Advanced Nanocarriers Based on Heparin and Its Derivatives for

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Advanced nanocarriers based on heparin and its derivatives for cancer management Xiaoye Yang, Hongliang Du, Jiyong Liu, and Guangxi Zhai Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm501532e • Publication Date (Web): 17 Dec 2014 Downloaded from http://pubs.acs.org on December 20, 2014

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Advanced nanocarriers based on heparin and its

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derivatives for cancer management

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Xiaoye Yang1, Hongliang Du1, Jiyong Liu2*, Guangxi Zhai 1*

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1. Department of Pharmaceutics, College of Pharmacy, Shandong University, Jinan 250012,

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China；

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2. Department of Pharmacy, Changhai Hospital, Second Military Medical University, Shanghai

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200433, China

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Keywords: heparin, heparin derivatization, nanocarriers, cancer management, photodynamic therapy, gene therapy.

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ABSTRACT

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To obtain a satisfying anti-cancer effect, rationally designed nanocarriers are intensively studied.

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In this field, heparin and its derivatives have been widely attempted recently as potential

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component of nanocarriers due to their unique biological and physiochemical features, especially

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the anti-cancer activity. This review focuses on state-of-the-art nanocarriers with heparin/heparin

ACS Paragon Plus Environment

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derivatives as backbone or coating material. At the beginning, the unique advantages of heparin

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used in cancer nanotechnology are discussed. After that, different strategies of heparin chemical

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modification are reviewed, laying the foundation of developing various nanocarriers. Then a

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systematic summary of diverse nanoparticles with heparin as component is exhibited, involving

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heparin-drug

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nanoparticles and heparin-coated organic and inorganic nanoparticles. The application of these

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nanoparticles in various novel cancer therapy (containing targeted therapy, magnetic therapy,

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photodynamic therapy and gene therapy) will be highlighted. Finally, future challenges and

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opportunities of heparin-based biomaterials in cancer nanotechnology are discussed.

conjugate,

polymeric

nanoparticles,

nanogels,

polyelectrolyte

complex

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1. INTRODUCTION

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As a major cause of mortality, cancer remains a global public health concern. To date, the most

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common treatment of cancer has been chemotherapy, the therapeutic effect of which is far from

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optimal due largely to the nonspecific toxicity of chemotherapeutics. That is why the idea of

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cancer nanotechnology is put forward, which provides a unique approach against cancer by

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applying nanotechnology in cancer management.1,

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designed nanofomulations such as Abraxane® and DOXIL® have been approved by FDA for

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cancer therapeutics and received considerable attention. Compared with conventional

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formulations, nanocarriers possess many advantages in delivering bioactive agents either

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encapsulated inside the core or absorbed onto the surface: (1) Utilizing both passive and active

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targeting strategies, nanocarriers may guarantee the safety and efficacy of cancer treatment by

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changing the distribution of loaded drugs in vivo (i.e., increasing amount of drugs in tumor sites

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Guided by nanotechnology, rationally


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while minimizing the accumulation in normal tissues); (2) In light of the biodegradability, pH,

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ion or temperature sensibility of materials, nanovehicles can be functionalized to release drugs in

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a controlled manner;3 (3) Nanovehicles can load multiple agents based on a rational design,

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thereby realizing combinatorial therapy of cancer;4 etc.

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Various synthetic and natural materials have been applied in constructing nanocarriers for

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cancer management. Superior to some synthetic materials, natural resourced polysaccharides

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represented by chitosan, hyaluronic acid, dextran and heparin are often described as non-

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immunogenic and non-toxic, driving the desire to employ them to nanoformulations.

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Unfractionated heparin (UFH), a highly heterogeneous sulfated glycosaminoglycan mainly

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isolated from porcine intestinal mucosa, is composed of repeating disaccharide units (~20

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disaccharide units per chain, responding to an average molecular weight of ~12,000 Da),

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primarily glucosamine and uronic acid residues with varying degrees of sulfation and N-

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acetylation.5,

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charge, mediating its electrostatic interactions with a myriad of proteins such as growth factors

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and chemokines.5 The application of UFH as a blood anticoagulant in clinical has a long history

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since 1937 based on its ability to bind with antithrombin.7,

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interaction of heparin and growth factors, it has been employed to not only stabilize these

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proteins but also increase their affinity to cell receptors for tissue regeneration 5. However, the

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problems thwart the clinical of UFH may lie in the side effects such as hemorrhagic

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complications and thrombocytopenia. To remedy this problem, low-molecular-weight-heparin

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(LMWH), which can be degraded from UFH with a better defined chemical composition, has

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been investigated as an alternative.9, 10 Compared to UFH, LMWH has been reported to exhibit

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similar or even better anticoagulant, anti-inflammatory, anti-angiogenesis, antitumor and anti-

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The abundant sulfate and carboxylate groups endow heparin a high negative

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Additionally, owing to the


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metastasis activities

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LWMH, they are collectively called heparin in this article.

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. Based on the similar biological and chemical properties of UFH and

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More recently, there has been a tendency to incorporate heparin into nanoformulations for

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cancer therapy in view of its unique biological and physicochemical properties, such as non-

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toxicity, anti-cancer activity12-17, protein-delivering capacity, and potential targeting ability18-19,

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which will be further discussed in the next part. Hence, various advanced heparin-based

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nanocarriers, such as nanogels, self-assemblies, polyelectrolyte complex nanoparticles and

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heparin coated nanoparticles, have been devised.

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Recently, several reviews about heparin and its derivatives as nanomaterials have been

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pubilished.5,

6, 20

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management were more or less involved in these articles, the authors put more emphasis on

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tissue engineering application. Hence, to better understand the role played by heparin and its

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derivatives in well-designed nanosystems for cancer therapy, a systematic review of this aspect is

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in need. This article systematically reviews various nanoparticles with heparin and its derivatives

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as both backbone and coating materials. Additionally, up-to-date applications of these heparin-

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based nanocarriers in novel cancer therapy (e.g., targeted therapy, magnetic therapy,

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photodynamic therapy and gene therapy) will be highlighted. Moreover, the challenges and

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future perspectives of heparin-based nanocarriers in the field of cancer nanotechnology are also

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discussed.

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2. ADVANTAGES OF USING HEPARIN IN CANCER NANOTECHNOLEGY

Although nanovehicles composed of heparin and its derivatives for cancer

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When incorporating heparin into nanoformulations for cancer management, there are basically

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four categories of advantages as following. (1) As an endogenous polysaccharide in body,


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heparin exhibits non-toxicity. (2) Heparin shows anti-cancer activity in the process of

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angiogenesis and metastasis, which differentiates heparin from many other polysaccharides.12-17

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It has been demonstrated in numerous studies that heparin can reduce metastasis of carcinoma

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cells in animal models of experimental metastasis and benefit human malignant desease.21-24 The

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underlying mechanism, which is not clear entirely, may be releated with the ability of heparin to

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interact with multiple molecules, such as heparinase, P- and L-selectins and growth factors.13

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Heparinase is an endoglycosidase site-selectively cleaving heparan sulphate chains which are

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expressed as proteoglycan componet on the surface of most cells.21 The overexpressed

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heparinase in tumors promotes the degradation of exreacellular matrix, thus facilitating the

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extravasation of tumor cells, which is critical in the metastatic cascade. Taking advantage of the

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interaction between heparin and heparinase, the activity of heparinase can be inhibited, resulting

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in an attenuated metastasis. Similarly, heparin also contributes to inhibiting P- and L-selectins,

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which have been proved to promote tumor metastasis by mediating cell-cell interactions.13,25 In

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addition, heparin was reported to inhibit the angiogenesis by binding with growth factors such as

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FGF and VEGF, which can induce the proliferation of endothelial cells.17 As an instance, a

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heparin-derived oligosaccharide phosphomannopentaose (PI-88), which is now in Phase Ⅲ

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clinical trials for cancer management, has been demonstrated to inhibit angiogenesis, metastasis

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and tumor growth.

