Surface modification strategies on mesoporous silica nanoparticles for anti-biofouling zwitterionic film grafting

    Surface modification strategies on mesoporous silica nanoparticles for antibiofouling zwitterionic film grafting Yit Lung Khung, Dario Narducci PII: DOI: Reference:

S0001-8686(15)00180-3 doi: 10.1016/j.cis.2015.10.009 CIS 1589

To appear in:

Advances in Colloid and Interface Science

Please cite this article as: Khung Yit Lung, Narducci Dario, Surface modification strategies on mesoporous silica nanoparticles for anti-biofouling zwitterionic film grafting, Advances in Colloid and Interface Science (2015), doi: 10.1016/j.cis.2015.10.009

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Yit Lung Khung1,2* and Dario Narducci1 1

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Department of Materials Science University of Milan-Bicocca, Via R. Cozzi 55, I-20125 Milan (Italy)

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Surface modification strategies on mesoporous silica nanoparticles for anti-biofouling zwitterionic film grafting

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Institute of New Drug Development China Medical University No. 91 Hsueh-Shih Road, Taichung 404 (Taiwan)

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Corresponding Author: Dr. Yit Lung Khung [email protected] phone + 39-02-6448-5143 fax +39-02-6448-5400

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*

Abstract

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In the past decade, zwitterionic-based anti-biofouling layers had gained much focus as a serious alternative to traditional polyhydrophilic films such as PEG. In the area of assembling silica nanoparticles with stealth properties, the incorporation of zwitterionic surface film remains fairly new but considering that silica nanoparticles had been widely demonstrated as useful biointerfacing nanodevice, zwitterionic film grafting on silica nanoparticle holds much potential in the future. This review will discuss the conceivable functional chemistries approaches, some of which are potentially suitable for the assembly of such stealth systems.

Content 1 2.

3.

Introduction Surface hydration and anti-biofouling properties 2.1 Polyhydrophilic 2.2 Polyzwitterionic Types of poly-zwitterionic layers 3.1 3.2 3.3

4.

Phosphorycholine (PC) Sulfobetaine (SB) Carboxybetaine (CB)

3.4 Mixed SAM (MS) Synthesis of zwitterion functionalized MS nanoparticles 4.1 Surface functionalization of Silica Nanoparticles (thoughts and considerations) 4.2 Si-OH surface functionalization

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1

Introduction

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5.

4.3 Zwitterionic “Graft to” strategies (common conjugation chemistry on the distal R group) 4.4 Examples of “Graft to” approach 4.5 Polymeric grafting on functionalized MS nanoparticles: “Graft from” strategies 4.6 Examples of “Graft from” approach Conclusion

Ever since Yanagisawa et al. reported on producing three-dimensional

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network of mesoporous (2-4 nm) silica films in 1990[1] and the subsequent development of the popular surfactant-templated synthesis by Kresge et al. in 1992[2], mesoporous silica (MS) nanoparticles has evolved to become an

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attractive material as nanoparticles for biological applications. The popularity was due to its excellent biocompatibility as well as its high thermal and Various types of mesostructures can be easily

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mechanical stability.

assembled through tailoring of the surfactant template (spheres, fibers, cubic and nanorod etc).

Being one of the mostly widely used materials in

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nanotechnology, its surface chemistry is well understood, highly flexible and its pore dimensions can be easily tuned.

MS nanoparticles can reach

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extremely high surface area (900m2/g)[1] and demonstrate good thermal and chemical stability, thus rendering it as an excellent medium for drug

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delivery/transfection agent[3, 4]. It had found a healthy repertoire in many bioimaging[4, 5], biosensing[6] and gene/drug delivery platform[7, 8].

While the in-vitro application of MS nanoparticles had been widely reported, one of the major challenges that researchers face is the maintenance of longevity of the circulating MS nanoparticles when administered in biological environment. This is because that in the serum-rich environment in the body, unmodified nanoparticles would undergo opsonization or coating on the surface by opsonin proteins almost instantly upon administration. This subsequently results in the attraction of phagocytic cells towards the nanoparticle target and trigger immunogenic responses that can ultimately lead to faster clearance from the body[9]. Furthermore, the phagocytes cannot

ACCEPTED MANUSCRIPT normally destroy non-biodegradable MS nanoparticles and this may further produce on-site accumulation of these particles in liver and spleen, resulting in many other adverse side effects[10, 11]. Hence, biofouling and particulate

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agglomeration of nanoparticles are by far some of the most pressing issues to

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address prior to commercialization of nanoparticle-based biosensors.

One of the ways to address the biofouling issue is to render nanoparticles “stealthy” (impervious to protein adsorption) by means of changing the surface In fact, surface modifications on MS nanoparticles is fast

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chemistry.

becoming an essential feature in nanoparticle research and considering the

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wide berth of biological based applications reported, ranging from drug delivery to biosensing/cell imaging, the importance of introducing an antibiofouling layer cannot be understated.

A wide range of methodologies for

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the nanoparticle design meant for biological applications had already been already discussed in literature but what is more important is the chemistry at

be addressed.

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this very top layer of the MS nanoparticle‟s surface that biofouling issues must That is where cellular materials and nanoparticles would

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interact at the very first instances. So far, anti-biofouling properties on the surface of nanoparticles are typically achieved by introducing specific organic

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layers that will alter some fundamental surface properties such as surface charges and hydration.

Of these, zwitterion-based surface modifications is fast becoming one of the most interesting and efficient ways of introducing highly efficient antibiofouling films on surfaces. There are numerous ways by which zwitterionic films can be grafted onto the surface of MS nanoparticles. In this review, we sought to discuss many of the common reaction mechanisms used on silica surfaces as well as some of the bioconjugation chemistries that can potentially be feasible on the surface on MS nanoparticles to form covalent linkage to the surface and for the subsequent development of a zwitterion layer on the surface. And prior to discussing the strategies of grafting zwitterion films on MS surface, it is important to examine some of the

ACCEPTED MANUSCRIPT fundamental aspects of anti-biofouling modifications.

Surface hydration and anti-biofouling properties

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In the past, many different types of anti-fouling chemical layers had been grafted on the surfaces to produce stealth-like behavior that can efficiently repel proteins binding. As early as the 1970s, several groups had reported a reduction in protein adsorption on OH-rich Hydroxylethyl methacrylate (HEMA)

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modified surfaces[12, 13] although it was only in the 1980s when Andrade et al. suggested the notion of surface hydration as a viable reason behind the

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repellent of proteins on surfaces[14] and this was heavily influenced by earlier studies on „non-freezing water‟ layers on phospholipids[15, 16]. The solvation interaction energy between proteins and polymer surface was also found to

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have a significant effect on protein dehydration due to the shedding of the hydrated layer, as proposed by Lu and his coworkers in 1991[17]. Tsuruta et

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al. later suggested in 1996 that the entrapment of proteins on surface was a physicochemical phenomenon arising from a readjustment of a perceived

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“water network” formed on surfaces of hydrophilic polymers and that fouling is also a time dependent process[18]. Over time, it became gradually accepted

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in mainstream literature that when a layer of water is tightly bound onto a surface, a physical/energetic barrier is formed and this barrier can subsequently discourages the adsorption of incoming proteins. It is also important to note that there are also two types of water-substratum interaction needed to consider, one forming from hydrogen bonding (typical of hydrophilic surface) and the other arising from ionic solvation (on zwitterionic surface). In order for proteins to be absorbed onto the surface, the water layer had to “make room” to facilitate for the protein adsorption. How nanoparticles can gain its anti-biofouling property is through retaining a surface hydrated layer so tightly that the energy required to displace this layer during protein adsorption is highly energetically unfavorable. The strength of water retention on the surface is strongly influenced by the physicochemical property of the material, packing density, as well as thickness of the material. The influence

ACCEPTED MANUSCRIPT of steric repulsion may also play an important role especially when dealing with long chain polymeric based stealth layers.

