therapeutic approaches for improving efficacy of brain-targeted drug delivery and have a great potential for vascular dementia treatment. 2


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Keywords: Polysialic acid, Polymeric micelle, Drug delivery system, Blood-Brain Barrier, Calmodulin antagonist, Vascular dementia

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Introduction With acceleration of aging process in human society, dementia has become a noteworthy factor which affects the health of people and causes a main burden on health care systems.1 Vascular dementia (VaD) is widely considered as the second most common cause of cognitive dysfunction following Alzheimer's disease.2-5 Unfortunately, there has been no approved drugs capable of attenuating or stopping the progression of VaD currently.1 Therefore, discovery of more efficient drugs for treatment of VaD is of great significance. We previously reported that a calmodulin antagonist, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1(4-imidazolylmethyl) -1H-indazole dihydrochloride 3.5 hydrate (DY-9760e), could reduce the infarct volume, diminish BBB breakdown and suppress the elevated signal intensities present in the cortical region of the ipsilateral hemisphere in T2-weighted magnetic resonance imaging (MRI) studies.6, 7 Moreover, DY-9760e was capable of reducing BBB breakdown in a brain ischemia model by inhibition of calpain activation, calcium/calmodulin-dependent nitric synthase activation and protein tyrosine nitration.6-8 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6dimethoxyindazole (DY-9836), a pharmacologically active metabolite of DY-9760e, does not interfere with metabolism of other drugs in liver,9 thus it seems more attractive than DY-9760e for clinical application with great potential for treatment of VaD. Nevertheless, low water-solubility and poor pharmakinetics of DY-9836 (DY) restrict its further application. Nano drug delivery systems (NDDS), featured with appropriate size and 4


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extended circulation time in blood in vivo can be used to effectively deliver the hydrophobic drug across the BBB.10, endocytosis and/or transcytosis.

12

11

Many NDDS fulfil the BBB penetrating by

Ligands/antibodies and thermal/mechanical force

were further utilized to enhance the BBB penetrating capacity of the NDDS, which were also used for drug molecule.

13-14

The materials composing these NDDSs are

usually exogenous including natural and synthetic polymeric materials. The usage of excess amount of exogenous carrier materials, even the alleged nonimmunogenic materials with good biodegradability, imposes safety issues inevitably.15 For example, accumulating evidences suggest that polyethylene glycol (PEG) is able to induce immune responses in humans as well as animals, particularly after repeated administration.16. Therefore, NDDS based upon endogenous and nonimmunogenic materials may be a strategy to avoid potential toxicity of nanomaterials. Endogenous materials feature lower immunogenicity, better biological degradability and lower potential toxicity compared with exogenous materials. Till now, NDDS based on endogenous materials, mostly focused on the application of albumin, has not been extensively studied.17, 18 Polysialic acid (PSA) is an endogenous carbohydrate polymer and mostly expressed together with neural cell adhesion molecule (NCAM).19-21 As a hydrophilic linear polymer, PSA plays a vital role in cell migration

22-24

volume leading to a weaker interaction between cells.

due to its large hydrated

25, 26

Furthermore, PSA is

competent to ameliorate the circulatory stability, extend circulation time by diminishing urinary excretion and hepatic uptake and improve therapeutic efficacy of 5



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existing therapeutics.27-29 Here, NDDS based on endogenous PSA was designed to improve the permeability to BBB and pharmacokinetics of DY-9836 in vivo. Firstly, PSA was conjugated with octadecylamine (ODA). The physicochemical properties of PSA-ODA such as self-assembly ability to form micelle in aqueous phase and the morphology and size of the formed micelle were then performed. After that, DY-9836 loaded PSA-ODA micelle (PSA-ODA/DY) was prepared. The characteristics of PSA-ODA/DY including size, drug content, encapsulation efficiency and drug release property in vitro were examined. The BBB penetrating capacity, pharmacokinetic property and therapeutic effects against VaD of the micelle delivery system were further investigated in detail.

Results and discussion Synthesis and structure confirmation of PSA-ODA copolymer The carboxyl group of PSA was activated by EDC and NHS, followed by conjugating with amino group of ODA to synthesize PSA-ODA copolymer.

27

ODA

was used as a hydrophobic chain for increasing the cellular uptake property and the membrane permeability of PSA. The reaction scheme was shown in Scheme 1. The structure confirmation of PSA-ODA copolymer was determined via 1H NMR spectra and the results were presented in Figure 1A. The characteristic peak of PSA, the acetyl (-NHCOCH3), was at approximately 2.0 ppm. The peak at ~0.9 ppm belonged to the terminal methyl (-CH3) in ODA. The 1H NMR spectra of PSA-ODA showed 6


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both peaks, indicating that ODA was grafted to PSA chains successfully. The degree of substitution (DS) of PSA-ODA copolymer was about 3.9%, calculated by comparing their characteristic peaks’ area.27 The DS here referred to the amount of ODA grafted to polysialic acid per 100 sialic acid monomer.