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transcription factors to induce apoptotic cell death.28,29 It is also remarkable that when used as a

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nanocarrier, the anti-cancer activity of heparin can be preseved, which will be specifically

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demonstrated later. (3) Based on the good affinity to various protein heparin may help to develop

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a platform for delivery of protein drugs as coating material or backbone of nanocarriers. (4)

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Since heparin exhibited high-affinity binding to tumors, it can also be introduced as a targeting

26,27

More recent research has suggested that heparin can also interact with


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functional moiety.18, 19 The basis of using heparin as a targeting moiety can be summarized in

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several aspects. First, a network of clotted plasma proteins (fibrinogen-derived product) in the

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tumor stroma and the vessel walls, which is absent in normal tissues, is expected to interact with

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heparin, thus causing the enhanced targeting ability19. In addition, heparin shows targeting ability

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to the abundant dividing vascular endothelial cells in tumors based on its high-affinity binding

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and internalization to these cells.18 Besides, due to presence of over-expressed heparin-binding

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angiogenic growth factors in tumors, heparin might be recruited to tumor sites.18,30

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Taking into account the considerations above, various advanced heparin-based nanocarriers, such

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as nanogels, self-assemblies, polyelectrolyte complex nanoparticles and heparin coated

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nanoparticles, have been devised for cancer management.

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3. HEPARIN DERIVATIVES SYNTHESIZED BY COVALENT MODIFICATION

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In most cases, heparin cannot be applied directly in constructing nanoplatforms for drug

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delivery, which has driven the desire to synthesize various heparin derivatives functionalized

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with unique features. Various derivable groups of heparin, such as carboxylic, amino and

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hydroxy groups, provide the possibility of derivatization via a chemical method, so that a

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diversity of heparin derivatives with different functional groups, hydrophobic chains and

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chemical probes have been synthesized (shown in Figure 1).31

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Among various chemical modification methods of heparin, derivation of the carboxyl groups,

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involving esterification and amidation reaction, is overwhelmingly used. As shown in Figure 1,

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multifarious amide derivatives of heparin have been reported. They were often synthesized via

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direct linkage between carboxylic groups of heparin and amino groups of other compounds,


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forming amide bonds. For example, aminated deoxycholic acid (DOCA) was covalently grafted

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to heparin chains in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide

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hydrochloride (EDAC), producing hydrophobically modified heparin amphiphiles, which could

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self-assembled into nanoparticles in contact with water, serving as a carrier of hydrophobic drugs

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or quantum dots.32, 33 By adopting similar methods, heparin could be conjugated with biocin 34, 35,

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fluorescent amines,

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acid ,37 tyramine,38 polyethyleneimine (PEI) 39, 40 and so on.

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aminated folic acid (FA),36 aminated retinoic acid,26 aminated lithocholic

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To make heparin derivatives more versatile platforms of drug delivery, various reactive

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functional groups have been introduced to heparin in its pendant groups (shown in Figure 1).

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Taking advantage of the functional groups, heparin derivatives were able to react with end

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groups of various polymers or crosslink into nanogels. For instance, to render heparin with

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additional amine groups, diamine such as adipic acid dihydrazide (ADH), cystamine have been

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mostly reported to be conjugated on heparin chains via amidation reaction.41, 42 It is noteworthy

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that if the molar ratio of the diamine to carboxyl group of heparin is much higher than 1/1, the

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diamine may be grafted as a pendant group, the free amino group of which can be further linked

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to various hydrophobic segments to form amphiphiles. On the contrary, if the ratio is lower than

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1/1, the diamine will act as a crosslinker to form hydrogel. Additionally, azido groups were also

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introduced to heparin chain via amidation reaction of heparin with N-(tert-Butoxycarbonyl)-N-

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(4-azido butyl)-ethylenediamine.43 Moreover, end-thiol modified heparin could be prepared with

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a similar method developed for hyaluronic acid modification.44 Simply put, via amidation

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reaction, cysteamine was grafted to the carboxyl of heparin, so that free thiol groups were

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introduced in its pendant groups. Excess amount of dithiotreitol (DTT) was always used to avoid

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oxidization of thiol groups. Alternatively, cystamine could be conjugated to heparin instead of


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cysteamine and the DTT afterward added would reduce the disulfide groups to produce end-thiol

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groups.45 Besides, thiol groups were also introduced to heparin chain via reductive amination

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reaction.46 These heparin derivatives with end-thiol groups have been applied in preparation of

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nanogels crosslinked by disulfide bonds and modification of gold nanoparticles.

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Besides carboxyl groups of heparin, derivatization of hydroxyl and amine groups has been

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studied in previous studies (shown in Figure 1). The modification of hydroxyl groups was always

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driven by the purpose of introducing more carboxyl groups onto heparin chains, the

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derivatization of which was vital to heparin modification.47-50 As an example, Wang and co-

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workers synthesized succinylated heparin by reacting succinic anhydride with the hydroxyl

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groups of heparin under the classical O-acylation reaction conditions. 47 The resulting heparin

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derivative with more reactive carboxyl groups enabled more conjugation of PTX onto heparin

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backbone via spacers. In addition, it was also claimed that the anticoagulant function of

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succinylated heparin was inactivated,49 presumably as a result of conformational change. To

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synthesize heparin-Pluronic conjugates, Choi and co-workers coupled carboxylated Pluronic

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F127

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(dimethylamino)propyl] carbodiimide (EDC) and N-hydroxysuccinimate (NHS) as coupling

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agents.51,

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formation between the primary amine group of heparin and the terminal hydroxyl group of F127,

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which was previously activated by para-nitrophenyl chloroformate (pNPC).18

to

the

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amino

group

or

hydroxyl

group

of

heparin

using

1-ethyl-3-[3-

Alternatively, Chung et al. conjugated heparin to F127 via urethane linkage

20


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Figure 1. Various heparin derivatives prepared by chemical modification. Derivatives in the red

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circle are obtained via derivation of the carboxyl groups, while those in the blue circle are

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prepared via modification of amine or hydroxyl groups. a. Heparin amide derivatives are

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synthesized via amidation reaction; b. Various reactive groups are introduced to heparin via

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amidation reaction; c. Heparin derivatives are synthesized by modification of amine or hydroxyl

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groups.

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4. NANOCARRIERS WITH HEPARIN AND ITS DERIVATIVES AS BACKBONE

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During the past years, different methodologies have been developed to prepare nanoparticles

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in which heparin/ heparin derivative chains take a leading role in the particles’ formation. As

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presented in Figure 2, heparin-drug conjugate (a) and heparin-based polymeric conjugate (b)

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nanoparticles are characterized with a hydrophilic shell (heparin) and a hydrophobic core

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(insoluble drug or hydrophobic segment, respectively). Formation of these nanoparticles usually


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relies on the self-assembling behavior of the amphiphilic heparin derivatives, mainly driven by

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the hydrophobic force. The differences between these two kind of nanocarriers mainly lie in

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different manners to introduce drug, either physical encapsulation or chemical conjugation.

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Obviously, both kinds of nanocarriers show great advantages as ideal depots for insoluble drugs.

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Besides, cross-linked by physical or chemical interaction, hydrophilic nanogels using heparin as

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backbone can be obtained with three-dimensional porous network not only serving as a reservoir

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for drugs, especially hydrophilic drugs, but also preventing the cargos from environmental

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degradation (Figure 2 c). Furthermore, taking advantage of the electrostatic attraction between

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heparin (polyanion) and another polycation, polyelectrolyte complex nanoparticles (PCNs) can

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be simply manufactured in a mild condition (Figure2 d), making them especially suitable for

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delivery of biological drugs like protein.

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Figure 2. Various nanocarriers with heparin and its derivatives as backbone. (a) Heparin-drug

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conjugate nanoparticle with hydrophobic drug as the core; (b) heparin-based polymeric

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conjugate nanoparticle with drugs encapsulated inside the core; (c) Chemically/physically

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crosslinked nanogel with drug loaded in the three-dimensional network; (d) Polyelectrolyte

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complex nanoparticles (PCN).

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4.1 Heparin-drug conjugates

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Great interest has been generated in developing self-assemblies of amphiphilic polymers as

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drug delivery systems mainly due to the absence of organic solvents or surfactants during

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preparation process.53 Heparin, as a highly hydrophilic polysaccharide with plentiful modifiable

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groups along the chain, provides a positive outlook as hydrophilic backbone of such


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amphiphiles.5 After grafting hydrophobic chains to heparin, amphiphilic copolymers are

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synthesized, which may self-assemble into micelles or micelle-like nanoparticles based on the

3

intra- or intermolecular associations between hydrophobic moieties upon contact with aqueous

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solution. According to different ways of introducing poorly water-soluble drugs to the

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nanosystem, the amphiphiles are classified into two categories in this article (shown in Figure 3).