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In terms of chemically modified surface stealth layer based on the principle of hydration on surface, there are currently two major classes of anti-biofouling

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layers commonly described in literature for the realization of anti-biofouling surface coating; polyhydrophilic and polyzwitterionic.

Polyhydrophilic

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2.1

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Traditionally, polyhydrophilic materials are the most widely used surface modification for anti-biofouling applications in literature. The most common characteristics of polyhydrophilic materials are their hydrophilicity as well as

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having region of rich hydrogen bond acceptor/donor sites.

Of the many

models in literature, the most popular in polyhydrophilic surface modification

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are poly(ethylene) glycol (PEG) and its structural derivatives.

PEG is typically synthesized via anionic ring opening polymerization of

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ethylene oxide that is initiated by nucleophilic attack of a hydroxide ion on the epoxide ring. At high molecular weights, PEG is not immunogenic as there are no known anti-PEG antibodies that had been generated in the body against Pegylated nanoparticles.

As early as 1994, PEG was used in

conjunction with biodegradable polymer (PLA) to form anti-biofouling nanoparticles[20].

However, the examinations were rather rudimentary

(involving the co-incubation with albumin) and the design was of a block copolymer type rather than the distinctive layers of core/shell. Nonetheless, the results were sufficiently encouraging to sprout a subsequent following[21, 22]. It must be said that while a copolymer based nanoparticle can help resist biofouling, there will definitely some issues pertaining to drug delivery and gene delivery, especially if the nanoparticle is required to „home‟ towards a particular cell type. This is due to the overall hydrophilicity of the nanoparticle

ACCEPTED MANUSCRIPT surface that not only rejects incoming proteins but would also reject its intended cell target as well.

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Nonetheless, PEG based materials had ever since been a mainstay candidate in literature for surface modifications due to its excellent protein repellency

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behavior, especially for those of high molecular weight and had consequently received much research coverage. The protein repellency effect has been broadly described as a formation of initial thin layer of water on surface that effectively inhibited proteins from attaching and this is namely due to the steric

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entropy arising from the unfavorable change in free energy if the surface film is to undergo dehydration as well as confinement of polymer. Monolayers of

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PEG derivatized films can conferred a high degree of monodispersity of nanoparticles but in the case of dense layer of PEG polymer, the conformational freedom can be highly restrictive and may limit the possibility

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for further performing further conjugation chemistry. Nonetheless, they had demonstrated excellent protein repellent properties with a major advantage of

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size dependent circulation time within the body, i.e. the higher the molecular weight, the longer the undetected circulation within the body[23]. However,

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the use of bulky hydrophilic PEG may be disadvantageous in this aspect as it had been reported that for efficient renal removal of spherical nanoparticle

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from the system, the optimal hydrodynamic size of nanoparticles should be lower than 5.5 nm[24]. Furthermore, despite being widely used in numerous research applications, another major weakness is that PEG tends to be unstable under prolonged oxygen exposure and also in the presence of transitional metals ions, especially from those circulating within biological environment. This may often result in a total loss of function over time. It is also important to highlight that the production of PEG had led to contamination by ethylene oxide and 1,4 dioxane components within the PEG polymer and these had been found to be carcinogenic in nature. Hence, while PEG had been highly successful in short-term experimental models, the stability and toxicity issues may outweigh many of its advantages in the long run.

ACCEPTED MANUSCRIPT Another major class of polyhydrophilic material that had attracted attention is Polyoxazolines and found much popularity in drug delivery applications due to its excellent biocompatibility[25, 26] and can also be designed to be

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thermoresponsive[27]. But one major disadvantage of polyoxazoline is the difficulty to achieving low polydispersity index during synthesis and this may

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impedes its wider use[28].

Certain groups had also classify polymer based HEMA as another important polyhydrophilic material but its performance had been too disappointing to

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garner sufficient support as a major class on its own[29, 30]. Some of the other notable examples of polyhydrophilic materials are polysaccharides[31],

Polyzwitterionic

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2.2

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acrylamides[32] and polyethersulfone[33].

Polyzwitterionic materials are attractive alternatives to PEG and had gained

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much interest in recent years and had been coined as the “next generation anti-biofouling system”. By passivating a surface with an array of zwitterionic

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moieties, a high level of anti-biofouling activity can be achieved. There are two major properties that had been thought to be the reasons behind this antibiofouling effect. Firstly, a zwitterionic surface containing both positive and negative charge can electrostatically bind water more strongly compared to hydrophilic materials while the free energy involving the dehydration will be too high at normal circumstances. Secondly, as proteins typically exhibit a variation of charge distribution on their surface, they can only effectively bind to surfaces when the surface exhibits only a single charge type.

In the

presence of varying charges on the surface, as in the case of zwitterion, proteins cannot effectively adhere due to the inability to compromise its mature structure to accommodate charge distribution they observed on the target surface.

This also contributes in an eventual repulsion from the

zwitterion surface.

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3.

Types of poly-zwitterionic layers

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Polyzwitterionic materials can be further subdivided into two categories; (1) monomers carrying a positive and negative charge on the same monomer

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(such as 2-methacryloyloxylethyl phosphorylcholine (MPC), sulfobetaine methacrylate (SBMA) or large polyampholytes); (2) A pool of mixed monomers of positive and negative charge passivated collectively onto the surface.

Similar to hydrophilic systems, the efficiency of polyzwitterionic

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material is also highly regulated by packing density of the chains as well as film thickness although the oxidative damage is less profound for zwitterionic

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systems compared to that of the more popular PEG systems.

Currently, betaines forms the mainstay as primary zwitterionic units and can

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be described as compounds having both anionic and cationic charge residing within the same repeat unit.

As with any other zwitterionic species, the

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classical betaines polymer commonly used in literature are neutrally charged compounds with a positively charged cationic group, typically in the form of

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quaternary ammonium or phosphonium cation, and a negatively charged group (either carboxylate or sulfonate). The persistent charge from the ionic

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species within the betaine molecule had permitted it to maintain a constant near-zero net charge across a wide spectrum of pH although it is possible to modulate surface charges by inducing copolymerization of different betaine species[34]. Furthermore, there had been attempts to render them pH responsive[35]. It is also the zwitterionic nature of the betaines that ultimately allowed for a tighter retention of a hydrated water layer on the surface. Generally, for protein rejection purposes, betaines in polymeric form are highly preferred due to their high density on the surface.

However, prior to further discussions into the characteristics of betaines, it is necessary to deliberate on that the mechanism behind protein resistant nature of zwitterions (such as phosphorylcholine), which actually remained relatively elusive to researchers well into the late 1990s where it was at this time when

ACCEPTED MANUSCRIPT the concept of hydration finally took root as the main causative effect for the anti-biofouling properties observed in polyhydrophilic materials.

