Covalent Guanosine Mimetic Inhibitors of G12C KRAS - ACS

Review Article Growth factor conjugation: Strategies and applications Mirhamed Hajimiri,1,2 Sheida Shahverdi,1 Golnaz Kamalinia,1,2,3 Rassoul Dinarvand1,3 1

Nanomedicine and Biomaterial Lab, Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 1417614411, Iran 2 Nano Alvand Co., Avicenna Tech Park, Tehran University of Medical Sciences, Tehran 1439955991, Iran 3 Nanotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 1417614411, Iran Received 30 January 2014; revised 17 March 2014; accepted 3 April 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35193 Abstract: Growth factors, first known for their essential role in the initiation of mitosis, are required for a variety of cellular processes and their localized delivery is considered as a rational approach in their therapeutic application to assure a safe and effective treatment while avoiding unwanted adverse effects. Noncovalent immobilization of growth factors as well as their covalent conjugation is amongst the most common strategies for localized delivery of growth factors. Today, immobilized and covalently conjugated growth factors are considered as a promising drug design and are widely used for protein reformulation and material design to cover the unwanted characteristics of growth factors as well as improving their functions. Selection of a suitable conjugation technique depends on the substrate chemistry and the

availability of functional reactive groups in the structure of growth factor, the position of reactive groups in growth factor molecules and its relation with the receptor binding area, and the intention of creating either patterned or unpatterned conjugation. Various approaches for growth factor reformulation have been reported. This review provides an overview on chemical conjugation of growth factors and covers the relevant studies accomplished for bioconjugation of growth facC 2014 Wiley Periodicals, Inc. J tors and their related application. V Biomed Mater Res Part A: 00A:000–000, 2014.

Key Words: growth factor, conjugation, bioconjugation, immobilization, covalent

How to cite this article: Hajimiri M, Shahverdi S, Kamalinia G, Dinarvand R. 2014. Growth factor conjugation: Strategies and applications. J Biomed Mater Res Part A 2014: 00A:000–000.

INTRODUCTION

Growth factors were initially discovered as a result of their ability to motivate continuous mitosis of quiescent cells in a nutritionally complete medium without serum. While nutrients and growth factors are both essential for mitosis, only growth factors know how to initiate mitosis of quiescent cells. A variety of cellular processes need growth factors as regulatory agents. The biology of these factors differs from the classical hormones as neither their site(s) of synthesis nor site(s) of action is limited to defined tissues.1,2 They are also known as intercellular signaling molecules, which promote cell proliferation, migration, differentiation and maturation depending on their type. Some growth factors act in a nondiffusible manner (e.g., juxtacrine and matricrine) but most of them act in a diffusible manner (e.g., endocrine, paracrine, autocrine, and intracrine; Fig. 1). It should be considered that each growth factor may have multiple mechanisms of action at the cellular level.2,3

The growth factors that act in a diffusible manner are generally unstable in their environment because of rapid degradation or inactivation prior to reaching their target.4,5 For instance recombinant platelet-derived growth factor (PDGF) can improve healing rates and reduce time to wound closure in diabetic neuropathic foot ulcers in the gel form.6,7 To overcome this disadvantage, large amounts of soluble growth factors are frequently needed to affect cellular outcomes. However, the delivery of such large quantities has the potential to damage cells and tissues. Many attempts have been made to maintain the activity and improve the performance of growth factors. The most common strategy to prolong growth factors retention in their environment is to generate growth factor modified substrates by covalent bonds for many applications including wound healing, tissue engineering, targeting, etc.8,9 Several studies have demonstrated that cancer cells overexpress growth factor receptors. Thus, the other application

Conflict of Interest: No benefit of any kind will be received either directly or indirectly by the author(s). Correspondence to: R. Dinarvand; e-mail: [email protected]

C 2014 WILEY PERIODICALS, INC. V

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FIGURE 1. Growth factors functioning in nondiffusible (juxtacrine and matricrine) and diffusible manners (endocrine, paracrine, autocrine, and intracrine). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

for covalent conjugation of growth factors is playing the role of a ligand substrate to target anticancer drugs. This approach could help to defeat many disadvantages of systemic administration of anticancer treatments.10,11 GROWTH FACTOR DELIVERY APPROACHES Localized growth factor delivery is considered as a rational approach in growth factor therapeutic application to assure a safe and effective treatment while avoiding unwanted adverse effects.12 Noncovalent immobilization of growth factors on an appropriate delivery system, as well as their covalent conjugation is amongst the most common strategies for localized delivery of growth factors. Non-covalent immobilization is usually achieved by growth factors physical entrapment, surface adsorption, affinity based binding and ionic complexation.13 The selection of each of these choices is totally dependent on the physicochemical characteristics of growth factor and carrier and any interactions between them.8,13 As an alternative to physical immobilization techniques, covalent conjugation of growth factors to various substrates was introduced as a method to enhance the stability of growth factors and their perseverance when delivered to different cells and tissues.8 As a result of chemical conjugation the drug molecule will not undergo passive diffusion and the growth factor release will take place as long as the lifetime of the matrix.14 These objectives are not achievable through simple growth factor physical adsorption, which is controlled by weak noncovalent forces. Therefore a full control over growth factor delivery retention, orientation, or desorption rate would be made possible through conjugation strategies.15 By the help of conjugation, strategies the amount of growth factor required may be reduced which subsequently decreases the cost of growth factor delivery system and

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increases its efficacy.8 Moreover, covalently bonded growth factors show a more prolonged release when compared with physically immobilized growth factors.16 When successfully conjugated, the growth factor can retain its activity on the related receptors but the degradation rate will decrease significantly and the growth factor is internalized in a lower amount.16 Various instances of successful conjugation practice have been reported in literature.17,18 For example, when epidermal growth factor (EGF) was covalently conjugated on glass surfaces, its mobility was retained and the conjugated form was more effective in cellular growth. Covalent conjugation technique for growth factor delivery stands along with a range of other strategies including encapsulation techniques.13 Other than controlled release of encapsulated growth factors, the encapsulation strategy is of special interest because it can protect the growth factor from unwanted enzymatic effect, which makes it superior when compared with conjugation strategy.13 The carrier system degradation kinetics, protein loading and diffusion and size and zeta potential of the used nanocarrier are the determinant factors on the growth factor release.13 To increase the encapsulation efficiency in such systems protein encapsulation is usually accompanied by a surface protein grafting technique. However, research is continued in this field to overcome the barriers exposed by the harsh and unwanted process conditions along with the low encapsulation efficiency.13 Immobilization of growth factors has physiological importance, as soluble and jointed growth factors show distinctive functions in the in vivo environment.19,20 A study was conducted by Sahni et al. to investigate whether there are specific interactions between basic Fibroblast Growth Factor (bFGF) and fibrinogen and fibrin that could play a role in vessel repair.19 It was found that bFGF binds specifically and saturably to fibrinogen and fibrin at tissue injury site and provides a temporary matrix to support the initial responses needed for vessel repair.19 Not only the covalent conjugation techniques may preserve the growth factor and enhance its stability, but also they can change the impacts of growth factors on cellular behavior and functioning.8 In fact, cells may respond differently to soluble and immobilized growth factors. This may be due to different interactions of cellular receptors to soluble and immobilized growth factors. Soluble growth factors are simply recognized by cellular receptors and are internalized as a substrate-receptor complex. The immobilization of growth factor usually results in a lack of internalization and sustains the signaling pathways inside the cells.8 This phenomenon is called “artificial juxtracrine signaling” and is kept responsible for distinctive cellular behavior when exposed to immobilized growth factors.3 Despite the fact that this sustained activation of signaling pathways has received a great deal of interest, there are still many concerns on the unwanted effects of such long term receptor activation. Most growth factors are naturally present transiently during tissue repair processes and regeneration. Their inappropriate presence during the regeneration may stop the healing process or interfere with the process.8 Another concern in covalently conjugating growth factors is that they should keep their biological activity after the