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(1) Hydrophobic drugs are chemically linked to heparin chains, producing heparin-drug

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conjugates. (2) After the formation of polymeric conjugates, drugs are added and encapsulated

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inside hydrophobic cores of the self-assemblies. The former approach will be herein discussed

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while the latter is involved in the next section.

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By grafting water-insoluble bioactive agents to hydrophilic macromolecular polymers directly

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or via biological response linkers, macromolecular conjugated prodrugs can be obtained,54 which

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may self-assemble into nanoparticles in water with drug as the core. The advantages of such

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prodrugs over their precursors may lie in: 1) increased solubility of the hydrophobic drugs; 2)

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improved bioavailability and a prolonged plasma half-life of drugs; 3) protection of drugs from

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degradation; 4) altered distribution in vivo due to enhanced permeability and retention (EPR)

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effect; 5) realization of a triggered release in response to pH or enzyme.55 Table 1 summarized a

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series of heparin-drug conjugates and their anti-cancer activity in vitro and in vivo.

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A classical mode of heparin-drug conjugates is composed of a heparin scaffold as the carrier,

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hydrophobic drug conjugated to heparin and a biological response linker.56 When used for cancer

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treatment, such conjugates mainly benefit from the EPR effect due to the appropriate size. As

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Figure 4 shows, the leaky and discontinuous blood vessels with fenestrations (the size may range

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from 200 to 2000 nm depending on tumor types), which are resulted from the rapid angiogenesis

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in tumor tissues, allow the accumulation of nanoparticles in tumor sites.57 For normal vessels,


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however, this extravasation is limited. However, the therapeutic effect of such binary heparin-

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drug conjugates remains far from optimal in view of the limited specificity. Hence, ternary

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heparin-drug-targeting molecule conjugates were developed by grafting targeting moieties to

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heparin backbone, which would exist on particles’ surface after self-assembly (shown in Figure

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3A).48,

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synthesized ternary heparin-folate-PTX conjugates and encapsulated additional PTX into the

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obtained nanoparticles, using binary PTX-loaded heparin-PTX conjugate nanoparticles as the

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control.50 It was expected that the conjugation of FA could facilitate the targeting of

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nanoparticles to folate acid receptor-positive tumor cells. The results of in vivo distribution

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study, however, demonstrated no significant difference between the accumulation of heparin-

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folate-PTX nanoparticles and heparin-PTX nanoparticles in xenograft tumors 48 h after

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injection.50 To illustrate this phenomenon, the authors went on to prove whether the

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nanoparticles accumulated simply outside or actually inside the tumor cells using fluorescence

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microscopy.50 The results demonstrated that most of the fluorescent dye-labeled heparin-PTX

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nanoparticles simply existed in the extracellular space of tumor tissue, maybe due to the EPR

16

effect. On the contrary, heparin-folate-PTX nanoparticles predominantly accumulated within

17

tumor cells via receptor-mediated endocytosis, which might guarantee the anti-cancer efficiency.

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Considering the anti-angiogenic activity of the heparin, Wang et al. hypothesized that besides

19

the cytotoxicity induced by PTX, heparin backbone may also contribute to the enhanced

20

antitumor activity by inhibiting the angiogenisis.48 To confirm the hypothesis, a matrigel-based

21

capillary tube formation assay was performed to observe the formation of tubes, indicating that

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both heparin-PTX and heparin-folate-PTX nanoparticles could attenuate the tube formation.48

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However, no significant difference was observed between the PTX-treated group and the control

50

As a good illustration, to exactly exert biological functions of PTX, Wang et al.


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group, confirming the suppressed tube formation resulted from the presence of heparin

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backbone.48 Besides, the anti-angiogenic activity induced by heparin has been proved in some

3

other articles, demonstrating the preserved anti-cancer activity of heparin when utilized as a

4

nanocarrier, which can be regarded as a strong point of heparin-based nanoparticles for cancer

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management. 32,43

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As shown in Table 1, only a few drugs could be directly conjugated to heparin, while in most

7

cases a series of linkers were used. For some conjugates, linkers with simple and short chemical

8

structure (such as ethylenediamine) were used with the only purpose of connecting the drug and

9

heparin. For other conjugates, the linkers also contributed to developing a stimuli-responsive

10

drug delivery system due to the formation of pH-labile hydrazone linkers.

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It is well known that due to the excessive acidic metabolites including CO2 and lactic acid in

12

tumor, the pH value in tumor sites may drop to 6.5 or less. After endocytosis, anticancer

13

conjugates end up inside endosomes which are converted progressively to lysosomes, whose pH

14

value was between 4.5 to 5.58 Based on this, if a nanosystem undergoes a rapid destruction in

15

acidic environment, while keeping stable in normal tissues (pH 7.4), it may be promising as a

16

smart, controlled release system for cancer treatment. Therefore, great interest has been

17

generated in developing heparin-drug conjugates encompassing pH-labile hydrazone linkers.41, 43

18

Li et al. reported a macromolecular prodrug strategy for the combinatorial delivery of

19

dexamethasone (DEX) and doxorubicin (DOX) with heparin derivative as a vehicle.41 They

20

firstly reacted DEX with heparin-ADH in DMSO/H2O solution, synthesizing amphiphilic

21

heparin-DEX macromolecular prodrugs with pH-sensitive hydrazone linkages, which were

22

formed by the reaction between hydrazide and carbonyl groups. Then DOX was then physically

23

loaded into the self-assembled micelles of heparin-DEX conjugates, realizing combinatorial


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delivery of the two agents.41 The results of in vitro release showed that during the first three

2

days, for example, the cumulative release amount of both DEX and DOX at pH 5.0 was

3

significantly larger than that in the case of pH 6.0 and pH 7.4, which confirmed the pH-sensitive

4

release behavior of the designed system.41

5

Besides, heparin-drug conjugates have been utilized for an efficient photodynamic therapy of

6

cancer, which will be presented in the next section along with the other kind of amphiphilic

7

conjugates.

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9 10 11

Figure 3. Self-assembly of two kinds of heparin-based amphiphilic conjugates. A. Heparin-drug-

12

targeting molecule conjugate self-assemble into nanoparticles in water with drug as the core (not

13

physical encapsulation) and targeting molecules on the shell. B. (a) Heparin-hydrophobic

14

segment-targeting molecule conjugate self-assembled into nanoparticles in water with targeting


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molecules on the shell. (b) After the formation of heparin-based polymeric conjugates, drug is

2

added and encapsulated in the hydrophobic core via a dialysis method.

3 4 5

Figure 4. The mechanism of passive (A) and active targeting (B). (a) Minimum extravasation of

6

nanoparticles to normal tissue; (b) Increased drug accumulating in tumor sites due to the EPR

7

effect; (c) Uptake of nanoparticles by fluid-phase pinocytosis; (d) Enhanced uptake of

8

nanoparticles mediated by receptors; (e) Release of drug in cytoplasm.

9


16

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1 2

Biomacromolecules

4.2 Heparin-based polymeric conjugates as carriers of drugs By grafting hydrophobic chains such as Poly(β-benzyl-L-aspartate) (PBCA)

59,

60

,

3

polycaprolactone (PCL) 61 and DOCA 32, 62, 63 to heparin, heparin-based polymeric conjugates are

4

synthesized, which may self-assemble into nanoparitcles with core-shell structure in aqueous

5

solution. The hydrophilic shells of these aggregations, as a physical shield preventing

6

nanoparticle-nanoparticle or nanoparticle-protein (opsonin) interactions, ensure the stability and

7

prolong the circulation time of the particles.64 And the hydrophobic cores can load insoluble

8

drugs via hydrophobic interactions which are thermodynamically driven.64 As to selecting

9

suitable hydrophobic inner core, non-immunogenicity and non-toxicity are always the main

10

prerequisites. In addition, the drug-loading ability of the core should also be considered, which is

11

largely relied on the compatibility between the core and drug.65 The compatibility can be

12

estimated by comparing the polarity of the drug molecules and the hydrophobic segment, similar

13

to the ‘like dissolves like’ rule to some extent.64 Additional targeting molecules, which will

14

present on aggregations’ surface, can also be conjugated to heparin backbone to realize specific

15

delivery of agents to tumor sites (shown in Figure 3B). Also, a commonly used dialysis method

16

to load water-insoluble drugs in the hydrophobic core of aggregations is depicted in Figure 3B.