The first

explanation offered by Ruiz and coworkers in 1998 for zwitterionic anti-

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biofouling properties suggested that the presence of a hydration layer occupied by the phosphorylcholine might also be responsible for protein This discussion was also in tandem with what Laibinis et al.

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repellency[36].

had proposed during their examination into hydration states of oligo(ethylene glycol) where water layer retention on the surface was thought to be responsible for rejecting proteins in solution[37]. In the same year, Ishihara

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and coworkers had also performed a series of well-controlled experiments on 2-methacryloyloxyethyl phosphorylcholine (MPC) to establish the importance

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of hydration layer for anti-biofouling[38] although they did not disregard the water network argument as forwarded by Tsuruta[18] in their argument. Experimental evidences was also provided by Murphy et al. using neutron measurements

which

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reflectivity

found

that

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phosphorylcholine layer comprised of 85% water[39].

top

2.5

nm

of

This concept of water

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layer retention by zwitterions was finally elaborated by Whitesides et al. in 2000 and 2001, using a mixed SAMs exhibiting both positive and negatively

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charge species on the surface and had also shown to effectively discourage protein fouling[40, 41]. This helped to significantly support the concept of surface hydration and hence generated an impetus towards research into

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zwitterions as a serious alternative for antifouling systems to replace the more conventional polyhydrophilic PEG.

While the key appeals of zwitterionic films and its polymeric derivatives are that they do formed a stronger water-surface retention layer (ionic solvation) as compared to polyhydrophilic surface[42], there is one significant drawback with zwitterionic system, especially in polymeric form. Polyzwitterions, being superhydrophilic, are unable to dissolve in most organic solvent, thus making them difficult to work with although this issue may be carefully circumvented using mixed solvent systems. Nonetheless we believe that the stability and appeal of zwitterionic surfaces have been gaining much ground in recent years and may be set to replace the traditionally popular PEG as the

ACCEPTED MANUSCRIPT mainstream anti-biofouling layer on nanoparticles in the coming future. Herein, in this review, we aim to discuss the various possible ways functionalization and fabrication of zwitterionic layers on MS nanoparticles can

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be attained and described some examples of zwitterionic based stealth MS nanoparticle with a major emphasis on the chemistry behind the approaches

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used in literature.

Figure 1. Graphic illustration of various aspects of zwitterionic based MS nanoparticles.

As mentioned earlier, there are two forms by which the zwitterion chain can be assembled on a surface, either collectively or separately as individual monomers of different charges. However, prior to discussing the virtues of

ACCEPTED MANUSCRIPT different surface arrangements, it is necessary to make certain distinctions of the various classes of zwitterion betaines that are widely reported in literature. These

can

be

broadly

divided

into

a

few

categories,

namely

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phosphorylcholine (PC), sulfobetaine (SB), carboxybetaine (CB)

Figure 2. Various zwitterionic groups incorporated in polymers as reported in literature[43]

3.1

Phosphorycholine (PC)

Of the many zwitterionic systems commonly available in literature now, the first pioneering work for such antifouling system was „discovered‟ during the late 1970s during the development of PC/haemocyte interfacing devices[44]. In brief PC compounds can be described as hydrophilic head groups derived from certain phospholipids and are basically comprised of a negatively charged phosphate couple to a positively charged choline group in the form of

ACCEPTED MANUSCRIPT a quaternary ammonium salt.

As early as 1984, Chapman et al. had

demonstrated that single charged surfaces was promoting thrombin biofouling while zwitterion moieties in the form of PC was able to dramatically reduce

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biofouling[45]. This had also led to subsequent developments of PC-based

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drug delivering implants that is currently used in cardiology.

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Figure 3. An example of phosphorylcholine betaine

Intentional use of PC as an antifouling layer was first described by Ishihara et

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al. early in 1990[46] and results had shown that PC was able to reduce thrombogenesis, thus making it a very useful candidate for designing

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bioimplants although the mechanism behind the anti-biofouling characteristics was not fully understood at that time. Nonetheless, the report was very wellreceived by the scientific community and Ishihara et al. further expanded their

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study on PC over the course of the next few years[47-49]. All results reported were consistent to the fact that PC can reduce blood thrombogenesis and

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activation of platelet on the surface. However it is important to note that the solubility of PC in organic system remained a primary issue during the

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fabrication, and strategies were undertaken to improve solubilization through the addition of longer alkyl methacrylate side chains[49]. Improving on this principle of branching the aliphatic chains from PC, a biodevice in the form of a glucose sensor was first conceptualized by Chen et al. in 1993 by using derivatised PC as surface coating for glucose sensing needles[50]. Sugiyama et al. in 1994 went on to produce copolymerized microspheres of PC and various vinyl derivatives under emulsifier-free conditions. They were able to demonstrate that, due to its enhanced hydrophilicity, PC in the copolymer would phase separate towards the external of the microsphere so as to interface with the water layer, while the more hydrophobic vinyl core remained within the microsphere. This also illustrated a dual functionality for PC, both as antifouling layer as well as a surfactant.

ACCEPTED MANUSCRIPT Up to 1994, much of the investigations on PC based materials had been rather confined to device fabrication and haemocompatibility studies. Ishihara et al. had initially proposed that the reasons why PC had been successful in

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reducing protein fouling was because PC on the surface tend to arrive into a biomembrane-like conformation and this reduced the change of biofouling[51,

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52]. However, this was extremely hard to prove from an experimental point of view and the subsequent years brought along an expansion on the physical examinations on the intrinsic surface properties of PC based materials. The notion of surface hydration as proposed by Andrade et al.[14] continued to

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resonate within the research communities. An excellent study performed by Ruiz et al. in 1998 on polymerized PC had highlighted the significance of

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chain folding and the subsequent presentation of polar groups at the interface and this had given certain impetus towards the hydration layer theory[36]. It is important to mention that while many of the examples cited in this review

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involved modeling on tertiary amines, it has also been shown that secondary and primary amines can also be used for anti-biofouling purposes[53, 54].

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However, levels of hydration may be lower for primary and secondary amines

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compared to tertiary amines[55] and this may reduced its biofouling efficiency. Sulfobetaine (SB)

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As the name suggest, this is a class of zwitterion with a sulfate group (SO 3-) acting as the negatively charged species coupled typically to a positively charged choline group in the form of a quaternary ammonium salt. Due to its haemocompatibility[56], polymerized sulfobetaine had found use in many applications in pharmaceutics as well as excellent dispersing and viscosifying agents. It also maintains its amphoteric properties at all pH values, making it an excellent candidate for adsorption style deposition strategies on charged surfaces.

Figure 4. An example of Sulfobetaine

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The solubility and physical properties of SB was first examined by Salomone et al. in 1978[57] although the synthesis of SB had already been known much

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earlier. It was in 2000 when Viklund et al. had initiated the preliminary study on SB interfacing with proteins[58]. In recent years, SB based polymers and

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monomers had shown excellent anti-biofouling properties[59-61] and its biocompatibility had further help raising its profile[62, 63] although there are certain arguments that SB are less efficient compared to PC[56].

An

attractive feature of SB is that the anti-biofouling properties is not influenced

Carboxybetaine (CB)

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3.3

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by modulations in pH[64] as mentioned earlier.