GROWTH FACTOR CONJUGATION

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conjugation process. Various studies have confirmed that covalently conjugated growth factors have maintained their desired biological effects after the chemical coupling reaction.21,22 However, the reaction environment and chemistry of reaction may adversely affect the growth factor and lead to its denaturation or incomplete conjugation especially when the receptor binding site gets involved in conjugation reaction or when the conjugation prevents the growth factor access to its receptor.14 As a result, a very critical consideration in designing conjugated growth factor systems is to ensure that the conjugation process does not impair the efficacy and biological function of the peptide. Additionally, it should be made sure that the cellular internalization of growth factor is not necessary for producing its desired effects.14 CONJUGATION CHEMISTRY Selection of a suitable conjugation technique depends on the substrate chemistry, the availability of functional reactive groups in the structure of growth factor, the position of reactive groups in growth factor and its relation with the receptor binding area, and the intention of creating either patterned or unpatterned conjugation. Various conjugation strategies differ in a range of features and may be chosen according to these needs. For example the possibility of involving different functional groups in growth factor and inclusion of spacer molecules are among the important aspects of any conjugation reaction. Covalent conjugation of growth factors generally uses the aqueous based chemistry, since most of the growth factors are insoluble in organic solvents or may get damaged or denatured in such environments.8 Carbodiimide mediated conjugation reaction Carbodiimide coupling reaction represents one of the most common approaches for covalently conjugating growth factors to other molecules. Among these coupling reagents, 1ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) is prevalently used for bioconjugation purposes where it mediates the conjugation reaction between the carboxylic functional groups with the amino groups resulting in the formation of stable amide bonds intermolecularly.23 Carbodiimide conjugation reaction may engage the amino groups of growth factor in its lysine residues and Nterminus. EDC may also employ carboxylic groups of aspartic acid and glutamic acid on the primary structure of growth factors or take place on the terminal carboxylic group of these molecules. EDC works through carboxylic group activation and forms an O-acylisourea intermediate which is highly reactive towards amino groups. This intermediate is also vulnerable to hydrolysis based destructive reactions in aqueous medium and will turn back to carboxylic group if hydrolyzed.8 To prevent such reverse reaction and to improve the efficiency of conjugation reaction, Nhydroxysuccinimide (NHS) or N-hydroxysulfosuccinimide as it is more hydrophilic derivative are used to convert this intermediate into an ester and provide a less susceptible molecule against hydrolysis.24 The esteric intermediate participates more readily in amide bond formation (Fig. 2).8

Various advantages have been mentioned for carbodiimide based coupling reaction including simplicity, low cost, high conjugation ratio and the mild reaction conditions, which makes this method as one of the most popular techniques for growth factor covalent conjugation.8,25 Photoimmobilization Photoimmobilization or photoinitiated reaction is another method of growth factors covalent conjugation to substrate. Generally, the growth factor is first functionalized by a photoreactive group, and the modified growth factor covalently binds to the target substrate upon exposure to a selected wavelength of light (usually ultraviolet irradiation).8 One of the significant advantages of photoimmobilization when compared to other conjugation techniques is the feasibility of creating patterns of growth factors. In this technique, photomasks and laser scanning light sources are used to immobilize the growth factors in a special position in two and three dimensional environments. Such patterns may provide a better control over growth factor effect on cellular functioning including the induction of directed cellular migration by the aid of growth factors gradients.26 Various photoreactive moieties are available among which phenyl azide and acrylate are considered as two of the most used moieties and will be introduced here. Phenyl azides One strategy in covalent conjugation of growth factors to various substrates is by the help of heterobifunctional crosslinkers, which have one terminus that can react with the selected functional group of growth factor, while the other end contains a photoreactive moiety, which can attach the substrate. Such crosslinkers usually have a phenyl azide, benzophenone, or diazo moieties as their photoreactive end. Phenyl azides have low activation energy and possess hydroxyl groups on their aromatic ring. This allows their activation with ultraviolet (UV) wavelength of 350 nm during short exposure times. UV light exposure results in the formation of a nitrene group which can initiate the addition reactions with double bonds and insertion into NAH and CAH sites. SulfoSANPAH (sulfosuccinimidyl-6-[40 -azido-20 -nitrophenylamino] hexanoate) is an example of phenyl azide photoreactive cross linkers, which holds a phenyl azide group on one terminus and an amine reactive NHS moiety on the other end.27 The NHS ester can efficiently react with the primary amines on growth factor to form stable amide bonds (Fig. 3).8 Acrylates Acrylate functionalization of polymers is another method for development of biomaterials, which can go through crosslinking by UV light exposure. Reactive centers including radicals can initiate the chain growth polymerization of acrylates which further results in the formation of a crosslinked network. These radicals are usually formed through the photocleavage of an initiator molecule and can propagate through the vinyl bonds present on the acrylated polymers. This will result in covalently crosslinked polyacrylate with a high molecular weight.8

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2014 VOL 00A, ISSUE 00

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FIGURE 2. Carbodiimide coupling reaction as one of the most common approaches for covalently conjugating growth factors to other molecules. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Primary amines in the primary structure of growth factor are generally used for acrylation reaction with various substrates such as monoacrylated polyethylene glycol (PEG)-NHS and monoacrylated PEG-succinimidyl carbonate, which will result in growth factor conjugation to an acrylate group by a PEG linker (Fig. 4). Monoacrylated growth factors can subsequently be conjugated on acrylate containing matrices via polymerization. PEG spacer arm can minimize the formation of noncovalent and nonspecific bonds between the growth factor and the substrate.8

Self assembled monolayers (SAM)and phosphonate and thiol anchors SAM is a rather new technique for growth factors conjugation on etalized or glass surfaces.22,28 In this regard, silane and phosphonic acid containing compounds can make chemical links between the growth factor molecules and titanium surfaces. To provide a SAM modified surface on titanium alloy, Kang et al. immersed titanium surface in an aqueous solution containing a phosphonic acid initiator agent, which was then used for surface polymerization and further conjugation of surface polymer with growth factor molecules.22 In fact, SAM altered the titanium surface in a way that it

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could be easily used for conjugation practice with growth factor molecules. The utilization of phosphonate and thiol anchors may actually expand the range of molecules, which can be attached to metalized surfaces like titanium alloy.29 In a variation of this method, Schliephake et al., used the special interaction between the two strands of oligonucleotides to overcome the restrictions of growth factor inactivation due to sterilization practice. In this method, the first strand is coated on titanium surface by the hel of phosphonate anchors and after the completion of sterilization step, the growth factor conjugated complementary strand is introduced to the system to be attached to titanium surfaces through double strands hybridization.29

Click chemistry Other than traditional esterification and amidation conjugation reactions, the concept of regiospecificity and its significance has led to a newly emerged field called click chemistry. Click chemistry first introduced by Kolb et al. in 2001, was aimed to produce materials by attaching small units together with hetero atom links and was recognized with some especial properties including simple reaction conditions, high yield, and stereospecificity. Although highly


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FIGURE 3. Sulfo-SANPAH (sulfosuccinimidyl-6-[40 -azido-20 -nitrophenylamino] hexanoate) as an example of phenyl azide photoreactive crosslinkers. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

reactive, the click reaction constituents are orthogonal and do not interact with a broad range of solvents and reagents resulting in their selectivity.30 Orthogonal chemistry is especially useful for growth factor immobilization. Moore et al. conjugated an osteogenic growth peptide (OGP) with an oxidized SAM gradient. For this purpose carboxylic functional group on SAM was first attached with a bifunctional polyethylene oxide spacer and was then conjugated to azide terminated OGP using click chemistry. The OGP immobilized surfaces exerted a higher cellular attachment and the osteoblastic response to various peptide concentrations was made possible due to presence of a concentration gradient.31 To improve the outcomes with immobilized growth factors, other macromolecules have been coimmobilized with them. He et al., coimmobilized Bone morphogenetic proteins-2 (BMP-2) with RGD peptide on a hydrogel substrate through click chemistry and found that the two peptides show synergistic effect in enhancing osteogenic differentiation and bone marrow stromal mineralization.32 Plasma treatment Plasma treatment is another useful surface functionalization approach, which has great potential for introducing func-

tional groups on various surfaces and may be a versatile method for metallic biomaterials functionalization. As an instance, plasma treatment by allyl alcohol and allyl amine introduces hydroxyl and amino groups into the material, respectively, which can participate in various immobilization chemistries.33 Puleo et al. used plasma polymerization by allyl amine to introduce functional groups on titanium surfaces. BMP-4 was subsequently conjugated on plasma modified metal surfaces containing a high density of amino groups via carbodiimide coupling strategy and showed that BMP-4 retains its biological osteoblastic activity in vitro after immobilization.34 Other methods Several other types of chemistries and reactive groups may be used to attach growth factors to polymeric substrates including thiol anchors that can bind to cysteine residues by maleimide thiol chemistry.35 sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC), which is a heterobifunctional crosslinker and contains a sulfhydryl reactive maleimide and an amine-reactive NHS group is an example of such chemistry related reagents (Fig. 5).36,37 Protein engineering is another useful technique for growth factor conjugation and many types of gene engineered