17

Over the years, various heparin-based polymeric conjugate nanoparticles (such as PBCA59

, PCL-heparin61 and DOCA-heparin62 conjugates nanoparticles) have been

18

heparin-FA

19

frequently fabricated to specifically deliver chemotherapeutic drug to tumor sites via both

20

passive and active targeting, similar to the heparin-drug conjugate nanoparticles discussed above.

21

Lately, heparin-based self-assembled nanoparticles have been used as a platform for

22

photodynamic therapy (PDT) of cancer. PDT is a clinically available therapeutic option for


17

Biomacromolecules

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Page 18 of 49

1

cancer treatment whereby cancer cells can be destroyed by combination of light and special

2

drugs, photosensitizers (PSs). The PSs comprised in PDT, inactive without irradiation, can be

3

activated by light of specific wavelength, generating reactive oxygen species, which can kill

4

tumor cells via various mechanisms.2 Although the damage of normal tissues can be limited by

5

selective illumination to tumor sites, the clinical use of PDT is still limited by the poor tumor

6

selectivity of photosensitizers; therefore, it is required to combine nanocarriers with spatial-

7

controlled light technique, utilizing the passive/active targeting ability of nanoparticles.2

8

Additionally, taking advantage of nanovehicles, the issue associated with the poor water-

9

solubility of PSs is also resolved. As an example, pheophorbide a (PhA), a second generation

10

PSs, has been physically encapsulated in heparin-PBCA self-assemblies for PDT of cancer.60 To

11

enhance the targeting efficacy of a PhA carrier, Tran and co-workers prepared ternary

12

amphiphilic heparin-folate-retinoic acid bioconjugates by linking hydrophobic retinoic acid and a

13

targeting ligand, FA, to heparin backbone.36 The targeting effect of heparin-folate-retinoic acid

14

nanoparticles was proved by the results of cellular uptake study in Hela cells (folate receptor-

15

possitive) and HT-29 cells (folate receptor-negative), in comparison with non-targeted heparin-

16

retinoic acid nanoparticles.36 As mentioned earlier, an efficient PDT was also anticipated by

17

conjugating photosensitizer to heparin chain, forming a heparin-drug conjugate. For example, Li

18

et al. developed a promising heparin-drug conjugate system by chemical conjugation of heparin

19

with PhA and a targeting ligand, FA.66 It was worth noting that no matter how PhA was

20

introduced to the nanoparticles, through chemical conjugation or physical encapsulation, the self-

21

photoquenching effect of the nanoparticles in PBS existed.36, 66 This effect might result from the

22

accumulation of PhA at inner core of the nanoparticles, leading to the fluorescence resonance

23

energy transfer (FRET) effect via hydrophobic and π-π stacking interactions, thus showing self-


18

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Biomacromolecules

1

quenching property.36, 66 Actually, this property could protect normal tissues by supressing the

2

phototoxicity until they arrived at target sites. Taking the heparin-PhA-FA conjugates just

3

mentioned as an example, after internalization of nanoparticles in tumor cells, a diversity of

4

biological activities might trigger the decomposition of the heparin skeleton and the cleavage of

5

amide bond between PhA and heparin— these interactions might induce dissociation of the

6

aggregated PhA and recover the phototoxicity.66

7 8 Drug

Table 1 Heparin-drug conjugates for cancer treatment Targeting molecules

Linker

Type of bond

Cell studies

In vivo studies (cell lines, route of administration)

Refs

All-trans-retinoid acid (ATRA); additional physical encapsulation of PTX

None

Ethylenediamine

Amide with ATRA, amide with heparin

Enhanced PTX-induced cytotoxicity to HepG2 cells than PTX solution

Extended circulation of PTX and ATRA comparing with PTX plus ATRA solution (i.v.)

67, 68

ATRA; additional physical encapsulation of PhA

FA

Ethylenediamine

Amide with ATRA, amide with heparin

Higher cellular uptake by HeLa cells；targeted anticancer effect of heparinATRA-folate conjugation in HeLa cells

None

36

PTX

None

Single amino acid (Val, Leu, or Phe)

Ester with PTX and amide with succinylated heparin

Better in inhibition of MCF-7 cells than free paclitaxel

Similar ovarian tumor growth inhibition comparing with PTX and no obvious body weight loss for Leu-linked conjugate (human ovarian serous cystocarcinoma cells, SKOV3; i.v.)

47

PTX; physical encapsulation of additional PTX

FA

Direct conjugation

Ester between PTX and succinylated heparin; amide with FA, amide with heparin

Superior to free PTX and PTX-heparin conjugate without FA in inhibiting proliferation of human epidermal carcinoma cell lines , KB-8-5cells

Markedly retarding tumor growth in a (KB-8-5 cells, i.v.)

48

PTX

None

Carbonate, ethylenediamine

Amide heparin, with PTX

Higher toxicity against KB cells than free PTX and enhanced cellular uptake compared with heparin

None

69

with ester


19

Biomacromolecules

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Page 20 of 49

DEX; additional physical encapsulation of DOX

None

ADH

Amide with heparin, hydrazone linkage with dexamethasone

Successfully internalized by human naso-pharyngeal carcinoma CNE1 cells and obvious antitumor activity

None

41

Docetaxel (DTX); additional conjugation of taurocholic acid onto heparin chain to enhance oral absorption

None

Ethylenediamine

Ester with DTX, amide with heparin

Effective anti-tumor activity in human breast cancer MDA-MB231 cells and epidermal carcinoma KB cells

Six-fold higher concentration of the conjugate than heparin alone; significant tumor growth inhibition activity (MDA-MB231 and KB cells, p.o.)

70

PhA

FA

Ethylenediamine

Amide with heparin, amide with PhA

Marked phototoxicity HeLa cells

None

66

on

1 2

4.3 Nanogels

3

Nanogels refer to cross-linked and swellable hydrophilic polymer nanoparticles with three-

4

dimensional network structure. They have become versatile tools in delivering a variety of drugs

5

because of their advantageous properties, which can be summarized as following. 1) Their

6

nanoscale size (50-200 nm) and high stability are favorable for intravenous and intracellular drug

7

delivery.45 2) The porous cross-linked networks of nanogels can not only serve an ideal reservoir

8

for drugs but also protect the cargos from environmental degradation.71 3) Their nanoscaled

9

dimensions ensure rapid response to environmental stimuli when nanogels are designed for

10

triggered drug delivery.72

11

Similar to their macrogel counterparts, nanogels can also be classified into physical and

12

chemical cross-linked nanogels based on different formation mechanisms.73 Heparin has been

13

used to construct both kinds of the nanogels.


20

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Biomacromolecules

1

Physical cross-linked nanogels are formed via non-covalent attractive forces, such as

2

hydrophibic forces, ionic interactions and hydrogen bonding.71,74,75 Compared with some other

3

nanocarriers, the mild preparation condition and simple synthetic process make them promising

4

vehicles for biological drugs (e.g., protein, nucleic acid) delivery. As a case in point, Choi and

5

coworkers reported heparin-Pluronic F127 nanogels crosslinked by hydrophobic force to deliver

6

a hydrophilic protein model, Ribonuclease A (RNase A).51 Although as an amphiphilic tri-block

7

copolymer composed of hydrophilic PEO and hydrophobic PPO, F127 could self-assemble into

8

micelles which would physically cross-linked into gels via enhanced hydrophobic interactions as

9

the temperature increased, the low capacity to encapsulate biological drugs like protein limited

10

its further use. Fortunately, this problem could be solved by addition of heparin, as a result of it

11

high affinity to various positively charged biological drugs. The authors prepared RNase loaded

12

heparin-F127 nanogels via a direct dissolution method where the lyophilized powder of heparin-

13

F127 conjugate along with RNase were firstly dissolved in water at 4Ⅲ and the temperature was

14

then increased to 37Ⅲ, followed by 24 h of stirring. As the temperature increased, the nanogels

15

were obtained mainly due to the enhanced hydrophobic interaction between the PPO chains of

16

Pluronic. RNase was loaded based on its high electrostatic affinity to heparin, resulting in

17

decreased size of nanogels compared to empty ones (from 89 to ~29nm).