A class of zwitterionic molecules containing a quaternary ammonium salt and

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terminating with carboxyl (RCOO-), CB had been found to have excellent protein repelling agents when deposited on surfaces[65]. In fact comparative

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studies had shown that CB polymers were able to resist biofouling for days longer than the conventional PEG based systems[66]. One advantage of CB over PC and SB is the ease in fabrication process, especially in the polymer

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form. There are arguments that the spacing groups as well as the distance between the positively charged quaternary ammonium and the negatively charged carboxyl may influence the overall property of CB[67, 68] and SB[69] although the correlation had yet been conclusive[70]. The ability to resist protein adsorption for CB polymers are actually pH dependent (pH 5-9)[64]. Nonetheless, the acid-base equilibrium characteristic of CB and the ability for its carboxyl groups (COO-) to be used as reactive intermediates that can confer further functional groups on the surface.

Figure 5. An example of Carboxybetaine

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Mixed SAM (MS)

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3.4

Mixed SAMs are arrangements styles on the surface of two separate

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molecules carrying opposite charges periodically to mimick the overall zwitterionic characteristics on the surface. They may also be arranged in polymeric form and are then termed “polyampholytes”[71, 72] although the

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distinction between polyzwitterionic betaine and polyampholytes remains relatively vague in literature. Lowe et al had attempted classification and subclassing of polyamopholytes on the basis of polymer responses to changes to

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pH in solution[71]. The grafting of separately charged molecular chains had been initially reported by Whitesides‟ group[73] and had been shown to be of a superior model compared to grafting of single molecular chains with both

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charges on surface as they had demonstrated that mixed SAM had been more effective in reducing protein adsorption compared to the alkyl chains

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that carry both charges simultaneously. While the anti-fouling properties had been reported to be excellent, one concern of using such systems is the

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uneven distribution of the charges on the surface considering that the monolayer comprises of two individual molecules mixed at a specific ratio

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rather than a single one.

This may result in producing inhomogeneous

surface patches of varying charges

Figure 6. An example of charged ampholyte on different residues

A few considerations should be taken note of when using this sort of mixed system in the form of monolayer. Firstly, the chain length of the two different

ACCEPTED MANUSCRIPT molecules should be accounted for, as it is more ideal to have the same chain length molecules grafting on the surface. In conjunction to surface curvature of the MS nanoparticles, chain length should be kept relatively similar so as to

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improve the maintenance of surface charge neutrality. On the issue of chain length, several studies had shown that twenty carbons and below for

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monolayer are the most ideal for producing stable monolayer[74] while well ten carbons and above are required to produce well-packed and ordered

4.

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SAM[75].

Synthesis of zwitterion functionalized MS nanoparticles

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Prior to discussing the different chemistries and functionalization routes, it is important to state that the synthesis and fabrication of MS nanoparticles had

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already been amply covered in the existing literature and will be no added impedus to further dwell on this topic in this review. Some of the more common approach include soft and hard templating[76], Stöber process[77,

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78] and sol gel strategies[79], just to name a few of them.

While the

conditions of producing MS nanoparticles may differ (temperature, pH,

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surfactant type), the building blocks remain essentially the same, i.e. silica species and organic templates serving as surfactants.

On the subject of

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production of MS nanoparticles, a good review by Wu et al. had highlighted the virtues and cons of each of the processes[80]. Nonetheless, the ultimate aim is to achieve a good suspension of MS nanoparticles in solution and this is often aided by the choice of the surfactant.

What is more important is that the intended use of the silica nanoparticles may predetermine the type of methodology by which the MS nanoparticles is produced. Yet, from the mechanistic point of view, regardless whether the intended use is for drug delivery or bioimaging, the actual interfacing of the nanoparticles with the biological protein in-vivo would be at the outermost surface and the interior nanoarchitecture is thought to play a relatively minor role in deciding if there will be ultimately biofouling toward the nanoparticles.

ACCEPTED MANUSCRIPT However, the selection of the fabrication method would still be considered on an ad hoc basis if the function of “stealth” is to be implemented on the surface of the nanoparticles for biological applications.

For example, if the

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subsequent surface modification for a drug-loaded nanoparticle may result in seepage of the drug into solution, a more suitable functionalization approach

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should be selected (i.e. choice of solvent) and designed in order to minimize

4.1

Surface

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the loss.

functionalization

of

Silica

Nanoparticles

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(thoughts and considerations)

In the highly ionic environment of body fluid, mere physisorption of organic

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layers onto the surface silica nanoparticles is deemed unstable and therefore, it is necessary to incorporate the organic layer covalently. The selection of the

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chemistry route is very much dependent on the type of chemical grafting required as well as intended use of the nanoparticles.

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As mentioned, while the choice of the chemistry functionalization route on the surface of the nanoparticles is dependent on how the silica nanoparticles are

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produced, often the starting surface to initiate the reaction is Si-OH. It is important to note that surface resident silanol groups that must be handled with caution due to the possibility of form irreversible Si-O-Si bridges with neighboring nanoparticles upon the removal of the organic surfactant phase. While many groups had used air-based calcination at elevated temperature to remove the surfactant, this may also compromise the „bioactiveness‟ of the MS nanoparticles, especially if there is drug loading via covalent attachment to the silica. This formation of interconnecting bridges would result in uncontrollable aggregation upon which is self-defeating. The removal of surfactant by extraction and with solvents at elevated temperature had help impede the problem of aggregation effect[81].

MS silica surfaces are

occupied with various types of silanol (Si-OH) species and siloxane bridges and the interaction with water will rehydroxylate the surface to form

ACCEPTED MANUSCRIPT predominantly Si-OH group that can then be used as a starting point for

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passivation.

Prior to examining the types of functionalization chemistry, it is important to

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state that unless the monolayer on the surface of the MS nanoparticle is well controlled (i.e. film thickness and homogeneity etc), most groups would opt to deposit a thick layer of polymer zwitterionic film to improve the anti-biofouling characteristics. With regards to forming thick polymer grafts on silica surface

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that are often used in zwitterionic system, two terms are often used in literature to describe the way the polymer/monolayer is introduce to the

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surface. The “graft-to” approach means having the entire monomer/polymer directly grafted to the silica surface while the “graft-from” terminology refers to propagation of the zwitterionic film using the surface as the starting point.

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From a holistic stand-point of view, grafting via a “graft from” strategy will confer a thicker and better stack polymeric layer due to the lesser steric

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hindrance compared to the “graft to” approach[82] although the latter remains relatively popular in literature. In actuality, many of the grafting approaches

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often rely on a combination of both “graft to” and “graft from” approach, for example, functionalizing the initial Si-OH layer with a spacer with a functional distal and subsequently initiating polymerization from this distal end, as shown

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by Sun et al[62].