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FIGURE 4. Monoacrylated PEG-succinimidyl carbonate will result in growth factor conjugation to an acrylate group by a PEG linker. Monoacrylated growth factors can subsequently be conjugated on acrylate containing matrices via polymerization. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

growth factors (fusion proteins) have been reported.3,38,39 A better control over growth factor oriented configuration has been achieved by the use of genetic fusion protein engineering.12 GROWTH FACTOR CONJUGATES

History Growth factor conjugation concept was first emerged in 1978 with the purpose of selective labeling of receptors in viable cells and to evaluate the receptor hormone complexes mobility. In this regard, a fluorescent labeled EGF analogue was developed by covalently conjugating the growth factor to rhodamine substituted a-lactalbumin molecules. The fluorescent labeled EGF molecule retained its activity in fibroblast receptor interactions and kept a 40% potency of the native growth factor in DNA synthesis stimulation.40 In another attempt, Haigler et al. prepared a bioactive conjugated species of EGF with ferritin, which maintained

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its binding affinity towards cellular receptors. For this purpose, EGF was activated by glutaraldehyde and was covalently attached to ferritin molecules to produce an EGFferritin conjugated form with a conjugation molar ratio of 1:1.41 In 1980, a pyridyl-dithiopropionate derivative of EGF was developed and was conjugated to ricin or diphteria toxin fragments (DTA) through disulfide linkage. The ricin conjugated EGF was found to be toxic in vitro with the same concentration levels at which EGF shows its bioactivity and with superior toxicity than ricin. A physical mixture of ricin toxin and EGF did not show any greater effect than ricin itself. In contrast to the ricin conjugated EGF, the EGF molecules conjugated with DTA were non toxic although EGF retained its receptor binding affinity.42 In 1986, K. Miller et al. used EGF conjugation with horseradish peroxidase and some antireceptor monoclonal antibodies to characterize the internalization pathways of


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FIGURE 5. Sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) is a heterobifunctional crosslinker. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

EGF and its receptor in human epidermoid carcinoma cells.43 Today, immobilized and covalently conjugated growth factors are considered as a promising drug design and are widely used for protein reformulation and material design to cover the unwanted characteristics of growth factors as well as improving their functions. Up to now, various strategies and approaches for growth factor reformulation have been reported. Many different purposes such as increasing the protein stability, pharmacokinetics improvement, targeting the growth factor toward a selected tissue or using the growth factor as a targeting moiety exist behind this material design. This part provides an over view of the relevant studies accomplished for bioconjugation of growth factors and their related application (Table I). Epidermal growth factor (EGF) EGF family, which is consisted from EGF, transforming growth factor-a (TGF-a), heparin binding EGF like growth factor (HB-EGF) and various other members is recognized by three major properties.1,44 First, they can create mitogenic responses in EGF sensitive cells.1 Second, they have a

high affinity toward EGF receptors (EGFR), and third, they share six conserved cysteine residues spaces.1,44 EGF family members can initiate a cellular signaling cascade when bounded to their receptors, which in turn, can activate other signaling pathways, which will subsequently result in cellular responses.1,44 EGF is the most significant member of EGF family, which was first isolated from submaxillary glands of mice and can stimulate the proliferation of epithelial tissues.45 EGF conjugation has been reported by various researchers for many different reasons from increasing its stability to cellular targeting. To increase EGF stability when exposed to proteolytic degradation conditions, Hardwicke et al. developed a dextrin growth factor conjugate as a bioactive nanomedicine to be used in tissue regeneration. EGF was conjugated to succinoylated dextrin via carbodiimide mediated coupling reaction. This reformulation was based on a hypothesis called polymer masking unmasking protein therapy (PUMPT), which uses biodegradable polymers to temporarily mask the protein during its transit to stabilize and inactivate it.46 Later, triggered polymer degradation will result in protein


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TABLE I. An Over View of the Relevant Studies Accomplished for Bioconjugation of Growth Factors and Their Related Application Growth Factor a

Substrate Succinoylated-dextrin

EGF

Hyaluronic acid LMWCa Gelatin nanoparticles PMBNa Oxidized dextran PEIa

PEGa-EGF EGF and IGFa

Polystyrene plates ADV PEI-DNA complex polystyrene plates PCLa-PEG nanofibers

BDNFa

PEG

PEG-BDNF NGF

OX26 OX26 PEG-PCL nanofibers

NGF-derived peptide FGFa

Plasma treated glass coverslips PEG-b-PCL polymersome nanoparticles PPy coated PLGA nanofibers PPy PDMS PEG-b-PCL polymersome nanoparticles p(SS-co-PEGMA)a Acryloyl-PEG Saporin OX26 PEG

BMPa-2

PLGA scaffold PCL scaffolds succinylated type I atelocollagen Bisphosphonate functionalized hydroxyapatite ceramic pPEGMAa coated titanium surfaces Dextran coated titanium SMA functionalized gold coated glass coverslips Hydrogel substrate PMMAa Titanium surfaces

BMP-2-derivative

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Alginate

Conjugation Strategy Carbodiimide mediated conjugation Carbodiimide mediated conjugation Carbodiimide mediated conjugation Avidin biotin strategy Amide bond formation Reductive amination SPDPa mediated conjugation Photoimmobilization Avidin biotin strategy Avidin biotin strategy Photoimmobilization Carbodiimide mediated conjugation Carbodiimide mediated conjugation Avidin biotin strategy PDPHa mediated conjugation Carbodiimide mediated conjugation GMBSa mediated conjugation Sulfo SMCCa mediated crosslinking Carbodiimide mediated conjugation reaction Photoimmobilization Photoimmobilization Sulfo-SMCC mediated conjugation Disulfide bond formation Amide bond formation SPDP mediated conjugation Avidin biotin strategy Maleimide thiol conjugation Acrylate-NHSa-PEG mediated conjugation Sulfo-SMCC mediated conjugation Carbodiimide mediated conjugation Carbodiimide mediated conjugation

Application

Ref.

Increasing stability

46–48


49–51

Increasing stability Targeting Targeting Targeting Targeted gene therapy (Non viral)

52 53 11 56

Wound healing Targeted gene therapy (Viral) Targeted gene therapy (Nonviral) Wound healing Wound healing

26 54 55 58 57


62

Targeting Targeting

63,65,66 67–69

Tissue engineering

72

Tissue engineering

73

Increased stability

70

Tissue engineering

38

Tissue engineering Tissue engineering Targeting

74 75 70

Increasing stability Tissue engineering Targeting

83 84,86 85

Targeting Targeting

87 88

Tissue engineering

91

Tissue engineering

94

Tissue engineering

98,99

Tissue engineering

101

Amide bond formation

Tissue engineering

22

Reductive amination Amide bond formation

Tissue engineering Tissue engineering

102 28

Click chemistry Avidin biotin strategy Oligonucleotide hybridization Carbodiimide mediated conjugation

Tissue engineering Tissue engineering Tissue engineering

32 103 29

Tissue engineering

21,95


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TABLE I. Continued Growth Factor BMP-4 TGFa-b1

TGF-b2

Substrate Plasma treated titanium surfaces PEG diacrylate hydrogel Tri-co-polymer of gelatin, hyaluronic acid and chondroitin-6-sulfate Collagen PDMS

TGF-b3 DIF-7c OGPa PDGFa PEG-PDGF Variant of IGF TNFa

VEGFa

VEGF and Anga

PLGA-GCH hybrid scaffold Nanocrystalline hydroxyapatite Oxidized SAM gradient coated glass coverslips Agarose hydrogel PEG diacrylate hydrogel Methotrexate PEG PEG Murine antibody ZME018 Transferrin Diphtheria toxin Collagen PEG diacrylate hydrogel Collagen PVLa-b-PEG-b-PVL Collagen

Conjugation Strategy

Application

Ref.