18

Chemically cross-linked nanogels are constructed with several cross-linking points throughout

19

the backbone of heparin chains due to the formation of chemical bonds such as disulfide and

20

amide bonds.39, 40, 46, 76

21

As an example of chemically cross-linked nanogels, heparin/PEI nanogels cross-linked by

22

amide bonds have been applied in gene therapy of cancer. Although heparin cannot be used to

23

condense and deliver gene alone due to its negative charge of heparin, several reports indicated


21

Biomacromolecules

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Page 22 of 49

1

that safe and efficient gene carriers could be developed by introducing heparin to the PEI/gene

2

complex.39, 76, 77 Cationic PEI has been extensively investigated as an efficient tool for gene

3

delivery and transfection since 1995 and its branched form with a molecular weight of 25,000 Da

4

(PEI 25K) has been widely used as a “gold standard” to estimate transfection efficiency of other

5

gene vectors.39, 78, 79 As the length of PEI chains increase, better transfection efficiency can be

6

obtained, however, along with undesirable increase of cytotoxicity of the non-biodegradable

7

PEI.39, 80 To overcome this inherent defect of PEI, short PEI chains were coupled into longer

8

ones by utilizing heparin, resulting in biodegradable heparin-PEI nanogels with high transfection

9

efficiency and low toxic.39, 76 The introduced heparin could also prolong circulation time of PEI

10

and hence improved its therapeutic potential. Gou et al. developed such nanogel of heparin and

11

PEI, which was formed by amidation crosslinking between carboxy groups of heparin and amine

12

groups of branched PEI.39 Under consistent stirring, the crosslinking reaction was performed by

13

dropping EDC/NHS activated heparin into PEI solution, wherein the activated carboxyl groups

14

of one heparin molecule might combine with the primary amine groups of several PEI

15

molecules, thus forming nanogel crosslinked by amide linkages. The cationic PEI/heparin

16

nanogels were used to translate anionic plasmid expressing vesicular stomatitis virus matrix

17

protein (pVSVMP) into C-26 colon carcinoma cells by electrostatic interaction with a

18

comparable transfection efficiency to that of PEI 25K. It was noteworthy that the cytotoxicity of

19

the nanogels was much lower than that of PEI 25K, even PEI 2K. It was also indicated that the

20

nanogels could inhibit the growth of colon carcinoma efficiently in vivo. Their findings

21

demonstrated the PEI/heparin nanogels were promising in constructing pVSVMP gene vectors

22

for colon cancer treatment with better therapeutic efficacy and lower toxicity. Next, their group

23

further studied the potential of this heparin/PEI nanogel in delivery other recombinant plasmid


22

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Biomacromolecules

1

carrying shRNA or filamin A interacting protein 1-like gene for ovarian cancer treatment and

2

reported promising results.76,

3

adenoviral vectors with the heparin/PEI nanogel to construct complexes and reported the

4

enhanced transfection efficiency in CT26 cells and targeting ability to lung.40

81

Adopting an analogous method, Wei et al. also modified

5

Another noticeable application of heparin-based nanogels was to develop redox-responsive

6

nanosystems to realize controlled intracellular release of drugs. Such systems usually

7

encompassed disulfide bonds, which were stable in the mild oxidizing extracellular environment

8

and prone to being cleaved to thiol groups in response to the reductive milieu in intracellular

9

region, resulting in a burst release of encapsulated agents in cytosol. The oxidation-

10

reduction property depends on the expression levels of reducing substance, especially glutathione

11

(GSH), which is ~2-20 µM in cytosol while ~2-10 mM in plasma.82-84 Importantly, the

12

concentration of GSH in tumor tissues is several times higher than that of normal tissues, making

13

redox-sensitive nanosystem ideal for cancer management.82, 85 Generally, two methods have been

14

adopted to introduce disulfide bonds to heparin-based nanogels. Figure 4 shows two cases of

15

disulfide bond-containing nanogels which can represent these two methods.

16

In the first approach, the disulfide linkages were newly formed by the adjacent thiol groups in

17

heparin chain and the formation of disulfide linkages, in some cases, could result in crosslinking

18

of nanogels. Such nanogels crosslinked via disulfide linkages could be obtained in a facile and

19

rapid manner via ultrasonic treatment, the process of which generated reactive free radical

20

species, promoting the oxidation reaction of free thiol groups. H2O2 was sometimes used to fully

21

oxidize the free thiol groups. As an example, Bae et al. prepared heparin-based nanogels

22

with two steps.46 Firstly, thiolated heparin was dissolved in DMSO with PEG as a condensing

23

agent, forming complexes of heparin and PEG linked with hydrogen bonds. Then, the obtained


23

Biomacromolecules

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Page 24 of 49

1

complexes were treated ultrasonically, generating disulfide linkages between adjacent thiolated

2

heparin chains. Their study was designed to realize intracellular delivery of heparin which might

3

induce apoptotic cell death and the construction of heparin redox-sensitive nanogels was mainly

4

based on the following considerations. First, compared to free heparin, the nanoparticulate form

5

was more preferred for transmembrane transport. Second, the redox-dependent cleavage of

6

disulfide linkages renders the nanogels the ability to be rapidly degraded and released free

7

heparin within cells. The designed nanogels were reported to enhance the internalization of

8

heparin and exhibit an improved apoptotic activity. By combining with various agents, the

9

nanogels might be potentially applied for tumor targeting therapy and diagnosis. In another

10

study, disulfide linkages were also introduced by reaction of thiol groups in heparin chain.

11

However, unlike the former example, nanogels were crosslinked by the hydrophobic force of

12

F127, instead of disulfide bonds. The decision of introducing disulfide bonds was based on their

13

ability to stabilize the physically crosslinked nanogels and intracellularly deliver RNase (a model

14

protein).45 RNase loaded Pluronic nanogels were prepared by using the direct dissolution

15

method, wherein Pluronic conjugate self-assembled into nanogels and the nanogels were further

16

oxidized with H2O2 and sonication to fully convent the thiol groups to disulfide bonds. Superior

17

to the physically crosslinked heparin-Pluronic nanogels, this kind of nanogels might serve as a

18

smart intracellular delivery vehicle, which protected the loaded protein in the network before

19

breakage of disulfide bonds by GSH in cytosol.45

20

The other approach took advantage of heparin derivatives with inherent disulfide bonds in the

21

chain. Utilizing this approach, Nguyen et al. firstly synthesized heparin-cystamine derivative

22

with disulfide bonds in its chain. Then, Pluronic with terminal vinyl sulfone groups was

23

conjugated to heparin-cystamine, producing heparin-Pluronic conjugate 42. To render the system


24

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Biomacromolecules

1

active targeting property, Cyclo(Arg-Gly-Asp-D-Phe-Cys) (cRGDfC) targeting ligand was

2

introduced to RNase loaded heparin/Pluronic conjugate at the terminal vinyl sulfone (VS) groups

3

of Pluronic via Michael-type addition. The in vitro release study showed that the release of

4

heparin and RNase increased instantly after adding GSH into dissolution medium (the final

5

concentration of 5 mM), indicating that the disulfide bonds in nanogels were exceedingly

6

sensitive to GSH and thus the nanogels could selectively release the loaded drugs in cells. It was

7

also addressed that the cRGDfC ligand contributed to higher cellular uptake than unmodified

8

nanogels.42

9

10 11

Figure 5. Preparation, intracellular uptake and destruction of nanogels: (A) Disulfide bonds are

12

introduced to heparin-based nanogel via reaction of adjacent thiol groups and, in this case,


25

Biomacromolecules

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Page 26 of 49

1

nanogel is crosslinked by the newly formed disulfide bonds. (B) Disulfide bonds are inherent in

2

the chain of heparin-F127 derivative. For both kinds of nanogels, disulfide bonds are cleaved by

3

excess amount of GSH in cells after endocytosis, resulting in destruction of nanogels and

4

intracellularly release of drug.