Nonetheless, regardless of how the anti-biofouling

zwitterionic film is grafted on the surface, the first steps often involve the direct modification of the very top Si-OH layer with a bifunctional/multifunctional organic intermediate chain that would decorate the surface with other functional groups for further conjugation or covalently grafting the zwitterion chains directly. Hence, it often involve a two-step process by which the native OH is passivated with an initial organic linker and the distal end of this linker is then responsible for subsequent surface grafting of the zwitterion film as exemplified by Quintana et al[83]. Also, depending on the way the MS nanoparticle is made, this functionalization of the Si-OH may not exactly be necessary if the silica material is made via co-condensation with alkylated species that confers active functional groups or even zwitterionic groups as demonstrated by Collila et al[84]. While this is very much a fast single step

ACCEPTED MANUSCRIPT fabrication route to form a viable zwitterionic surface, one major drawback may be the presence of existing or unreacted OH species from the silica that may alter the surface charges and this could affect the outcome of zwitterion‟s

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anti-biofouling performance. Still, it is frequently reported in literature that researchers would initiate the actual grafting of MS surfaces from the Si-OH

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surface groups. The forthcoming section shall discuss some of the possibilities for working with Si-OH groups and their subsequent grafting strategies and all citations of the reaction schemes as discussed were cited

4.2

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with silica surface in mind.

Si-OH surface functionalization

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Silanization is, by far, the most popular technique for surface modification on MS nanoparticles. Especially for sol-gel methodology for preparation of the

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MS nanoparticles, the Si-OH surfaces are often functionalized under hot ethanolic solution of an appropriate silane immediately after nanoparticle had

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been fabricated during the surfactant removal step[85]. This helps to serve two important purposes. Firstly, the degree of aggregation is much reduced

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by avoiding the formation of Si-O-Si bridging of the nanoparticles after the surfactant from the fabrication step is removed. Secondly, it will immediately confer the next functional group at the distal end for further reaction.

Scheme 1. Silanisation on silica surface

While the process of silanisation on the surface is fast, one potential disadvantage is the cross-linking effect between the silane chains, which may result in uneven distribution of the R group on the surface. This inequality in

ACCEPTED MANUSCRIPT distribution may pose a problem if the zwittterionic system is the mixed SAM system whereby good distribution of the alternating charges is sought for. Furthermore, the inability for betaines to dissolve in organic solvents had

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mostly confined the use of silanisation mainly as a „preparatory‟ step prior to polyzwitterion betaine grafting. Hence, many papers in literature had been

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restricted to using silanes only as initial starting points to introduce additional functional groups that can help in the subsequent grafting of zwitterions chain

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to silica nanoparticle surface[62, 82, 86].

Liu et al. had shown that it is feasible to react with epoxy in the presence of

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Tin Chloride[87] although care must be taken during the reaction as it is possible for the epoxy group can be converted to dihydroxyl under acidic conditions[62].

This is a relatively uncommon method compared to

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silanisation but the major advantage is that a monolayer is almost guaranteed as each epoxy will reach only with one –OH group and packing density is only

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limited by the steric hindrance of the carbon chain. Unlike silanisation, it does

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not cross-link with itself and hence the film thickness can be well controlled.

Scheme 2. Reaction with Oxirane with Si-OH

Kim et al. had shown that it is possible to directly abstract hydrogen from the Si-OH group to form a radical site for reaction[88] using a photosensitizer, which in this case was Benzophenone. The method involved the absorption of UV at 250 nm that in turn forms free radicals that allows for the abstraction of hydrogen from the silica surface. The radicalized surface then proceeds to form a Si-O-C bond with unsaturated carbon ends on the polymer. There may be certain complications with such system such as the removal of

ACCEPTED MANUSCRIPT photosensitizer after the reaction as well as the degradation of the polymer over prolonged periods of UV irradiation[89]. Benzophenone O

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OH

Hydrogen abstraction

O

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UV irradiation

R

Radicalized state

O

O R

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OH

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Silica Nanoparticles

Scheme 3. Abstraction of hydrogen by radical generated from photosensitizer

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Catechol, or 1,2 dihydroxylbenzene, are useful in interfacing with –OH groups to form a multivalent type linkage to the surface. Its adhesiveness towards

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silica surface OH is useful especially in “graft to” approach, by which entirely “pre-synthesized” polymer film may be directly grafted to the silica surface[90,

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91]. While the linkage via a multivalent surface bond is strong, one major concern would be the issue of steric hindrance that can impede the level of

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surface coverage.

Scheme 4. Catechol attachment to Si-OH handle

ACCEPTED MANUSCRIPT Isocyanates can also react directly to silica Si-OH surface and this was as exemplified by Sugimoto et al[92] with the advantage being that the reaction does not liberate alcohol or water from the bond formation. However, the

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major concern with using isocyanate is the highly reactive nature towards a wide range of different common functional group such as amide, anhydride

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and epoxides[93], thus making it rather difficult to work with.

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Scheme 5. Isocyanate reaction to silica surface

Thionyl chloride can be used to react with the OH group to form a Cl terminus

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can be used for further reaction[94, 95] and this may prove to be useful considering that the Cl in place of –OH is a much easier leaving group which

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allows for the other reactions to take place subsequently on the surface. This

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is especially useful for polymeric grafting[94].

Scheme 6. Thionyl chloride interfacing with Si-OH for the surface chloronation

Hydrosilylation is a well-established technique on flat silicon based substrates[96] although there had been some attempts to use on silica nanoparticles[97]. Although the Si-C is more stable than Si-OR type bond, the use of HF to remove the OH group to form silicon hydride (Si-H) will reduce the size of the silica nanoparticles that might downgrade its usefulness as the time-dependence size reduction of the nanoparticles from the HF

ACCEPTED MANUSCRIPT etching could pose a challenge. On the other hand, Si-C linkage on the silica nanoparticle is extremely stable for subsequent surface grafting under harsh

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chemical conditions.

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Scheme 7. Hydrosilylation chemistry for formation stable Si-C linkage

Zwitterionic “Graft to” strategies (common conjugation

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chemistry on the distal R group)

“Grafting to” involves the direct reaction of either an organic spacer with a

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functional group “R” that is meant for subsequent polymerization or the actual zwitterionic chain to the surface (as in the case of Mixed SAM). The surface

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may either be passivated with an organic linker or directly towards the native Si-OH group. Typically, if the intention is to graft an aliphatic chain of betaine

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nature, it is imperative to understand that most betaine/polyzwitterionic betaines are mainly soluble in polar protic solution and therefore all reactions must take the solubility of betaine into account, especially for users wanting to graft betaine directly on the first chemically passivated layer on silica. Considering that the chemical possibilities for conjugation are relatively extensive, only some of the most significant ones will be discussed here.

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Illustration 1. Graphical representation for “graft to” approach and the possible zwitterionic combinations

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Amine (-NH2) terminated chemistry is by far the most popular terminus group reported in literature. The accessibility and ease of amine to form covalent Isocyanate and

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bond with incoming organic chains is well established.

thioisocyanate are grouped together as shown below as being able to react

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with primary amine to form urea and thiourea linkage[98]. While isocyanate derivatives are often used in fluorescence probes to form direct linkage to amino-terminated surfaces[99], its use on silica nanoparticle is complicated by the reactive nature to many functional groups that subsequently impedes its wider use.

Scheme 8. Surface amine reacting to isocyanate and thiocyanante

ACCEPTED MANUSCRIPT The interfacing of 1,3 propane sultone with an amino group results in the ring opening event that produces a positively charged NH2 and a negatively charged SO3 on the same chain residue[62, 100], hence producing a While this methodology had not been widely

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zwitterionic surface graft.

explored in literature due to the general preference of grafting thick

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zwitterionic brush, in view of the developing generic “monolayer”, this approach is believed to hold much promise for forming “true” ultrathin

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monolayer on the surface.

