Journal of Controlled Release 245 (2017) 27–40

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Journal of Controlled Release

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Review article The principles and applications of avidin-based nanoparticles in drug delivery and diagnosis

Akshay Jain, Kun Cheng ⁎

Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri Kansas City, Kansas City, MO 64108, United States article info abstract

Article history: Avidin-biotin interaction is one of the strongest non-covalent interactions in the nature. Avidin and its analogues Received 7 October 2016 have therefore been extensively utilized as probes and affinity matrices for a wide variety of applications in bio- Accepted 7 November 2016 chemical assays, diagnosis, affinity purification, and drug delivery. Recently, there has been a growing interest in Available online 16 November 2016 exploring this non-covalent interaction in nanoscale drug delivery systems for pharmaceutical agents, including small molecules, proteins, vaccines, monoclonal antibodies, and nucleic acids. Particularly, the ease of fabrication Keywords: Nanotechnology without losing the chemical and biological properties of the coupled moieties makes the avidin-biotin system a Avidin versatile platform for nanotechnology. In addition, avidin-based nanoparticles have been investigated as Neutravidin diagnostic systems for various tumors and surface antigens. In this review, we will highlight the various Streptavidin fabrication principles and biomedical applications of avidin-based nanoparticles in drug delivery and diagnosis. Non-covalent interaction The structures and biochemical properties of avidin, biotin and their respective analogues will also be discussed. Drug delivery © 2016 Elsevier B.V. All rights reserved. Imaging Diagnosis

Contents

1. Introduction...... 27 2. Biochemicalinsightsofavidin,biotinandanalogues...... 29 2.1. Structureandphysical-chemicalpropertiesofavidin...... 29 2.2. Avidinanalogues...... 29 2.3. Biotinandanalogues...... 29 3. Applicationsinnanoscaledeliverysystems...... 29 3.1. Nucleicaciddelivery...... 29 3.2. Proteinandpeptidedelivery...... 31 3.3. Vaccinedelivery...... 31 3.4. Monoclonalantibody(MAb)delivery...... 32 3.5. Smallmoleculedelivery...... 33 4. Applicationsofnanoscaleavidinsystemsindiagnosisandbiotechnology...... 33 4.1. Surfaceantigendetection...... 33 4.2. Imaginganddiagnosis...... 34 4.3. Tissueengineering...... 36 5. Conclusion...... 37 Acknowledgment...... 37 References...... 37

1. Introduction

⁎ Corresponding author at: Division of Pharmaceutical Sciences, School of Pharmacy, Nanotechnology holds the greatest potential and promise for bio- University of Missouri-Kansas City, 2464 Charlotte Street, Kansas City, MO 64108, United fi States. medical research. Signi cant progress has been achieved in nanotech- E-mail address: [email protected] (K. Cheng). nology across a wide spectrum of fields from applied physics to