5

4.4 Polyelectrolyte complex nanoparticles (PCNs)

6

Based on the electrostatic interaction between oppositely charged polyelectrolytes, PCN can

7

be manufactured in an extremely simple and mild condition.86 As such, polyanion heparin can

8

spontaneously associate with polycations like proteins and chitosan, so that polyelectrolyte

9

complexes are formed. The simple and mild condition makes heparin an ideal candidate for

10

biological drug delivery. To efficiently deliver tumor necrosis factor-related apoptosis inducing

11

ligand (TRAIL) and protect this protein, Kim et al.

12

simply mixing them and indicated that heparin was a promising tool to deliver protein used in

13

cancer therapy. Analogously, heparin/protamine PCNs were also reported.88, 89 In another case,

14

Bae et al. designed chitosan-g-poly (ethylene glycol) (chitosan-g-PEG)/heparin polyelectrolyte

15

complex micelles, wherein heparin was not only an important component of the carrier, but also

16

the agent to promote the apoptosis cancer cells.90 Cores of the micelles were formed by the

17

neutralization of positively charged chitosan-g-PEG and negatively charged heparin and PEG

18

chains grafted on chitosan were anticipated to form the hydrophilic shell. Due to the increased

19

intracellular uptake of heparin in micelles, the PCNs were reported to induce more apoptosis of

20

cancer cells. The author assumed that if co-encapsulated with negatively charged nucleic acid

21

agents like siRNA related with cell apoptosis, the apoptotic effect for cancer cells could be

22

further promoted.90

87

prepared PCNs of heparin and TRAIL by


26

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1

Biomacromolecules

5. NANOPARTICLES MODIFIED WITH HEPARIN AND ITS DERIVATIVES

2

Over the past decade, together with the above progress in developing nanocarriers with

3

heparin as backbone, conveniently designed nanoparicles with heparin coating have also been

4

extensively investigated. Heparin modification of biomaterials was originally explored to reduce

5

the thrombogenicity of materials in blood, which has been has been performed for over 50

6

years.20 More recently, heparin and its derivatives have been applied to modify advanced

7

nanovehicles for cancer management and the main advantages are: 1) improving the stabilization

8

of colloidal solution; 2) preventing nanoparticles from being cleared by reticuloendothelial

9

system (RES) cells; 3) targeting tumor sites with heparin as a functional moiety of nanoparticles;

10

4) improving the drug loading of nanoparticles; 5) making use of the attractive biological

11

activities of heparin (e.g., anti-inflammation, anti-angiogenesis, anti-tumor cell proliferation

12

properties); 6) increasing cellular uptake of nanoparticles.5

13

As shown in Table 2, the modified cores could be various inorganic or organic nanoparticles

14

such as liposomes, magnetic iron oxide nanoparticles, and gold nanoparticles. Actually, in the

15

cases of hydrophobically modified heparin self-assembled nanoparticles, due to the particles’

16

micelle-like structure with heparin as the shell, heparin served as not only a building block but

17

also a modification material of nanoparticles. Therefore these heparin-based aggregations could

18

also be regarded as nanoparticles with heparin coating. To be coated onto nanoparticles, heparin

19

could be attached to inner nanoparticle core either via forming covalent bonds with appropriate

20

functionalities or electrostatic interaction.

21

5.1 Heparin-modified inorganic nanoparticles

22

5.1.1 Heparin-modified magnetic nanoparticles


27

Biomacromolecules

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1

Due to the unique properties of magnetic nanoparticles, they have been intensively applied in

2

imaging, cell labeling, drug delivery, gene delivery, and hyperthermia.91 In order to overcome

3

their inherent defects, there is an urgent need to improve the stability, compatibility, targeting

4

ability, as well as drug loading efficacy of the magnetic nanoparticles. To that end, heparin and

5

its derivatives have been widely applied in magnetic nanotherapy as coating materials based on

6

various considerations.

7

Firstly, to improve the biocompatibility of magnetic nanoparticles, heparin has been widely

8

used as coating materials. Iron oxide nanoparticles coated with heparin as recycling

9

anticoagulant have been prepared by adopting both chemical and physical modification

10

methods.92, 93 Liu et al. modified polyvinyl alcohol-shell magnetic nanoparticles by covalently

11

conjugation of heparin onto them using aminotrimethoxysilane (ATMS) and 4, 4-diphenyl

12

methane diisocyanate (HMDI) as spacer.93 Alternatively, taking advantage of the electrostatic

13

interactions between heparin and positively charged poly(L-lysine) (PLL), Khurshid et al. coated

14

heparin onto iron oxide nanoparticles which were pre-coated with PLL.92 Their studies indicated

15

that when further used for chemotherapeutic cancer treatment, the heparin coating could improve

16

the biocompatibility of magnetic nanoparticles and particles’ magnetization would be retained

17

after modification, making it possible to targeting tumor sites and controlling the metastasis of

18

tumors manipulated by magnets.

19

In addition, heparin coating was introduced to render magnetic nanoparticles the ability to load

20

bioactive agents. As the dense solid cores of magnetic iron oxide are not porous to accommodate

21

cargo molecules for specific delivery, the use of magnetic iron oxide nanoparticles as a drug

22

delivery platform was limited. Therefore, to efficiently load and deliver bioactive agents, surface

23

modification of iron oxide nanoparticles was necessary. To make magnetic nanoparticle a protein


28

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Biomacromolecules

1

drug delivery platform, the particle surface was reported to be modified with heparin to

2

guarantee effective loading of proteins based on the electrostatic attraction. Zhang et al. prepared

3

PEGylated iron oxide magnetic nanoparticles modified with heparin to deliver model protein

4

protamine to subcutaneous induced tumors of 9L-glioma taking advantage of an external

5

magnetic field.94 In another study, electropositive conjugation of PEI and β-Galactosidase was

6

electrostatically and reversibly loaded onto magnetic nanoparticles coated with negatively

7

charged heparin.95

8

Besides, heparin was reported to render magnetic nanoparticles targeting ability based on the

9

different amount of fibrinogen-derived products expressed in normal tissues and solid tumors,

10

which could interact with heparin.96,

97

11

nanoparticles are expected to be specifically localized at tumor sites, showing great advantages

12

for cancer therapeutics and diagnostics. For specific diagnostic purpose, glycol chitosan/heparin

13

immobilized iron oxide nanoparticles were developed by Yuk et al. as novel magnetic resonance

14

imaging (MRI) agents with specific tumor-targeting ability.99 Besides, their group also used this

15

coating material to decorate PTX-loaded Pluronic nanoparticles to enhance their targeting

16

capability to tumors.98 By applying near-infrared fluorescence imaging technology, tumor-

17

bearing mice treated with heparin-coated nanoparticles showed a stronger NIR fluorescence

18

signal in tumor area compared with those treated with bare nanoparticles, proving the desirable

19

targeting ability of heparin. 98

20

5.1.2 Heparin modified gold nanoparticles

On account of this interaction, heparin modified

21

Gold nanoparticles, an emerging type of nanocarriers, show their promising applications in the

22

field of cancer nanotechnology owing to their unique features such as low toxicity, good


29

Biomacromolecules

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1

biocompatibility, large surface area and ease of preparation.99 In particular, another attractive

2

feature of gold nanoparticles lies in their interaction with thiols, giving rise to an effective

3

method of heparin modification.99

4

As an example of this modification method, Lee et al. developed multifunctional gold

5

nanoparticles chemically modified by heparin for imaging and apoptosis induction of metastatic

6

cancer cells.100 They conjugated end-thiol modified heparin (fluorescent dye labeled) to surface

7

of gold nanoparticles via gold-thiol interaction, achieving the fluorescence resonance energy

8

transfer (FRET). FRET refers to the quenching property of gold nanoparticles at a separation

9

distance of 100 Å to between the dye and the nanopartiles.101 That is to say, gold nanoparticles

10

with surface modification cannot emit strong fluorescence signals in circulation until the

11

cleavage of the dyes in tumor sites. The incorporation of heparin into the gold nanoparticles was

12

based on the following considerations. On the one hand, it could detect a metastatic stage of

13

cancer cells since heparin-cleavage enzyme was over-expressed under tumor metastasis

14

condition. Upon the immobilized heparin was specifically cleaved by the enzyme, fluorescence

15

signal would recover, so that tumor metastasis was diagnosed. On the other hand, after

16

specifically released in cancer cells, heparin was expected to induce apoptosis death of the cells.