Carbodiimide mediated conjugation Amide bond formation Carbodiimide mediated conjugation

Tissue engineering

34

Tissue engineering Tissue engineering

18 97

Bifunctional PEG spacer mediated conjugation Bifunctional PEG spacer mediated conjugation Carbodiimide mediated conjugation Aminosilane chemistry


92

Tissue engineering

93

Tissue engineering

96

Tissue engineering

100

Click chemistry

Tissue engineering

31

Photoimmobilization Photoimmobilization Carbodiimide mediated conjugation Amide bond formation Amide bond formation SPDP mediated conjugation Maleimide PEG spacer mediated conjugation Disulfide bond formation Carbodiimide mediated conjugation Amide bond formation Amide bond formation Amide bond formation Carbodiimide mediated conjugation

Tissue engineering Tissue engineering Targeting

106 105 109

Targeting Targeting Targeting

111 113,114 110,115

Targeting

116

Targeting Tissue engineering

118,122

Tissue Tissue Tissue Tissue

124 125 126 23,123

engineering engineering engineering engineering

List of abbreviations: Ang: Angiopoitin; BDNF: Brain derived neurotrophic factor; BMP: Bone morphogenetic proteins; EGF: Epidermal growth factor; FGF: Fibroblast growth factor; GMBS: N-y-maleibutyryloxy succinimide ester; IGF: Insulin like growth factor; LMWC: Low molecular weight chitosan; NGF: Nerve growth factor; NHS: N-hydroxysuccinimide; OGP: Osteogenic growth peptide; p(SS-co-PEGMA): Poly(sodium 4styrenesulfonate-co-poly(ethylene glycol) methyl ether methacrylate); PCL: Poly(E-caprolactone); PDGF: Platelet-derived growth factor; PDMS: Poly(dimethyl siloxane); PDPH: S-(2-thiopyridyl) mercaptopropionic acid hydrazide; PEG: Polyethyleneglycol; PEI: Polyethylenimine; PMBN: Poly[2-Methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate-co-p-nitrophenyloxycarbonyl poly (ethylene glycol) methacrylate]; PMMA: Poly(methyl methacrylate); PPy: Polypyrrole; PVL: Polyvalerolactone; SPDP: N-succimindyl 3-(2-pyridyldithio)proprionate; sulfo-SMCC: Sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate; TGF: Transforming growth factor; TNF: Tumor necrosis factor; VEGF: Vascular endothelial growth factor.

unmasking and the restoration of its bioactivity (Fig. 6). In this study, dextrin section is degraded by the addition of aamylase, which results in an extended and sustained release of EGF.46 When chronic wounds were incubated with EGF conjugated dextrin, EGF was released by endogenous a-amylase and an enhanced cellular migration was observed with both free EGF and a-amylase activated dextrin-EGF conjugates and increased fibroblasts proliferation was achieved.47 It is noteworthy that topically applied conjugates significantly accelerated the wound healing in vivo.48 PUMPT concept was also used by Ferguson et al. using hyaluronic acid conjugates to enhance protein stability. In contrast to Hardwicke et al. results, conjugated EGF was not effective on cellular proliferation, even when the conjugated species were exposed to hyaluronidase (HAase). It was suggested that oligosaccharide residues attached to the growth factor may be

responsible for these results which is due to the cleavage mechanism of HAase that cleaves HA along its main chain instead of cleaving the polymer and peptide bond.49 To enhance EGF stability, Son et al. suggested EGF conjugation to low molecular weight chitosan via carbodiimide mediated reaction. EGF conjugated chitosan was more stable than free EGF both thermally and proteolytically and exerted a stronger biological effect than EGF on proliferation of fibroblasts.50,51 Another reason behind EGF reformulation through conjugation reactions is for targeting purposes (Fig. 7). Tseng et al. developed a core shell structure by grafting avidin molecules on the surface of gelatin nanoparticles, which were then coupled with biotinylated EGF through avidin biotin strategy.52 EGF played the role of a targeting moiety for lung adenocarcinoma.52 The EGF decorated nanoparticles showed a higher entrance in EGFR positive adenocarcinoma cells


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FIGURE 6. Polymer masking unmasking protein therapy (PUMPT) hypothesis, which uses biodegradable polymers to temporarily mask the protein during its transit to stabilize and inactivate it. Later, triggered polymer degradation will result in protein unmasking and the restoration of its bioactivity. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

when compared with normal lung cells. Furthermore, EGF decorated nanoparticles were administered in vivo as an aerosol and confirmed the in vitro observations.52 Shimada et al. conjugated EGF to paclitaxel loaded poly [2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate-co-p-nitrophenyloxycarbonyl poly(ethylene glycol) methacrylate] (PMBN).53 EGF conjugation resulted in a stronger cytotoxicity in EGFR over expressing cells when compared to EGFR deficient cells.53 Bue et al. conjugated EGF to periodate activated dextran molecules through reductive amination reaction to target urothelial carcinoma tissues, which commonly over express EGFR.11 These results suggested that radiolabeled EGFdextran conjugates have the potential to be used as tools for local treatment of recurrent bladder carcinoma. Furthermore, with appropriate modifications they can also be used for systemic radiotherapy.11 EGF conjugation strategy has also been applied in gene therapy. To prevent the nonspecific cellular transduction during gene therapy by adenovirus (ADV), pegylated EGF was immobilized on the surface of ADV via avidin biotin interaction to target ADV toward EGFR over expressing tumor cells and it was observed that EGF targeted complexes provided an enhanced cellular uptake (Fig. 8).54

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These scientists have also developed an efficient nonviral gene delivery system conjugated with pegylated EGF via avidin biotin strategy. The complex exerted a higher transfection efficiency, which was suggested to be related to their elevated cellular uptake via receptor mediated transcytosis.55 In another study by Wagner et al., EGF was covalently conjugated to polyethylenimine (PEI), which once again resulted in increased transfection efficiency.56 Due to the fact that shielded particles have a special place in systemic gene delivery, the EGF conjugated complexes were found to be more efficient than simply pegylated complexes without EGF.56 Wound healing is a common application of EGF based drug delivery systems. Wound healing acceleration not only reduces patient suffering and decreases the cost of treatment, but also minimizes scarring development and leads to formation of a better and more stable healed wound. EGF was covalently conjugated on the functional amino moieties of an electrospun surface by carbodiimide mediated coupling reaction to manage diabetic ulcers. EGF conjugated nanofibers were found to be superior in the in vivo wound healing when compared with EGF solutions.57 Cell migration is regarded as a significant key factor in wound healing, and the management of the direction of


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FIGURE 7. Targeting purposes are one of the important reasons behind EGF reformulation through conjugation reactions. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

cellular migration will result in an accelerated wound closing. As a result, some researchers have prepared chemically functionalized surfaces with EGF gradients to create a platform to direct cell migration. It was observed that human epidermal keratinocytes migrate toward higher EGF concentrations.26 Insulin growth factor-1 (IGF-1) gradients immobilization also directed and accelerated keratinocyte migration but the migration was not dependant on gradient patterns. Furthermore, a combination of EGF and IGF-1 did not result in an accelerated migration when compared to EGF alone.58 Neurotrophins Neurotrophins including nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) are a group of key regulators of neural development and functioning and play a major role in neuronal survival. Neurotrophins can activate tyrosine kinase receptors, which results in neuronal proliferation and survival and regulates the neuronal extensions growth and remodeling. Furthermore, they are involved in cytoskeleton assembly, synapse formation and its functioning and membrane transport and fusion.59 Neurotrophins, which are considered as potential neuropharmaceuticals in various neurologic disorders including neurodegenerative disorders, peripheral neuronal injury and neuropathies have been subjected to conjugation techniques

for various reasons.60,61 One of the most significant reasons behind reformulation of neurotrophins via conjugation techniques is their inherent instability in the blood stream. Neurotrophins are cationic molecules, which are rapidly cleared from the blood circulation after their intravenous administration. This fast removal is related to the rapid hepatic uptake of these cationic molecules.60 To address this problem, BDNF was conjugated on its carboxylic functional groups to terminal hydrazide moieties of PEG molecules using carbodiimide based coupling reaction. BDNF pegylation with PEG molecules with a molecular weight of 2000 Daltons resulted in its reduced systemic clearance while its biologic activity was not altered.62 In addition to their rapid removal from the systemic circulation, neurotrophins suffer from their negligible blood brain barrier (BBB) transport and permeability and many studies have focused on making these proteins more permeable through BBB to achieve their potential pharmacological effects in central nervous system. For this purpose, various researchers have worked on the attachment of these nontransportable peptides to different brain targeting carriers including peptides, modified proteins and monoclonal antibodies to provide chimeric peptides with higher brain permeability.63–66 The brain targeting vector binds the receptors on the BBB and triggers the receptor mediated