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Scheme 9. Formation of basic “betaine” from ring opening reaction between amine and 1,3 propane sultone

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One of the most popular methods of functionalizing primary amine terminated surface is via the reaction via the hydrolysis of N-Hydroxysuccinimide (NHS)

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and had been reported quite extensively for silica nanoparticles to form an amide link[101, 102]. The major advantage with resorting to the NHS route is

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that sulfonated forms of NHS permit for reaction in water, which is a major advantage for polyzwitterionic betaine chains. Hydrolysis of NHS ester is pH dependent; in basic conditions (pH >8), the reaction can be completed in a matter of minutes. Another advantage with NHS is that its derivatives are widely available commercially.

Scheme 10. NHS ester reacting with primary amine to form amide linkage

Sulfonyl chloride can react with silica surface amine to form covalent sulfonamide linkage[103] via the nucleophilic attack of the amine by the

ACCEPTED MANUSCRIPT sulfonyl chloride in basic polar solvent[104] such as pyridine and can be done under room temperature within minutes[105]. Furthermore, sulfonamide had been shown to have very potent pharmaceutical bioactivity[106, 107] and this

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may be an added incentive to form a sulfonamide bond on silica nanoparticle for polyzwitterionic grafting although more studies would be required on this

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topic.

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Scheme 11. Sulfonyl chloride reacting with primary amine to form sulfonamide bond

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Surface amine from silica nanoparticle and aldehyde can also condense to form a strong covalent N=C linkage under aqueous conditions at room

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temperature[108, 109]. The same reaction can also proceed via carbodiimide

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intermediates[110].

Scheme 12. Amine-aldehyde condensation reaction

Epoxides are useful in reacting to amino terminated silica surface[110, 111]. While epoxy groups are often used to produce network structure of nanocomposite[112, 113], epoxy affinity to other functional group such as thiol and hydroxyl (Si-OH) may have limit its function as a „chemoselective‟ ligation towards amine, especially on silica nanoparticles having unreacted surface OH sites and these sites are also susceptive towards the epoxy.

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Scheme 13. Epoxy reaction to amine via the amine reactive hydrogen. In principle, each primary amine can form two linkage with different epoxy.

Maleic anhydride can also be used to form a reversible amide bond with primary amine functional group[114, 115].

Under basic conditions, ring

opening will occur but the reaction is reversible under acidic conditions. This

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may impose a challenge if there are fluctuations in pH although it must be said that the reaction with primary amine is very spontaneous in aqueous

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conditions.

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Scheme 14. Reversible ring opening reaction between Maleic Anhydride and primary Amine

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The carbodiimide chemistry route for forming an amide linkage between an amine and a carboxylic acid in solution is a very well established and popular methodology[116-119]. In brief, carbodiimide derivatives such as n-ethyl-N‟(3- dimethylaminopropyl)carbodiimide (EDC) will react with carboxylic acid to form an active Acylisourea intermediate and will subsequently be displaced by primary amine present in the reaction system.

In presence of N-

hydroxysuccinimide ester (NHS), the intermediate Acylisourea reacts with the NHS to form an amine reactive NHS intermediate which rapidly expedite the reaction with primary amine. EDC and sulfonated form of the NHS is water soluble, making the reaction very compatible with polyzwitterionic betaines especially for the “graft to” strategy. Carbodiimide assisted coupling also works equally well with epoxy and carboxylic acid as shown by Sun et al[62].

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ACCEPTED MANUSCRIPT

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Scheme 15. EDC/NHS assisted coupling between primary amine and carboxylic acid to form an amide bond

One of the most important functional groups in literature is thiol (SH) and its

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conjugation chemistries are very well understood. However, passivating the silica surface with unprotected thiol is unadvisable due to the susceptibility

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toward oxidative dimerization, hence creating issues of crosslinking. Nevertheless, conjugation with thiol groups is feasible, as shown in Scheme 16. One of the most popular methods reported in literature to form disulphide bonding is via the use of a protective pyridyl disulfide group that will be liberated to form a disulfide bridge in the presence of thiol carrying species in solvent[120, 121]. Epoxy can react with thiol under relatively mild conditions (water/acetone solvent at room temperature) and is very rapid (1 hour) as demonstrated by Grazu et al[122]. Maleimide can also be used to conjugate with thiol[123, 124] and the reaction conditions are relatively mild (buffers without thiolated species at pH 7-7.5) although the solubility in water is poor. Solubility of the maleimide can be improved by either a co-solvent (DMF, DMSO) or using the sulfonated version. Another advantage of maleimide is its wide commercial availability.

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Scheme 16. Thiol reaction with (1) pyridyl disulfide, (2) epoxy group and (3) maleimide

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Apart from reacting with thiol, maleimide can be made to undergo Diels-Alder reaction with a furan group[125] and had also been demonstrated on silica

maleimide-furan

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nanoparticle surface by Engel et al[126]. conjugate

is

thermally

It must be note that while the reversible,

the

detachment

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temperature of the furan is above 100°C but this is well beyond the operation temperature for biological applications.

Furthermore, as long as both the

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maleimide and furan bearing residues possesses sufficient polar groups, it is also possible to perform the reaction in water under very mild temperatures without catalyst, as shown by Garcia-Astrain et al[127].

Scheme 17. Diels Alder reaction between Maleimide and Furan

Schiff-base condensation between an aldehyde and a hydrazine can also occur on silica nanoparticles to form a hydrazone bond[128] although the

ACCEPTED MANUSCRIPT bond itself is relatively pH sensitive, i.e. degradation occurs at low pH. The bond stability, however, can be improved using an aromatic aldehyde[129]. One precaution regarding the use of hydrazine based linker is that the

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functionalization of hydrazine onto the surface of silica nanoparticle is strongly discouraged due to the explosive nature of hydrazine and its methylated

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derivatives in solid state[130].

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Scheme 18. Schiff-base condensation between Aldehyde and hydrazine

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Click chemistry is very popular and this is no different for silica nanoparticles. Copper assisted Alkyne-Azide Cycloaddition (CuAAC) had recently been

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reported in several papers[131, 132] which is intended for subsequently radical polymerization[133], an approach well suited for the grafting of

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polyzwitterionic betaines. The main attraction of using click chemistry is that the 1,2,3-triazole formed is very stable chemically and does not interference with subsequent reactions. However, the presence of copper catalyst may

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not be the most ideal of situation considering that alkyne may form complexation with copper catalyst[133, 134] as well as the mesoporous structure of silica.

However, the emergence of newer copper-free click

reaction may resolve this issue adequately[134].

Scheme 19. Click chemistry on silica nanoparticles

ACCEPTED MANUSCRIPT 4.4

Examples of “Graft to” approach

First and foremost, zwitterionic grafting on silica surface is relatively new in literature and in most of the „graft to” strategies as reported in literature for

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silica nanoparticles, silane chemistry had dominated the scene due to the ease and accessibility of the surface modifications.

Nonetheless, it is

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important to state that many of the conjugation chemistries as discussed above are feasible considering the all reported citations in this review originates from chemistry performed on silica surface. While “grafting from”

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strategies are highly popular due to the desire to saturate the surface with thick polymer brush in hope of maximizing the function of zwitterionic, several papers still reported on the “graft to” approach. A notable example for „graft to‟

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strategies was demonstrated by Estephan et al[135] who functionalized a sulfobetaine siloxane to the silica nanoparticle surface through silanisation chemistry.