17

The specific intracellular release of heparin in cells was based on the aforementioned redox-

18

sensitive property. Furthermore, to achieve active targeting ability of the nanoparticles to cancer

19

cells, arginine-glycine-aspartic acid (RGD) was additionally conjugated to nanoparticles’

20

surface.

21

For an efficient PDT, heparin coated gold nanoparticles have also been reported by Li and co-

22

workers.102 The authors proposed a novel pattern of nanoparticles composed of gold

23

nanoparticles as an energy quencher and a heparin coating.102 In their study, heparin-PhA


30

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Biomacromolecules

1

conjugate was used to modify the gold nanoparticles via gold-thiol interaction, preparing hybrid

2

nanoparticles with an average size of 40 nm. Due to the strong photoquenching through FRET of

3

gold nanoparticles, after excited at 405 nm, PhA showed no fluorescent and photo activity in the

4

circulation. However, after localized in tumor sites via the EPR effect and internalized into the

5

tumor cells, the abundant GSH facilitated the release of PhA from gold nanoparticles by cleaving

6

the gold-thiol linkage and recovered its photo activity. As such, PhA could specifically kill the

7

tumor cells upon light irradiation. The in vivo therapeutic efficacy of the novel nanoparticles was

8

verified and the results showed that compared to free PhA, the prepared nanoparticles

9

significantly reduced the size of tumor in A549 tumor-bearing mice, which might be associated

10

with the efficient accumulation and cytotoxic singlet oxygen production in tumor cells. In their

11

study, the incorporation of heparin into the nanocomplex was based on the following

12

considerations. Firstly, by coating with water-soluble heparin, gold nanoparticles can be

13

solubilized and stabilized under physiological conditions, preventing colloidal aggregation.

14

Additionally, the advantageous anti-inflammation, anti-angiogenesis biological functions make

15

heparin attractive as an ingredient of the nanoformulation.

16

5.2 Heparin-modified organic nanoparticles

17

Obviously, to prolong the circulation of nanoparticles in vivo, measures should be taken to

18

attenuate the opsonization, which may cause the rapid clearance of nanoparticles by the

19

reticuloendothelial system (RES) cells mainly located in the liver and spleen. Consideration this,

20

nanoparticles are often modified with electrically neutral hydrophilic polymers such as

21

polyethyleneglycol (PEG), which is regarded as stealth coating to avoid opsonization due to its

22

hydrophilicity and steric hindrances. Besides the widely studied PEGylation of nanoparticles,

23

heparin

has

recently

been

proved

stealth-coating

material

acting

as

inhibitor

of


31

Biomacromolecules

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Page 32 of 49

1

opsonization.103,104 It has been envisaged that the effect of heparin can be basically attributed to

2

two aspects.103 First, heparin coating can be depicted as a biomimetic method because the surface

3

of cells and pathogens are naturally coated with glycosaminoglycan, which may avoid

4

phagocytosis by inhibiting the activation of complement at different stages, an important process

5

mediating the phagocytosis of the RES.103 Second, nanoparticles covered with heparin would

6

have a dense brush-like structure, creating steric barrier thus preventing interactions with the

7

proteins in blood and phagocytosis by immune system.

8

As an example, Han et al. conjugated negatively charged heparin onto the surface of DOX-

9

loaded cationic liposomes via the strong ionic interaction by simply incubating the liposomes in

10

heparin solution for 2 h, converting the zeta potential from 14 mV to -70 mV.105 They claimed

11

that the half-life of DOX loaded in heparin-liposomes significantly higher than the control

12

liposomes. Besides the simple modification based on physical interaction, chemically modified

13

nanoparticles have also been reported by researchers.103, 104 Passirani et al. prepared heparin-poly

14

(methyl methacrylate) (PMMA) amphiphile which self-assembled into nanoparticles of ~80 nm

15

aqueous medium. By incorporating non-biodegradable fluorescent N-vinyl carbazole (NVC) in

16

the nanoparticles, the authors followed the in vivo fate of nanoparticles after intravenous

17

injection into mice. They found that compared to bare PMMA nanoparticles, the circulation half-

18

life in blood of heparin-PMMA/NVC nanoparticles was dramatically increased from 3 min to

19

more than 5 h. It was envisaged that nanoparticles coated with heparin avoided the activation of

20

complement in a biomimetic way, like natural cells and pathogens cover with

21

glycosaminoglycans.104 In addition, after the self-assembling process, the PMMA cores were

22

covered with heparin having a dense brush-like structure which created steric barrier to the

23

proteins in blood, so that the opsonization was avoided to some extent. 104


32

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Biomacromolecules

1

In addition, due to the high-afﬁnity binding and internalization of heparin to vascular

2

endothelial cells, which are abundant in tumor sites, heparin and its derivatives are utilized to

3

target these cells.18 As an example, Chung et al. reported PLGA nanoparticled coated with

4

heparin-Pluronic conjugation as functional moiety. They prepared PLGA nanoparticles by the

5

nanoprecipitation/solvent diffusion method with the previously synthesized heparin-Pluronic

6

conjugation or bare Pluronic as stabilizers, which would exist on particles’ surface. Compared to

7

the control, bare Pluronic coated PLGA nanoparticles, the heparin modification enhanced both

8

the uptake of nanoparticles in vitro and the accumulation in tumors in vivo.

9 10

Table 2 Summary of nanovehicles modified with heparin or its derivatives for cancer

11

management Nanovehicles

Coating methods

Function of heparin coating

Refs

Liposomes

Ionic interaction between liposomes and anionic heparin

cationic

Reducing the uptake of RES

105

Liposomes

Ionic interaction between liposomes and anionic heparin

cationic

Exhibiting high antiangiogenic efficacy

106

Poly-εcaprolactone/polymethacryla te nanoparticles

Electrostatic interactions

Loading peptide or protein

107

PAMAM/DNA nanocomplex


Improving the biocompatibility and

108

partially shielding the positive charge of PMAMA/DNA complexes. Self-assemblies of heparinpoly (methyl methacrylate) (PMMA) conjugates

Covalent bonding of PMMA and heparin

Reducing the uptake of RES

104

PLGA nanoparticles

Physical coating (heparin-Pluronic conjugation was added as stabilizer in

Improving the tumor-targeting ability of PLGA nanoparticles as a functional moiety (due to the high-

18


33

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 49

the preparation of PLGA via a nanoprecipitation/solvent diffusion method)

afﬁnity binding and internalization of heparin to dividing vascular endothelial cells, which are plenty in tumors)


Targeting purpose

98

Iron oxide nanoparticles

Ionic interaction between cationic golddeposited iron oxide nanoparticles and anionic heparin

Improving the tumor-targeting ability of iron oxide nanoparticles (due to a meshwork of clotted plasma proteins, fibrinogen-derived product only present in tumors)

19


Amidation reaction between aminated and PEGylated commercial Starchcoated with heparin

Loading cationized protamine as a model protein via electrostatical interaction

94


Not mentioned

Loading cationized model proteins β-Galactosidase via electrostatical interaction and delivering proteins to brain tumor via magnetic targeting technology

95

Gold/silver nanoparticles

Reducing silver nitrate and gold chloride with heparin

Serving as both reducing and stabilizing agents

Gold/silver nanoparticles

Reducing silver nitrate and gold chloride with diaminopyridinyl -derivatized heparin

Inhibition basic fibroblast growth

Gold nanoparticles

Immobilizing end-thiol terminated, fluorophore-labeled heparin onto surface of gold nanoparticles through a saltaging process via a gold-thiol bond

Detecting a metastatic stage of cancer cells (due to the overexpression of heparinase in tumor sites) ; triggering apoptosis-induced cancer cell death after intracellular delivery

100

Gold nanoparticles

Immobilizing end-thiol terminated, PhAlabeled heparin onto surface of gold nanoparticles via gold-thiol bonds