http://dx.doi.org/10.1016/j.jconrel.2016.11.016 0168-3659/© 2016 Elsevier B.V. All rights reserved. 28 A. Jain, K. Cheng / Journal of Controlled Release 245 (2017) 27–40 biotechnology to medicine. Naturally occurring interactions are the crux especially biochemical assays and affinity purification, over four de- for such innovations and have been explored for various nanoscale cades. Tremendous efforts have also been converged to utilize the in- applications to achieve uncountable scientific goals. As one of the stron- herent properties of avidin in biotechnology medicines, and some of gest non-covalent interactions in the nature, the avidin-biotin interac- them have been evaluated in clinical studies. Recently, the avidin-biotin tion has been utilized in nanoscale drug delivery systems for technology underwent a renaissance in nanoscale drug delivery and di- pharmaceutical agents, including small molecules, proteins, vaccines, agnostics. Targeting ligands or imaging agents can be easily coupled to monoclonal antibodies, and nucleic acids (Fig. 1). nanocarriers via the avidin-biotin linkage. For example, PMA hydrogel Avidin is a basic tetrameric glycoprotein composed of four identi- capsule functionalized with biotin forms a stable nanocomplex with av- cal subunits, each binds to biotin with high specificity and affinity idin-coupled antibodies and improved its cellular uptake in cancer cells −15 (Kd ~10 M). Avidin is originally derived from the eggs of aves, [7]. Liposomes modified with biotinylated polyethylene glycol can at- reptiles and amphibians. Avidin-biotin interaction is considered tract a layer of neutravidin on the surface to resist nonspecificbinding one of the most specific and stable non-covalent interactions, to serum proteins, thus leading to prolonged circulation time [8]. which is about 103 to 106 times higher than an antigen-antibody in- Microbubbles coupled with RGD peptide via avidin-biotin linkage teraction [1]. Several genetically and chemically engineered avidin were developed for the detection of Hep-2 related tumor angiogenesis and its analogues have been studied to improve the functional and [9]. Neutravidin conjugated superparamagnetic iron oxide nanoparticle structural characteristics of avidins [2]. The biggest advantage of has also been explored as an imaging agent for rhodopsin degeneration this system is its high affinity interaction, which is robust and stable [10]. More recently, the avidin-based nanotechnology has found its ap- against manipulation, proteolytic enzymes, temperature, pH, harsh plications in tissue engineering and cellular regeneration [11,12].Inone organic reagents, and other denaturing reagents [3–6]. Therefore, such study, an avidin-biotin system was used to improve osteoblast-like the avidin-biotin interaction serves as a great tool in the biomedical cell adhesion to a highly porous calcium phosphate glass scaffold for and nanotechnological applications. On the other hand, biotin-based bone tissue engineering [12]. conjugates are easy to synthesize and have less impact on the activ- The aim of this review is to highlight the unprecedented advantages ity of the biomolecules. of avidin and its analogues in nanotechnology. We will critically evalu- Compared to other covalent and non-covalent interactions, the avi- ate a wide variety of applications that have been recently explored for din-biotin system provides enormous advantages such as amplification drug delivery and diagnosis. It is our hope that this review will serve of weak signals, efficient operation, robust stability. Therefore, avidin as a one-stop reference for investigators who are interested in exploring has been a very versatile modality in the field of biotechnology, the avidin-based nanotechnology in their fields.

Avidin

Fig. 1. Avidin-based nanoscale systems in various applications. A. Jain, K. Cheng / Journal of Controlled Release 245 (2017) 27–40 29

2. Biochemical insights of avidin, biotin and analogues biotinylated moieties and biotin-coated surfaces for the detection of protein-specificantibodies[36]. 2.1. Structure and physical-chemical properties of avidin Bradavidin II is a relatively new avidin analogue that was isolated from Bradyrhizobium japonicum, a nitrogen-fixing bacteria found in Avidin is a basic (pI ~10), highly stable, tetrameric glycoprotein the root nodules of the soybean plant [37].BradavidinIIshowsamod- (molecular weight 66–69 kDa) that contains terminal N-acetyl glucos- erate amino acid similarity with avidin (38%) and streptavidin (32%), amine and mannose moieties [13]. Each of the four subunits contains but exhibits the same biotin binding affinity as avidin. Compared to 128 amino acids and binds to biotin with high specificity and affinity streptavidin, bradavidin II could be a better choice for therapeutic −15 (Kd ~10 M) [14,15]. The subunit is composed of eight antiparallel applications because it is less immunogenic. The physical-chemical β-strands that form a β-barrel, whose wide end binds to biotin [16]. properties of avidin, neutravidin, streptavidin and bradavidin are sum- The avidin-biotin interaction is approximately 103 to 106 times higher marized in Table 1. than an antibody-antigen interaction. However, avidin may have a Most of the avidin analogues have a similar tetrameric structure, high degree of nonspecificbindingin vivo due to its basic pI and glyco- which is beneficial to high amplification of a desired signal. On the sylation. Rigorous efforts have been made to study the structural prop- other hand, the tetrameric assembly may affect the accuracy of binding erties of avidin using X-ray analysis of its 3D structure to improve its quantitation due to the uncertainty of the precise binding stoichiometry stability and functional properties [17,18]. Investigators have success- and possible crosslinking. As a result, avidin analogues, such as fully generated several chimeric avidin analogues with better thermal hoefavidin and rhizavidin, which have dimeric arrangement, are of in- stability and resistance toward proteolytic enzymes [18,19]. terest [38,39]. Their tertiary topologies remain the same as avidin. On the other hand, the strong interaction between avidin and biotin Other recombinant or naturally occurring avidin include Tamavidin may pose a limitation in releasing the tagged biomolecules from the 2(Pleurotus cornucopiae) [40], Shwanavidin (Shewanella denitrificans) biotin or avidin. Reversibility of the avidin-biotin interaction can be [41], Switchavidin (chicken avidin mutant) [42], Zebavidin (zebrafish) achieved by addition of a highly concentrated biotin solution. [43]. Researchers have also developed biotin analogues that have slightly low affinity toward avidin in comparison to biotin. For example, 2.3. Biotin and analogues desthiobiotin can be easily released from avidin by addition of a moder- ately concentrated biotin solution [20]. Another method is to insert a Biotin is a vitamin also known as vitamin H, vitamin B7 or co-en- cleavable linker, such as a stimuli-responsive linker, between biomole- zyme R. Biotin is composed of a tetrahydrothiophene ring fused to a cules and the biotin or avidin. It is noteworthy to mention that chemical ureido (tetrahydroimidizalone) ring. It plays a key role in cell signaling modification of the biomolecules may compromise their activities [21]. and acts as a cellular growth promoter. Biotin receptor (sodium-depen- dent multivitamin transporter and high-affinity biotin transporter) is widely expressed in nearly all living cells. Moreover, its expression in di- 2.2. Avidin analogues viding cancer cells is higher than in normal cells, making biotin a poten- tial targeting moiety for cancer therapeutics [44]. Extensive effort has Despite its enormous advantages and wide applicability, avidin has therefore been made to develop biotin-based platforms for tumor several limitations including non-specific binding and possible immu- targeting and diagnosis [45]. nogenicity. To circumvent these limitations, tremendous efforts have The functional groups of biotin have been chemically modified been devoted to discovering and engineering superior variants of avidin to synthesize biotin analogues, such as iminobiotin, ethylbiotin, by genetic modification or discovering a completely new source, e.g., a desthiobiotin, biotin-carbamate, and biotin-carbonate for various appli- different species. cations [46]. Chemical modification of biotin may affect its affinity to-