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FIGURE 8. To prevent the nonspecific cellular transduction during gene therapy by ADV, pegylated EGF was immobilized on the surface of ADV via avidin biotin strategy to target ADV toward EGFR over expressing tumor cells. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

transcytosis (RMT) of the chimeric peptide through BBB. Avidin biotin technology has been vastly used for this purpose. OX26 monoclonal antibody is one of the most common used targeting vectors for brain delivery of growth factors, which targets transferrin receptors and subsequently results in RMT of the chimeric peptide through BBB. Pegylation technology is usually combined with this technique to increase the growth factor stability and half life and to optimize its plasma pharmacokinetics.63–66 This is especially important because BDNF biotinylation on its surface carboxyl moieties without the pegylation technique followed by the attachment of OX26 was not likely to achieve a maximal brain uptake of the growth factor. This is related to the higher hepatic uptake of not pegylated BDNF chimeric peptide, which results in its lower plasma area under the curve and subsequently its lower brain uptake.60 A very important consideration in BDNF chimeric peptide molecular reformulation is to ensure the retention of dual functionalities of the chimeric peptide where it should

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be able to bind to both tyrosine kinase receptor B (TrkB) and transferrin receptors. BDNF has multiple lysine amino acid residues on its surface, which develops a “cationic” groove, which is actively involved in its binding to TrkB receptors. Any manipulation on these lysine residues will result in a loss of BDNF biological activity.63 Therefore, BDNF was coupled with PEG molecules through its surface carboxylic functional groups of glutamate and aspartate residues. In this chimeric peptide, PEG molecules serve as a bridge, which is sterically separating BDNF and OX26 and removes any steric hindrance between BDNF and OX26.63 NGF has also been covalently conjugated to OX26 to enhance its BBB transport. Carboxylic functional groups on NGF molecules were activated by EDC followed by the addition of S-(2-thiopyridyl) mercaptopropionic acid hydrazide (PDPH) as a heterobifunctional cross linking agent. PDPH has a carbonyl reactive hydrazide moiety on one terminus and a sulfhydryl reactive group on the other end and can be used to covalently conjugate thiolated OX26 to NGF


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FIGURE 9. Growth factor conjugation practices are employed for tissue engineering purposes. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

molecule via PDPH mediated conjugation reaction. An increased intraocular tissue growth was achieved after the peripheral administration of conjugated NGF.67 Conjugated NGF was further administered intravenously to aged rats to determine their potential in central nervous system disorders treatment. Conjugated NGF resulted in a significant improvement in animal spatial learning ability in previously impaired rats.68 Intravenous administration of OX26-NGF conjugates was able to restore the nucleus basalis perikarya size in cerebral cortex lesions induced by epidural application of N-methyl-D-aspartic acid.69 In addition to targeting vector introduction for neurotrophin reformulation, neurotrophin molecules have been used as targeting moieties themselves. Roy et al. investigated the utility of NGF derived peptide functionalized polymersome nanoparticles prepared from PEG-b-polycaprolactone (PCL) block copolymers to target cells in the inner ear.70 PEG-bPCL polymer contains an amine group at the end of PEG unit which allows the surface functionalization by sulfoSMCC to enhance the survival of vestibular and cochlear neuronal cells. NGF-derived peptide was conjugated to the end of the hydrophilic part of polymersome nanoparticles surface in a way that they may extend outward. Conjugated nanoparticles presented an increased uptake into cells within the inner ear.70 Soumen et al. further used these nanoparticles to activate receptors in vitro and proposed that polymersome nanoparticles act as a scaffold and that the nanoparticles size and their PEG containing surface can increase in vivo retention time and growth factor half life.71 Tissue engineering is one other purpose behind neurotrophin conjugation practices (Fig. 9). Electrospun fibrous meshes resemble natural extracellular matrix (ECM) and have been used to prepare tissue engineering scaffolds, which mimic the natural topography of extracellular microenvironments.

As a result, various growth factors were introduced within these nanostructures. Cho et al., covalently conjugated NGF molecules on the surface of nanopatterned fibrous matrices. To prepare aligned nanofibrous meshes, amine terminated PEG molecules were conjugated to PCL and the resulting block copolymers were electrospun. NGF was chemically immobilized on the amine groups of PEG molecules in the surface of electrospun nanofibrous meshes via a carbodiimide based coupling reaction. NGF modified nanofibrous meshes were able to significantly increase neuronal differentiation in mesenchymal stem cells when compared with NGF physically adsorbed on nanofibrous meshes.72 Achyuta et al. proposed a coimmobilization of laminin, an ECM protein, and NGF to evaluate the presence of any synergistic effects on neurite outgrowth.73 Laminin and NGF were covalently conjugated onto plasma treated glass coverslips through N-y-maleibutyryloxy succinimide ester (GMBS) as a heterobifunctional crosslinking agent where immobilized laminin showed a synergistic effect in neurite extension induction by NGF.73 Combining NGF as a biological cue with electrical cues is another approach for achieving better outcomes in tissue engineering. Poly(lactic acid-co-glycolic acid) (PLGA) nanofibers prepared through electrospinning procedure were coated with polypyrrole (PPy) as an electrical conducting polymer which contains numerous carboxylic acid functional groups. NGF was then immobilized through a carbodiimide coupling reaction on the surface of the nanofibers. The resulting NGF immobilized nanofibers were found to be able to support neurite extension and the electrical stimulation was able to enhance neurite outgrowth.38 Gomez et al. immobilized NGF molecules on PPy polymer via photoimmobilization using a layer of polyallylamine coupled to an


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aryl azido group.74 Neurite extension was achieved with the same levels for immobilized NGF when compared to free NGF and the electrical stimulation was able to increase the neurite outgrowth in vitro.74 Biological cues have also been used in combination with other physical stimulating factors. As an instance, NGF was covalently conjugated to poly(dimethyl siloxane) PDMS surfaces where the substrate was subjected to microtopography in the form of microchannels. A combination of NGF as the biological cue with the physical cue resulted in a synergistic increase in axonal length.75 Fibroblast growth factor (FGF) FGFs are mitogenic growth factors, which belong to heparin binding growth factors (HBGF) group and were first extracted from bovine pituitary gland. FGFs are ubiquitously spread through the tissue and contribute in adult tissue homeostasis and development where FGF impaired expression is involved in cancer pathogenesis.76–79 bFGF is one of the remarkable members of FGF group and regulates cellular proliferation, migration, and differentiation. bFGF can stimulate tissue regenerating and may be used for wound healing in addition to bone damages management and ischemic brain and heart injuries.80 Acidic fibroblast growth factor (aFGF) is another remarkable cytokine used for wound healing which is a potent mitogen for various cells in skin.81,82 Like many other growth factors, FGF family members suffer from instability during their delivery and storage. To overcome this instability, bFGF was conjugated to a heparin mimicking polymer and the more stable conjugated growth factor was able to retain its bioactivity.83 Another reason behind FGF conjugation is to prepare biofunctionalized scaffolds. DeLong et al. coimmobilized bFGF and Arginyl glycyl aspartic acid (RGD) peptide on an acrylated PEG hydrogel scaffold where the conjugated growth factor retained its bioactivity. Biofunctionalized hydrogels increased vascular smooth muscle cells proliferation and migration. When bFGF gradient platform was used cells aligned on the functionalized hydrogels in the direction of bFGF concentration gradient and were able to migrate up to the gradient.84 FGF is also applied for tumor targeting where several cancer cells may express FGF receptors. bFGF conjugation with saporin, a ribosome inactivating protein showed cytotoxic effects in FGF receptor positive human melanoma, neuroblastoma, and teratocarcinoma, cell lines. In the in vivo environment a significant tumor growth inhibition was achieved along with low toxicity.85 Unfortunately, systemic administration of bFGF conjugated saporin was related to liver toxicity in therapeutic doses.86 Although bFGF is a notable targeting moiety itself, it was conjugated to OX26 monoclonal antibody via avidin biotin strategy to target transferrin receptors in BBB and increase its brain delivery. Conjugated bFGF retained its bioactivity toward its receptor and exerted an enhanced brain uptake.87 bFGF has a great potential in the management of spinal injuries due to its angiogenic and trophic properties. bFGF