More importantly is that upon functionalization the SB silane,

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incubation with lyzosomes shows no change in hydrodynamic ratios over a course of 30 hours while untreated nanoparticles shows an instantaneous

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increment in hydrodynamic ratios within minutes. This in turn suggested that silanization of a thin film on the surface help reduce protein non-specific

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adsorption on the nanoparticle surface.

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ACCEPTED MANUSCRIPT

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Figure 7. “Graft to” approach by which zwitterionic betaine is directly silanised onto silica nanoparticle surface and demonstrated very little increment in hydrodynamic radius under incubation of proteins[135].

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Another example for “graft to” approach was shown by Rosen et al[136]. Silica nanoparticle surface was first silanised to present an epoxy terminal group

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and this was then coupled to cysteine via thiol group (Scheme 16-2). While cysteine exhibits zwitterionic properties from its terminus NH2 and COOgroups, it is generally not considered to be a strong zwitterion for repelling protein adsorption as evidenced by the varying performance of adsorption between different protein types.

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Figure 8. Cysteine functionalized onto the surface via an epoxy-thiol reaction and the different hydrodynamic profile for (a) lysosome and (b) albumin[136].

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Zhu et al. utilized a robust catechol attachment (scheme 4) to the surface[90] to introduce a thick polymeric zwitterionic film on silica surface. Instead of

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approaching it via the “graft from” strategy, polymerization of the zwitterionic was first completed with a catechol functional group at the terminal end and

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the whole polymer film is functionalized to the surface in entirety. Building from a carboxybetaine formulation, the silica nanoparticle was shown to be

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resistance towards cellular uptake after 4 hours of incubation. Interestingly, as mentioned earlier, the authors also perform EDC/NHS coupling of the COO- groups in the film with a RGD peptide and this results in a reversal in trend of nanoparticle cellular uptake, hence demonstrating the „extra‟ functionality of carboxybetaine polymers. This result is extremely important considering that specific antibodies can also undertake this route to attach to the zwitterionic film so as to form a stealth/smart nanoparticle system.

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Polymeric grafting on functionalized MS nanoparticles:

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4.5

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Figure 9. Grafting of polymerized carboxybetaine to silica nanoparticle via catechol attachment and the subsequent coupling of RGD to COO group on the polymer residue to improve cellular uptake[90].

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“Graft from” strategies Using surfactant templating, co-condensation methodologies can certainly

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fabricated nanoparticles decorated with polyzwitterionic film on the surface such shown by Hankari et al[137].

However, these approaches may be

unsuitable for grafting nanoparticles with good size disparity without the high temperature calcination step that would in turn may thermally degrade the organic polyzwitterionic betaine[81].

Hence, some of the most important

procedures for acquiring biologically compatible zwitterionic film on silica surface involve working from the outmost surface of silica nanoparticles and direct grafting of polymer on silica surface is not straightforward from the starting silanol surface group.

While physisorption of zwitterionic film is

straightforward, the interfacing with the surface is not deemed stable enough for biological environment.

Therefore, when addressing polyzwitterion

polymerization on the surface, it is important to state that discussion below focus solely on the grafting chemistry initiated from the very top most layer of

ACCEPTED MANUSCRIPT silica surface.

Many excellent reviews had already covered on the

mechanisms of different types of polymerization and can serve a good background material in understanding the forthcoming discussion [138-140].

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As the amount of content in literature regarding polymerization techniques does not realistically permit for the discussion of every single possibility in this

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review, only some of the major mechanisms that can potentially be used in the formation of zwitterion anti-biofouling layer shall be discussed. Of these, free radical polymerizations had dominated the literature due to the tolerance of the zwitterionic betaines towards radicals. Three of the more popular

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reversible deactivation radical polymerization methods are Nitroxide-mediated Polymerization (NMP), Atomic Transfer Radical Polymerization (ATRP) and

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Reversible Addition Fragmentation chain-Transfer (RAFT) reactions.

ACCEPTED MANUSCRIPT Illustration 2. Graphical representation for “graft from” approach and the possible zwitterionic charge combinations. Although NMP represent the earliest

of

all controlled/living radical

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polymerization, it had not been used extensively for grafting and polymerizing betaines for the simple reason that it requires higher temperatures to work

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and that can often hydrolyze and degrade many betaines[139]. However, there are several obvious advantages in using NMP. Firstly, NMP does not require cytotoxic organometallic catalyst or produce a Bromide end group as in the case of ATRP and the radical is relatively stable. In fact, the absence of

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catalyst could be crucial for the MS nanoparticles in biological applications as removal of organometallic catalyst would be daunting task in porosified

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nanoparticles. Secondly, it also allowed for better control of the polymerization and hence better polydispersity for nanoparticles. So far, only Sinoj et al. had demonstrated that it is feasible to obtain zwitterionic films via NMP using a

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modified alkoxyamine initiator[70, 141].

Scheme 20. NMP polymerization on silica nanoparticle

Surface grafting via ATRP is another controlled/living radical polymerization technique that can be useful for grafting and growing polyzwitterionic chain on the silica surface[142, 143]. This generally requires a bromoester initiator on the surface and copper bromide catalyst to initiate the polymerization. While ATRP would give the highest surface grafting densities of all the living radical polymerization techniques[144], there are a few concerns regarding its use with MS silica. Firstly, bromoester itself is relatively hydrophobic and may not work well with silica nanoparticles dispersed in aqueous solution. Secondly,

ACCEPTED MANUSCRIPT the removal of the copper bromide catalyst may be a concern in a higher porosified surface and in biological applications and the retention of this

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catalyst species may be hazardous in the long run.

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Scheme 21. ATRP polymerization on silica nanoparticle

RAFT is a popular method of grafting zwitterionic layer on silica nanoparticle Its tolerance of a wide range of unprotected

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surface[62, 86, 145, 146].

functional groups in its monomer chains (OH, NH2 and COOH etc) as well the as the ability to perform the polymerization in aqueous conditions renders it as

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a very attractive candidate in “grafting from” strategies.

Furthermore, the

absence of organometallic catalyst means that lower probability of surface

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contamination. Another advantage with using RAFT is the ability to undergo polymerization under water, which is very attractive for biological intended

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applications. Nonetheless, there are some weaknesses in RAFT that may impede its broader use, such as the inability of initiator agents to graft certain monomers as well as lower chain density and slower reaction time than compare to ATRP which may pose a challenge for drug loaded MS nanoparticles. N N

N N

Azobisisobutyronitrile Radical Initiator

O

R

Z

S n

S

R R

Silica Nanoparticles

O

Z

S

Monomer

R

S

ACCEPTED MANUSCRIPT Scheme 22. RAFT polymerization on silica nanoparticle starting off from a thiocarbonylthio group

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Generally, monomeric and polymeric betaines are relatively insoluble in nonpolar solvent and should be handled under protic solutions. Solubility is crucial

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in the polymerization step and the solubility profiles of polymeric betaines are thought be to limited to solvents that has ability to disrupt ionic interaction, hence solvents with hydrogen-bond donating properties. As such, radical free polymerization of polyzwitterionic betaines is often performed in almost

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exclusive aqueous solvent such as water or a water/polar solvent mix[59, 147]. Zwitterion chains such as those from polysulfobetaines had exhibited strong

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electrostatic interaction between the sulfobetaine groups arising from the high dipole[148] moment. They also exhibit an upper critical solution temperature (UCST) and this in turn result in the chain expansion being ionic strength

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dependent. In salt-free pure water, polyzwitterion betaine remains insoluble and shrink into a condense state until the addition of salts and only with an

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increase of salt concentration, the polyzwitterionic increases its solubility in water. This behavior had been coined in literature as the “anti-polyelectrolyte”

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effect[71]. It is thought that the salt electrolyte permeates the ionic network and masks the electrostatic attraction within the polymeric chain and therefore

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increases the solubility.