Increasing colloidal stability of gold nanoparticles under physiological environment; exhibiting antiinflammation,anti-angiogenesis, and anti-tumor cell proliferation activities

102

Gold nanoparticles

Physical coating

Introducing an anti-biofouling, and liver-specific surface to gold nanoparticles

112

Amidation reaction between Ndeacetylated heparin and functionalized MSNs

End-capping agent to seal the mesopores of MSNs, preventing potential drug leakage; providing free amino groups for conjugation of targeting ligand

113

Pluronic nanoparticles

Mesoporous silica nanoparticles (MSNs)

109, 110

111

factor (FGF-2)-induced angiogenesis

1 2

6. CONCLUSIONS AND FUTURE PERSPECTIVES

3

Heparin, an endogenous polysaccharide ubiquitous in body with non-toxicity, has been used in

4

clinic as an anticoagulant for many years. Superior to other polysaccharides, heparin possesses


34

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Biomacromolecules

1

excellent biological properties such as anti-angiogenesis and anti-metastasis, making it attractive

2

for cancer management. In addition, it can also be utilized as a targeting moiety considering the

3

affinity to tumors and enhanced internalization in dividing endothelial cells.5 Therefore, to

4

combine the unique features of heparin and nanoparticles, recently there has been an increasing

5

attention to incorporating heparin into nanoformulations for cancer management.

6

From a pharmaceutical point of view, heparin is a highly hydrophilic molecule with abundant

7

modifiable groups on its chain, providing the possibility to construct amphiphilic drug delivery

8

systems, including heparin-drug conjugates and polymeric conjugate nanoparticles. Also,

9

hydrophilic nanogels with heparin as backbone can be prepared via physical or chemical cross-

10

linking. Besides, due to the high electronegativity of heparin, polyelectrolyte complex

11

nanoparticles can be easily manufactured. Analogously, taking advantage of chemical

12

modification or electrostatic interaction, heparin can be introduced to modify nanoparticles for

13

various purposes. These advanced nanocarriers have been applied in research of novel targeted

14

therapy, magnetic therapy, photodynamic therapy and gene therapy of cancer, achieving

15

considerable therapeutic effects. Compared with existing nanoformulations, these nanocarriers

16

exhibited numerous attractive advantages such as the inherent anti-cancer activity of nanocarrier,

17

potential targeting ability to tumors, non-toxicity, and ease of preparation. In future research of

18

heparin-based nanocarriers for cancer management, rational design to make them accommodate

19

to more bioactive agents and combining heparin with other nanomaterials may become

20

research focus.

21

Despite the achievement in research of heparin-based nanoparticles, there is still no anti-

22

cancer nanoformulation with heparin as a component in clinical trials. This situation may be

23

caused by the following aspects, requiring ongoing effort to realize the clinical transition. The


35

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 49

1

anticoagulant activity of heparin and its derivatives, which may result in adverse effects like

2

hemorrhagic complications, is always the priority. Although it has been proved that after

3

chemical modification, the anticoagulant activity of heparin can be significantly reduced (maybe

4

due to the change in conformation), while the anti-cancer activity being preserved, the exact

5

mechanism remains uncertain.69 Therefore, to make heparin safe and effective component of

6

nanocarriers for clinical use, it is beneficial to precisely separate the anticoagulant activity and

7

anti-cancer activity of heparin, which is based on a deeper understanding of the structure-activity

8

relationship of heparin. More specifically, the following questions should be considered in future

9

work: What kind of chemical structure on heparin chain contributes to its anti-cancer and

10

anticoagulant activity, respectively? How to precisely predict and control the anticoagulant and

11

anti-cancer activity of heparin? Is it possible to separate these two kinds of activity (i.e.,

12

decreasing its anticoagulant activity of heparin while maintaining or even increasing its anti-

13

cancer activity) by chemical modification or synthesis of heparin analogue? Another hurdle may

14

reside in the difficulty to analyze and control the quality of heparin considering its polydisperse,

15

polycomponent and polypharmacologic properties.114 Learning from the lessons of heparin

16

contamination, which caused allergy and even death due to the adulteration of synthetic

17

oversulfated chondroitin sulfate, effective assays are required to detect potential contaminants

18

and monitor the quality of heparin.114 For this purpose, NMR remains the most reliable method,

19

and other up-to-date assays have also been developed, which have been well reviewed in other

20

articles.114 From a long-term view, to guarantee the safety of using heparin, alternative methods

21

are urgently in need to detect both known and unknown contaminants, as well as control the

22

identity and/or purity of heparin.


36

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Biomacromolecules

1

As to future perspectives, to make the clinical application of heparin-based nanoparticles for

2

cancer management into reality, besides overcoming the hurdles illustrated above, the following

3

aspects should also be considered. To begin with, reasonable evaluative methods for controlling

4

the quality of heparin-based nanoparticles are needed. Also, the pharmacology and toxicology of

5

drug-loaded heparin-based nanoformulations need to be evaluated guided by reasonable indexes

6

and methods. Besides, aspects such as desired stability, cost-effective and reproducible

7

manufacture on a large-scale should also be considered

8

AUTHOR INFORMATION

9

Corresponding Authors

10

*Guangxi Zhai, E-mail : [email protected]

11

*Jiyong Liu, E-mail: [email protected]

12

Notes

13

The authors declare no competing financial interest.

14

ACKNOWLEDGMENTS

15

This work was partly supported by the Science Research Program of Jinan, China (201303032)

16

and grants from Shanghai Municipality Science and Technology commission, China

17

(14JC1491300).

18

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Title: Advanced nanocarriers based on heparin and its derivatives for cancer management

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Authors: Xiaoye Yang, Hongliang Du, Jiyong Liu*, Guangxi Zhai *

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Figure 1. Various heparin derivatives prepared by chemical modification. Derivatives in the red circle are obtained via derivation of the carboxyl groups, while those in the blue circle are prepared via modification of amine or hydroxyl groups. a. Heparin amide derivatives are synthesized via amidation reaction; b. Various reactive groups are introduced to heparin via amidation reaction; c. Heparin derivatives are synthesized by modification of amine or hydroxyl groups. 99x80mm (600 x 600 DPI)


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Figure 2. Various nanocarriers with heparin and its derivatives as backbone. (a) Heparin-drug conjugate nanoparticle with hydrophobic drug as the core; (b) heparin-based polymeric conjugate nanoparticle with drugs encapsulated inside the core; (c) Chemically/physically crosslinked nanogel with drug loaded in the three-dimensional network; (d) Polyelectrolyte complex nanoparticles (PCN). 353x247mm (150 x 150 DPI)


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Figure 3. Self-assembly of two kinds of heparin-based amphiphilic conjugates. A. Heparin-drug-targeting molecule conjugate self-assemble into nanoparticles in water with drug as the core (not physical encapsulation) and targeting molecules on the shell. B. (a) Heparin-hydrophobic segment-targeting molecule conjugate self-assembled into nanoparticles in water with targeting molecules on the shell. (b) After the formation of heparin-based polymeric conjugates, drug is added and encapsulated in the hydrophobic core via a dialysis method. 520x245mm (150 x 150 DPI)


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Figure 4. The mechanism of passive (A) and active targeting (B). (a) Minimum extravasation of nanoparticles to normal tissue; (b) Increased drug accumulating in tumor sites due to the EPR effect; (c) Uptake of nanoparticles by fluid-phase pinocytosis; (d) Enhanced uptake of nanoparticles mediated by receptors; (e) Release of drug in cytoplasm. 101x126mm (150 x 150 DPI)


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Figure 5. Preparation, intracellular uptake and destruction of nanogels: (A) Disulfide bonds are introduced to heparin-based nanogel via reaction of adjacent thiol groups and, in this case, nanogel is crosslinked by the newly formed disulfide bonds. (B) Disulfide bonds are inherent in the chain of heparin-F127 derivative. For both kinds of nanogels, disulfide bonds are cleaved by excess amount of GSH in cells after endocytosis, resulting in destruction of nanogels and intracellularly release of drug. 250x200mm (150 x 150 DPI)


Advanced Nanocarriers Based on Heparin and Its Derivatives for

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