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was conjugated to PEG molecules to improve its penetration into spinal cord via passive targeting due to a reduced clearance and a better immunogenic shielding. Intrathecally administered pegylated bFGF doubled the growth factor concentration in the injured spinal cord.88 Transforming growth factor-b (TGF-b) super family TGF-b superfamily consists from a group of members, which play a significant role in cellular proliferation and differentiation, development of embryonic tissue and tissue morphogenesis.13 Since first introduced by Urist in 1960s, several members of TGF-b superfamily have been found to be associated with bone induction related biological processes including cellular recruitment and proliferation along with ECM production.89 BMPs are among the most extensively used growth factors of TGF-b superfamily in bone tissue engineering and have a strong efficacy in bone formation.90 Immobilized TGF-b superfamily members not only have improved bone tissue engineering, but also are used for the osseointegration of various orthopedic implants. Synthetic and natural polymeric substrates as well as ceramics and metal alloys have been subjected to these growth factors immobilization. Pegylation technology is commonly applied for TGF-b superfamily conjugation. Liu et al. conjugated pegylated BMP-2 to PLGA scaffold to prolong its retention and extend its fibroblastic response in addition to making its localized release possible in bone tissue engineering. For this purpose, a heterobifunctional acrylate-NHS PEG spacer was used to immobilize BMP-2 on PLGA where BMP-2 retained its mitogenic activity.91 Bentz et al., pegylated TGF-b2 with a bifunctional PEG spacer, which was then covalently attached to fibrillar collagen to prevent its rapid clearance from the implant site and increase its stability. Covalent conjugation of TGF-b2 to collagen was able to potentiate it is in vivo responses as well as its in vitro outcomes.92,93 TGF-b1 has also been covalently conjugated to PEG and was able to retain its activity.18 PCL is another synthetic polymer, which is used for bone tissue engineering. BMP-2 was immobilized on threedimensional scaffolds of aminated PCL by sulfo-SMCC. Conjugated BMP-2 was released from the prepared PCL scaffolds with a significantly slower rate than BMP-2 simply adsorbed on the scaffolds. Osteogenic markers gene expression was also upregulated with BMP-2 conjugated scaffold in bone marrow stromal cell cultures.94 Natural polymers or a combination of natural and synthetic polymers have also been subjected to growth factor immobilization via covalent conjugation. Suzuki et al, covalently conjugated a BMP-2 derived oligopeptide to alginate molecules via a carbodiimide based coupling reaction and the resulting hydrogel composites resulted in ectopic bone formation when implanted in rat calf muscle.21 Furthermore, BMP-2-derived oligopeptide conjugated alginate hydrogel showed an extended ectopic calcification in calf muscle of rats.95 Fan et al., immobilized TGF-b3 on PLGA, gelatin, chondroitin sulfate and hyaluronic acid hybrid scaffold via a


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carbodiimide based conjugation reaction to be used for cartilage regeneration. The scaffold was fabricated by incorporating gelatin, chondroitin, and hyaluronic acid micro sponges into PLGA network, which was further conjugated with TGF-b3 to play the role of natural ECM. The immobilized growth factor was able to induce chondrogenic differentiation in mesenchymal stem cells. Furthermore, in vivo implantation of these scaffolds was able to regenerate and repair condral defects in rabbit models.96 In another effort, a hybrid of gelatin, chondroitin sulfate, and hyaluronic acid was conjugated to TGF-b1 and the immobilized sponges were able to suppress chondrocytes differentiation toward hypertrophic chondrocytes and osteoblasts and have the potential of organizing chondrocytes maturation.97 Tsujigiwa et al., immobilized BMP-2 on succinylated atelocollagen via carbodiimide coupling reaction and found an augmented cellular activity in vitro. The authors have suggested that BMP-2 conjugation to atelocollagen results in an increased molecular size and the conjugated growth factors create a substrate-receptor complex on cellular membrane, which is not internalized into the cells and will continuously send cellular signals.98 Furthermore, in vivo studies showed that immobilized BMP-2 was able to promote bone like structures formation when implanted into the backs of rats.99 BMP-2 has also been covalently conjugated on collagen coated PLGA scaffolds and was able to prolong cellular signal transduction and increase cellular outgrowth and differentiation.91 In contrast to polymeric substrates, ceramics and alloys and even some especial types of polymers, which are commonly used for orthopedic and dental implants, possess rather few and unreactive functional groups for growth factor immobilization and require a surface activation procedure prior to conjugation reaction. Aminosilanization technique is one of the methods, which is commonly used for surface activation and covalent conjugation of growth factors to various surfaces, which provides amines for covalent conjugation of proteins. Liu et al. explored biodegradable ceramic polymer nanocomposites exploitation for orthopedic implants to extend the local release of a BMP-7 derived peptide called DIF-7c and increase growth factor effectiveness. DIF-7c was covalently conjugated on nanocrystalline hydroxyapatite through aminosilane chemistry. In the next step, N-succinimidyl-3maleimido propionate was used as a hetero-bifunctional cross linker for the terminal amine and DIF-7c was subsequently immobilized on the nanocomposite and the resulting nanoparticles were dispersed into PLGA matrix.100 Bisphosphonates are also among the substances, which may be used for hydroxyapatite ceramic surfaces functionalization. Bisphosphonates show high affinity toward calcified tissues, which enables their utilization for guiding the conjugated growth factor to bone. The structure of bisphosphonates is composed of two phosphonates, which are attached together by a carbon atom, which is responsible for this high affinity toward bone mineral and their derivatives. Schuessele et al., used two different aminobisphosphonates, pamidronate, and alendronate for surface immobilization of BMP-2 on hydroxyapatite ceramic via carbodiimide coupling

chemistry on the amino moiety of bisphosphonates. Immobilized BMP-2 was able to create an enhanced stimulation of osteoblastic differentiation in vitro when compared with growth factors on unmodified surfaces.101 Puleo et al. used plasma polymerization by allyl amine to introduce functional groups on titanium surfaces. BMP-4 was subsequently conjugated on plasma modified metal surfaces containing a high density of amino groups via carbodiimide coupling strategy and showed that BMP-4 retains its biological osteoblastic activity in vitro after immobilization.34 When titanium surfaces are functionalized, both their activity and nonbiofouling property should be considered at the same time. Nonbiofouling property is especially important because of the possibility of nonspecific adsorption of biological molecules on medical devices and implants in the physiological environments. Non specifically adsorbed biological molecules may interfere with the desired role of medical devices, therefore, nonbiofouling property or so called bioinertness is a very important aspect in design of these devices.22 Kang et al. fabricated bioactive titanium surfaces by conjugating BMP-2 on nonbiofouling poly(poly(ethylene glycol) methacrylate) (pPEGMA) films coated on titanium surfaces while maintaining its bioinertness. N,N-disuccinimidyl carbonate was used to activate the hydroxyl functional groups of pPEGMA coating to make it reactive towards the amine groups. BMP-2 conjugated surfaces showed both nonbiofouling property and bioactivity in mesenchymal stem cells.22 Shi et al, coated titanium surfaces with oxidized dextran that inhibits bacterial adhesion. BMP-2 was then conjugated on dextran surfaces via a reductive amination reaction. It was observed that bacterial adhesion is significantly reduced in both dextran and dextran-BMP-2 functionalized titanium surfaces where BMP-2 immobilized surfaces were able to enhance osteoblast distribution.102 SAM represents another approach for covalent immobilization of growth factors on various substrates. Pohl et al. coated glass surfaces with a gold layer, which was subsequently decorated with SAM containing 11-mercaptoundecanoyl NHS ester as a hetero-bifunctional linker. BMP-2 was then covalently conjugated on its free amine groups on these functionalized surfaces and was able to induce cellular responses in vitro.28 Spatial conjugation to simulate the naturally occurring concentration gradients has received a great deal of attention, which provides topological cues for cellular differentiation and is particularly important in embryogenesis early phases. Lagunas et al. used a continuous gradient platform of BMP-2 immobilized on poly(methyl methacrylate) (PMMA) by avidin biotin strategy and observed that immobilized BMP-2 retained its biologic activity.103 Platelet-derived growth factor (PDGF) PDGF is a mitogenic factor in a range of cell lines including fibroblasts smooth muscle cells. PDGF was initially identified as a constituent of whole blood serum and was subsequently extracted from human platelets. PDGF has four known isoforms including A, B, C, and D. A and B isoforms form AA