An excellent review by Lowe et al. had further

elaborated on this mechanism[71].

While solubility in organic solvent is

limited for the polybetaines, both PC and CB exhibit higher degree of solvation in organic solvent compared to SB. While some other methods such as ring opening metathesis polymerization (ROMP) with its mild reaction conditions are potential possibilities, they had not been extensively evaluated for grafting polyzwitterionic betaine on silica nanoparticles Laibinis‟s group had shown that it is possible to initiate ROMP from SiO2 surfaces.

Subsequently, Colak et al. had produced a

polyzwitterionic betaine using ROMP with functional silanol that can be grafted on silica surface in principle under polar THF solvent and mild temperature conditions[149]. However, the synergy of polyzwitterionic betaines and silica nanoparticles via the ROMP process had yet been

ACCEPTED MANUSCRIPT demonstrated in literature. Nonetheless, the use of the Ruthenium complex

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may pose a challenge at the removal stage.

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Scheme 23. ROMP polymerization via norbenene initiator on the surface

Other methods such as anionic, cationic and photopolymerization are not

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considered for various reasons. Firstly, anionic/cationic based polymerization may be incompatible due to the need for having protic solvent as well as salt

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present in the system[150] which could interfere with the polymerization event. Photopolymerization, on the other hand, may not work well on spherical

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nanoparticles for producing good surface coverage.

4.6

Examples of “Graft from” approach

“Graft from” strategy for producing thick zwitterionic films are very popular in literature and this is not an exception for silica nanoparticle. Huang et al. had started with silanisation and subsequently graft with RAFT and the schematic of the reaction is as shown in figure 10[86]. In brief, through a silanisation, the authors introduce a thiocarbonylthio group on the surface, which in turn permits for RAFT polymerization to initiate. 4,4'-Azobis(4-cyanovaleric acid) was added as the radical initiator for chain propagation and subsequently used as a hydrophilic interaction chromatography medium for the enrichment of glycopeptides. Results had shown that nanoparticles grafted with the

ACCEPTED MANUSCRIPT zwitterionic brush via the RAFT method was able to identify a higher number of unique N-glycosylation sites compared to other grafting methods.

The

authors also graft single zwitterionic monolayer on the silica nanoparticle

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using the “graft to” approach but the dense polymeric was shown to be the

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most effective.

Figure 10. Schematic overview of “graft from” strategy as employed by Huang et al. for the enrichment of glycopeptide[86]

Dong et al. had also shown the feasibility of “graft from” approach for polymerizing sulfobetaine via ATRP[151]. In brief, silanization was first performed to ultimately present the surface with alkyl bromide that was

ACCEPTED MANUSCRIPT subsequently utilized for ATRP at room temperature. While the resultant nanoparticle had shown resilience in resisting protein adsorption, the authors had elaborated on an important point pertaining to the temperature dependent

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phase transistion. Under the USCT, nanoparticle aggregate and can only be individualized at temperature above the USCT point. This is an important

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finding that strongly highlighted the necessity to maintain the USCT for the zwitterionic polymeric graft below 37°C if the nanoparticles are targeted for

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biological applications.

Figure 11. ATRP on silica nanoparticles as demonstrate by Dong et al[151]

Sun et al. had shown the possibility of using “graft from” approach to produce stealth MS nanoparticles as drug carriers[62].

MS nanoparticles were

functionalised with silanes terminated with an epoxy moiety and this underwent carbodiimide mediated coupling with the terminal carboxylic group in S-1-dodecyl-S'-( α , α '-dimethyl- α ''-acetic acid) trithiocarbonate.

The

trithiocarbonate subsequently serve as initiating sites for RAFT polymerization of polysulfobetaine. Rhodamine B was then loaded into the MS nanoparticles

ACCEPTED MANUSCRIPT by diffusion in aqueous conditions. Drug release was monitored at different temperatures corresponding to the transition temperatures. Authors showed that the diffusion of drug out of nanoparticle system is temperature dependent Further cytotoxicity

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due to chain collapse at higher temperature (50°C).

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studies of the nanoparticle to cell had only shown minimal toxicity.

5.

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Figure 12.RAFT on MS silica nanoparticle and subsequent drug delivery at different temperature[62]

Conclusion

The advantages of zwitterion compared to polyhydrophilic films are well accepted although much challenges lie ahead due to the complex functionalization chemistry involved. Nonetheless it is in the authors‟ opinion that zwitterionic films modifications on silica nanoparticles can truly supersede many of the existing polymers to achieve anti-biofouling targets. In this review, many of the common reaction routes that had been under taken for MS materials had been described in the to serve a large and diversified community of future applicants for this chemistry. Although the choice of the synthetic methodology clearly depends upon the nature of the user‟s

ACCEPTED MANUSCRIPT application, it is in the authors‟ opinion that living polymerization such as RAFT will become the mainstay for such surface modifications as thicker surface layer is often more preferred. Nonetheless when choosing a suitable

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chemistry approach for inferring a stealth layer on the surface of silica nanoparticles for biological applications, it is imperative to consider a number

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of fundamental aspects, namely:

1. Ease of functionalization chemistry: whether the surface layer can be easily incorporated without any interference with the intended use of the nanoparticle

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2. Whether to undertake the “Graft to” or the “Graft from” approach in the synthesis

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3. The influence of transition temperature in view of biological application

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intentions.

As mentioned, silica nanoparticles and zwitterion films are relatively new in

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literature and there is certainly much room and avenue for exploration in the future considering that silica nanoparticles is one of most widely

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utilised nano-platform for many of today‟s challenging biological questions.

References

[1] Yanagisawa T, Shimizu T, Kuroda K, Kato C. The preparation of alkyltrimethylammonium-kanemite complexes and their conversion to microporous materials. B Chem Soc Jpn. 1990;63:988-92. [2] Kresge CT, Leonowicz ME, Roth WJ, Vartuli JC, Beck JS. Ordered mesoporous molecular-sieves synthesized by a liquid-crystal template mechanism. Nature. 1992;359:710-2. [3] Slowing II, Vivero-Escoto JL, Wu C-W, Lin VSY. Mesoporous silica nanoparticles as controlled release drug delivery and gene transfection carriers. Adv Drug Deliver Rev. 2008;60:1278-88. [4] Argyo C, Weiss V, Braeuchle C, Bein T. Multifunctional Mesoporous Silica Nanoparticles as a Universal Platform for Drug Delivery. Chem Mater. 2014;26:435-51.

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In this review, we described the various chemical strategies for grafting zwitterions based anti-biofouling film on mesoporous silica nanoparticles. We also give examples of the latest development in using zwitterionic films to create stealthy mesoporous silica nanoparticles oblivion towards protein adhesion.