Scheme 1. Synthetic scheme of PSA-ODA

Fig 1. Characteristics of PSA-ODA copolymer, PSA-ODA micelle and PSA-ODA/DY 7



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micelle. (A) 1H NMR spectra of PSA, ODA and PSA-ODA. The important peaks were pointed out. (B) Variations of fluorescence strength ratios for I1/I3 as the change of logarithm of PSA-ODA concentration. (C) TEM images and size distribution of PSA-ODA micelle (left) and PSA-ODA/DY micelle (right) (D) In vitro drug release behaviors of free DY and PSA-ODA/DY micelle. Data are presented as mean ± S.E.M., (n=3).

Preparation and characteristics of PSA-ODA polymeric micelle and PSA-ODA/DY micelle Critical micelle concentration (CMC) represents the self-assembly ability of amphiphilic polymer to form micelle in aqueous phase.30 The CMC of PSA-ODA copolymer was determined by fluorescence spectrophotometry using pyrene as a probe. As shown in Figure 1B, the values of the intensity ratios of I1/I3 (I1 = 374 nm, I3 =385 nm) stayed unchanged around at 1.8 when concentration of the copolymer was low, while as the concentration increased, the values of I1/I3 reduced quickly. The concentration at the turning point was determined as CMC. The CMC value of PSA-ODA was determined as 120.0 µg/mL. Compared with low molecular weight surfactants with much higher CMC values, PSA-ODA copolymer exhibited good capacity to form micelles.31 Then DY was encapsulated into PSA-ODA micelle. When drug feeding amount was 4.0%, Drug Loading (DL%) and Encapsulation Efficiency (EE%) were 3.6% and 90.0%, respectively. It was reported that drug delivery systems with hydrophilic surface had capacity of avoiding clearance by macrophages leading to a long circulation time when particle size was smaller than 200 nm.32 PSA-ODA micelle’s morphology and size were 8


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evaluated by transmission electron microscopy (TEM) and dynamic light scattering (DLS). TEM images showed a uniform distribution of both PSA-ODA micelle and PSA-ODA/DY micelle with size at around 50 and 30 nm, respectively (Figure 1C). The sizes were also investigated by DLS. The results in Figure 1C showed that the number average size of PSA-ODA micelle was 145.7 ± 2.1 nm, while the size decreased to 107.0 ± 4.0 nm after DY loading. The reduced size was mainly attributed to the enhanced hydrophobic interaction between ODA segments and hydrophobic DY. The bigger size obtained by DLS than that obtained by TEM might be due to the shrinkage of the shell during drying procedure of TEM samples preparation. PSA with carboxyl groups has negative charge. The Zeta potential was determined as -33.4 ± 2.56 mV. After modification with ODA, the zeta potential of PSA-ODA micelle decreased to -21.2 ± 1.01 mV. The reason was that partial carboxyl groups were replaced by ODA chains. The zeta potential of PSA-ODA/DY micelle was about -20.8 ± 1.40 mV, similar as that of PSA-ODA micelle. The relatively high zeta potential was a significant factor for increasing the stability of micelles in aqueous medium through repulsion interaction. The in vitro drug release behavior of PSA-ODA/DY micelle was studied by dialysis method. As presented in Figure 1D, the DY release behavior from the micelle showed a typical biphasic pattern composed of an initial fast release in the first 12 h and sustained release for a prolonged time up to 72 h. Cumulative DY release percentage of drug loaded micelle was about 69.1% in 72 h, while diffusion of free drug through membrane of dialysis bag only delayed to 12 h. 9



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In vitro cytotoxicity assay of PSA-ODA micelle Toxicity of blank micelle, which was a vital factor for its further application, was tested by methyl thiazolyl tetrazolium (MTT) method against bEnd.3 cells. The cells were mice-derived immortalized brain vascular endothelial cells with biological properties similar to brain endothelial cells. The results in Figure 2A displayed that cell viability was still above 65% even when concentration of PSA-ODA micelle reached up to 1000 µg/mL, which manifested a relatively high biocompatibility and low toxicity.

Fig 2. In vitro study of PSA-ODA micelle on bEnd.3 cells. (A) Cell viability after 48 h incubation with PSA-ODA at various concentrations (n=3). (B) Confocal microscopy images of cellular uptake of RITC-labeled PSA-ODA micelle for 2, 4, 6, 8, 12, 24 h, respectively. All pictures shown were merged images, which included the nuclei (blue) and micelle (red). Scale bars represent 20 µm in all images. (C) Fluorescence intensity inside cells measured by flow cytometry when cells pre-treated with different inhibitors and incubated with RITC-labeled PSA-ODA micelle. (D) Papp of DY and PSA-ODA/DY after incubation with cell monolayer as increase of transport time. Data are presented as mean ± S.E.M. (n=3). *P

Endogenous Polysialic Acid Based Micelles for Calmodulin Antagonist

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