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and BB homodimers and AB heterodimer by disulfide bonding. PDGF is biologically active during the embryogenesis, and is especially important for the development of kidneys, blood vessels, lungs, and central nervous system especially with regard to connective tissue like cells, which are mainly dependent on PDGF, This particular role in the formation of connective tissue makes PDGF a potential key factor in wound healing.104,105 Immobilized PDGF on cell-adhesive matrices was used to control neural stem-progenitor cells differentiation specifically to oligodendrocytes.106 PDGF has also been applied in tissue engineering where hydrogel containing covalently immobilized PDGF was able to improve the formation and development of functional microvasculature.105 Insulin like growth factors (IGF) IGF family is constituted from a complex system of peptide hormones including IGF-1 and IGF-2, cell surface receptors and circulating binding proteins. IGF-1 and 2 are mitogenic factors, which play a major role in regulating cell proliferation, differentiation, migration, and apoptosis and are extensively expressed in fetal and prenatal developmental stages in mammals. The mature IGF-1 and IGF-2 peptides consist of A, B, C, and D domains.107,108 IGF receptors are overexpressed in various cancer cells and have been implicated in metastasis occurrence and cancer cells resistance to apoptosis. As a result, IGF has been used as a conjugated moiety to target cancer cells, which over express IGF receptor by Mctavish et al. Covalent conjugation of methotrexate with a variant of IGF was able to increase the specific drug accumulation in tumor tissues and resulted in enhanced therapeutic effects.109 Tumor necrosis factor (TNF)-a TNF is a nonglycosylated macrophage derived, peptide, which shows a variety of biological and immunological functions including various cytotoxic and antiviral activities.110 TNF potentially displays antitumor properties, including direct cytotoxic effect on tumor tissue. Furthermore, an indirect cytotoxic effect is achieved through its role in activating the host immune response against tumors and its selective impairment in the microcirculation of tumor tissue capillaries.111 TNF family contains about 19 cytokine members based on their sequential, functional, and constructural similarities.112 PEG based modification of TNF-a is one of the most common conjugation practices in this family, which is used to enhance the anti tumor potency of TNF-a while reducing its adverse effects through passive targeting. TNF-a was covalently conjugated to monomethoxy PEG by a succinimidyl spacer and was subjected to coupling reaction with lysine amino groups on TNF-a primary structure. PEG related modification of TNF-a markedly improved its bioavailability and enhanced its potential as a therapeutically used anti tumor agent.111 Val-Cit moiety, a cathepsin B-sensitive dipeptide, was inserted into the conventional structure of pegylated TNF-a, which facilitates the release of TNF-a and improves its clini-

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cal use as an anti tumor agent. TNF-a in vitro release from these conjugated species was regulated by cathepsin B and was inhibited by the presence of a cathepsin B inhibitor in vitro. Such pegylated TNF-a species showed a greater in vitro and in vivo bioactivity in comparison with simply pegylated TNF-a.113,114 To actively target TNF-a, the protein was conjugated to a murine antibody called ZME-018, which targets a glycoprotein expressed on the majority of melanoma cells population. This conjugation reaction was accomplished by a heterobifunctional crosslinking reagent called N-succimindyl 3-(2-pyridyldithio)proprionate (SPDP). Similar tumor localization was found for the covalently linked TNF-a with the immunotoxin when compared with the free antibody. Furthermore, the rate of clearance for conjugated TNF-a was reduced in comparison with free TNF-a and a higher cellular sensitivity was achieved.110,115 A combination of pegylation techniques with active targeting was used by Jian et al. who coupled a PEG-TNF-a with transferrin and made benefit from the advantages of both pegylation and active targeting. Transferrin conjugated pegylated TNF-a provided a long circulating conjugated species with improved pharmacokinetic behavior along with specific transferrin receptor targeting resulting in enhanced antitumor effect.116 Vascular endothelial growth factor (VEGF) and angiopoietin (Ang) VEGF has a promising position due to its special role in angiogenesis and vasculogenesis and the formation of new blood vessels. Angiogenesis is an essential step in tumor growth and metastasis. This special property is involved in a variety of physiological processes including embryogenesis and wound healing as well as some pathophysiological processes such as tumor growth, myocardial ischemia, ocular neovascular diseases, etc. VEGF also regulates multiple endothelial biological functions, which impinge upon different aspects of cardiovascular homeostasis.117–119 Ang is another angiogenic growth factor family, which consists of Ang1, Ang2, Ang3, and Ang4, which are all considered as a family of endothelial cell specific growth factors. Ang3 is over expressed in response to hypoxia in various organs of rats including lung, liver, cerebellum, and heart. Thus, it has been proposed that the regulation of Ang3 expression can be used in physiological and pathophysiological angiogenesis.120,121 Like other growth factors, VEGF has been the subject of chemical conjugation for achieving a better targeting. For this reason, VEGF was chemically conjugated with a truncated species of diphtheria toxin as the targeting moiety. VEGF-toxin conjugate showed a selective toxicity against the endothelial cell lines and was well tolerated when evaluated in vivo for its effects in solid tumor growth by selective targeting of tumor neovasculature without any apparent toxicity.118,122 Developing scaffolds and hydrogels is an interesting tool in promoting the formation of vascular structures. Covalent immobilization of angiogenic growth factors is especially


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important in this regard because of their localized effect, their more stability and prolongation in signaling when compared with soluble growth factors.123 For this reason, VEGF and Ang1 were coimmobilized onto a threedimensional porous collagen scaffold by carbodiimide coupling strategy. Endothelial cells showed an increased proliferation on immobilized growth factor scaffolds. An improved tube formation was further observed for immobilized growth factor when compared to the soluble growth factor. It should be noted that coimmobilization of VEGF and Ang1 resulted in a more pronounced cellular infiltration compared with single growth factor immobilized scaffolds.123 One of the major problems in designing appropriate engineered tissues for clinical practice is their vascularization. VEGF and Ang1 were coimmobilized by carbodiimide coupling reactions to induce rapid vascularization in engineered tissues.23 In another interesting study, high and low degree levels of VEGF has been immobilized on porous collagen scaffolds, to replace the right ventricular free wall defect in the heart organ of rats. Endothelial and bone marrow cells showed an enhanced growth when seeded onto VEGF scaffolds and the high VEGF immobilization degree resulted in higher blood vessel formation density. Angiogenesis by VEGF scaffolds contributed in an improved cell survival and a better tissue formation.24 PEG hydrogels has also been evaluated in developing vascularized tissue engineered constructs. The covalent conjugation of pegylated VEGF in PEG based hydrogels promoted the endothelial cell migration in addition to the cellcell contact and tubulogenesis.124 To enhance the angiogenic properties of collagen matrices, VEGF was covalently incorporated in the matrix by a homobifunctional cross linker disuccinimidyldisuccinatepolyethyleneglycol (SS-PEG-SS). The angiogenic properties of the developed conjugated species were assessed in the in vivo environment with the help of chicken embryo chorioallantois membrane. A significant effect was observed on the microvessels formation in this membrane as well as a tissue growth into the implant. The covalent conjugation only showed minor unwanted effects on VEGF mitogenic activity.125 Angiogenic growth factors incorporated scaffolds made of biodegradable materials can even be injected for cardiac recovery after a myocardial infarction (MI). Wu et al. developed a VEGF conjugated temperature-sensitive polyester hydrogel to stabilize a MI. Post MI injection of such hydrogels in rats attenuated cardiac remodeling and provided a better ventricular function.126

CONCLUSION

Covalent conjugation of growth factors is a promising approach in regenerative medicine and targeted delivery systems design. Tissue engineering is one of the most significant fields which requires the development of novel biomaterials that are able to support living cells structurally in their scaffold while play the role of a bioactive system outside the body environment. This bioactivity can be achieved

by introducing growth factors into such systems and one of the most common approaches for this purpose is through covalent immobilization of growth factors. Covalent conjugation of growth factors has been fulfilled for arrange of other purposes such as targeting and pharmacokinetic improvement. Various studies have claimed that covalently conjugated growth factors are able to retain their biologic activity or have even found to be superior to free and soluble growth factors. However, the approach still suffers from a variety of obstacles and limitations especially with regard to bioactivity of growth factors. There are a range of available strategies for conjugating growth factors with different substrates from traditional methods including amidation and esterification to more novel techniques such as orthogonal click chemistry or creating an oriented configuration and gradient concentration of growth factors. New strategies are continuously emerging but the need for developing an optimized system still exists. Cellular response to conjugated growth factor may be affected by a complex of other signals inside the body so the scaffold and substrate composition finds a special role, which may change the efficacy of conjugated growth factors in the in vivo environment. Most of the growth factors are naturally transiently available during tissue regeneration and their consistent presence as a result of their immobilization may bring some concerns, which should be further covered in future researches. REFERENCES 1. Carpenter G, Cohen S. Epidermal growth factor. J Biol Chem 1990;265:7709–7712. 2. Tada S, Kitajima T, Ito Y. Design and synthesis of binding growth factors. Int J Mol Sci 2012;13:6053–6072. 3. Ito Y. Covalently immobilized biosignal molecule materials for tissue engineering. Soft Matter 2008;4:46–56. 4. Papanas D, Maltezos E. Benefit-risk assessment of becaplermin in the treatment of diabetic foot ulcers. Drug Saf 2010;33:455–461. € gelin E, Jones NF, Huang JI, Brekke JH, Toth JM. Practical illus5. Vo trations in tissue engineering: Surgical considerations relevant to the implantation of osteoinductive devices. Tissue Eng 2000;6: 449–460. 6. Margolis DJ, Bartus C, Hoffstad O, Malay S, Berlin JA. Effectiveness of recombinant human platelet-derived growth factor for the treatment of diabetic neuropathic foot ulcers. Wound Repair Regen 2005;13:531–536. 7. Embil JM, Papp K, Sibbald G, Tousignant J, Smiell JM, Wong B, Lau CY. Recombinant human platelet-derived growth factor-BB (becaplermin) for healing chronic lower extremity diabetic ulcers: An open-label clinical evaluation of efficacy. Wound Repair Regen 2000;8:162–168. 8. Masters KS. Covalent growth factor immobilization strategies for tissue repair and regeneration. Macromol Biosci 2011;11:1149– 1163. 9. Bennett NT, Schultz GS. Growth factors and wound healing: Biochemical properties of growth factors and their receptors. Am J Surg 1993;165:728–737. 10. Ciardiello F, Tortora G. A novel approach in the treatment of cancer: Targeting the epidermal growth factor receptor. Clin Cancer Res 2001;7:2958–2970. rquez M, Malmstro € m PU, Westlin JE, Nilsson S, 11. Bue P, Ma Holmberg AR. The potential of radiolabeled EGF-dextran conjugates in the treatment of urinary bladder carcinoma. Cancer 1997; 80:2385–2389. 12. Luginbuehl V, Meinel L, Merkle HP, Gander B. Localized delivery of growth factors for bone repair. Eur J Pharm Biopharm 2004;58: 197–208.


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13. Vo TN, Kasper FK, Mikos AG. Strategies for controlled delivery of growth factors and cells for bone regeneration. Adv Drug Delivery Rev 2012;64:1292–1309. 14. Willerth SM, Sakiyama-Elbert SE. Approaches to neural tissue engineering using scaffolds for drug delivery. Adv Drug Delivery Rev 2007;59:325–338. 15. King WJ, Krebsbach PH. Growth factor delivery: How surface interactions modulate release in vitro and in vivo. Adv Drug Delivery Rev 2012;64:1239–1256. 16. Lee K, Silva EA, Mooney DJ. Growth factor delivery-based tissue engineering: General approaches and a review of recent developments. J R Soc Interface 2011;8:153–170. 17. Kuhl PR, Griffith-Cima LG. Tethered epidermal growth factor as a paradigm for growth factor–induced stimulation from the solid phase. Nat Med 1996;2:1022–1027. 18. Mann BK, Schmedlen RH, West JL. Tethered-TGF-b increases extracellular matrix production of vascular smooth muscle cells. Biomaterials 2001;22:439–444. 19. Sahni A, Odrljin T, Francis CW. Binding of basic fibroblast growth factor to fibrinogen and fibrin. J Biol Chem 1998;273:7554–7559. 20. Ferrara N. Binding to the extracellular matrix and proteolytic processing: Two key mechanisms regulating vascular endothelial growth factor action. Mol Biol Cell 2010;21:687–690. 21. Suzuki Y, Tanihara M, Suzuki K, Saitou A, Sufan W, Nishimura Y. Alginate hydrogel linked with synthetic oligopeptide derived from BMP-2 allows ectopic osteoinduction in vivo. J Biomed Mater Res 2000;50:405–409. 22. Kang SM, Kong B, Oh E, Choi JS, Choi IS. Osteoconductive conjugation of bone morphogenetic protein-2 onto titanium/titanium oxide surfaces coated with non-biofouling poly (poly (ethylene glycol) methacrylate). Colloids Surf B 2010;75:385–389. 23. Chiu LL, Weisel RD, Li RK, Radisic M. Defining conditions for covalent immobilization of angiogenic growth factors onto scaffolds for tissue engineering. J Tissue Eng Regen Med 2011;5:69–84. 24. Miyagi Y, Chiu LL, Cimini M, Weisel RD, Radisic M, Li R-K. Biodegradable collagen patch with covalently immobilized VEGF for myocardial repair. Biomaterials 2011;32:1280–1290. 25. Liu L, Deng D, Xing Y, Li S, Yuan B, Chen J, Xia N. Activity analysis of the carbodiimide-mediated amine coupling reaction on selfassembled monolayers by cyclic voltammetry. Electrochim Acta 2013;89:616–622. 26. Stefonek TJ, Masters KS. Immobilized gradients of epidermal growth factor promote accelerated and directed keratinocyte migration. Wound Repair Regen 2007;15:847–855. 27. Png R-Q, Chia P-J, Tang J-C, Liu B, Sivaramakrishnan S, Zhou M, Khong S-H, Chan HS, Burroughes JH, Chua L-L. High-performance polymer semiconducting heterostructure devices by nitrenemediated photocrosslinking of alkyl side chains. Nat Mater 2010; 9:152–158. 28. Pohl TL, Boergermann JH, Schwaerzer GK, Knaus P, CavalcantiAdam EA. Surface immobilization of bone morphogenetic protein 2 via a self-assembled monolayer formation induces cell differentiation. Acta Biomater 2012;8:772–780. € tel C, Fo € rster A, Schwenzer B, Reichert J, 29. Schliephake H, Bo Scharnweber D. Effect of oligonucleotide mediated immobilization of bone morphogenic proteins on titanium surfaces. Biomaterials 2012;33:1315–1322. 30. Kolb HC, Finn M, Sharpless KB. Click chemistry: Diverse chemical function from a few good reactions. Angew Chem Int Ed 2001;40: 2004–2021. 31. Moore NM, Lin NJ, Gallant ND, Becker ML. The use of immobilized osteogenic growth peptide on gradient substrates synthesized via click chemistry to enhance MC3T3-E1 osteoblast proliferation. Biomaterials 2010;31:1604–1611. 32. He X, Ma J, Jabbari E. Effect of grafting RGD and BMP-2 proteinderived peptides to a hydrogel substrate on osteogenic differentiation of marrow stromal cells. Langmuir 2008;24:12508–12516. 33. Zhang Z, Hu J, Ma PX. Nanofiber-based delivery of bioactive agents and stem cells to bone sites. Adv Drug Delivery Rev 2012; 64:1129–1141. 34. Puleo D, Kissling R, Sheu M.-S. A technique to immobilize bioactive proteins, including bone morphogenetic protein-4 (BMP-4), on titanium alloy. Biomaterials 2002;23:2079–2087.

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