The most widely used analogue of avidin is streptavidin. Derived ward avidin. For example, iminobiotin shows pH-dependent Kd −11 −3 from Streptomyces avidinii, streptavidin is a ~56 kDa non-glycosylated values. Its Kd is 3.5 × 10 M at basic pH but lower than 10 Matacid- −14 tetrameric protein that binds to four biotins with a Kd of ~10 M ic pH (3–4) [47]. [22]. Homologs of streptavidin have been discovered from other species, Apart from biotin, strep-tag (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) is a −4 including fungus, bacteria, chickens and frogs [23]. Similar to avidin, peptide that shows high affinity (Kd 2.7 × 10 M) to streptavidin streptavidin is also resistant to denaturing agents, temperature, pH [48]. It has been used in a variety of nanotechnology and biotechnology and proteolytic enzymes. Despite having a tertiary/quaternary structure applications [49,50]. Recombinant proteins of interest can be easily and amino acid arrangement similar to those of avidin, streptavidin only fused to strep-tag for protein purification and other applications. Re- shows a moderate sequence homology level of ~30% sequence identity searchers have also discovered other variants of strep-tags, strep-tag I and 40% similarity with avidin [24,25]. Moreover, streptavidin is non- and strep-tag II, with higher affinities. The strep-tag I (Ac-Trp-Ser-His- glycosylated and has a slightly acidic pI of ~5–6 [26,27]. Due to its differ- Pro-Gln-Phe-Glu-Lys) and strep-tag II (Trp-Ser-His-Pro-Gln-Phe-Glu- −6 ent physical-chemical properties, streptavidin shows an in vivo tissue Lys) peptides bind to streptavidin with a Kd of 37 × 10 Mand distribution and clearance profile very different from those of avidin 72 × 10−6 M, respectively [51]. [27]. Furthermore, streptavidin protects the biotinyl esters from hydro- lysis, whereas avidin augments this hydrolysis [24]. A variety of geneti- 3. Applications in nanoscale delivery systems cally engineered streptavidins such as Strep-Tactin have been developed to exploit the outstanding specificity of the genetically 3.1. Nucleic acid delivery encodable peptide Strep-tag II [28]. Strep-tag II has been used for protein purification and detection [29] as well as in numerous in vivo Nucleic acids, including plasmid DNA, siRNA, miRNA, aptamers, and applications [30–32]. oligonucleotides have been extensively explored as therapeutic agents Neutravidin is another commonly used avidin analogue. It is the de- for a wide variety of diseases, and some of them have been applied clin- glycosylated derivative of avidin and has a molecular weight of ~60 KDa. ically. Their negative charge and poor stability are the two major hur- In the absence of the carbohydrate moieties, the pI of neutravidin is only dles that prevent nucleic acids from reaching their full potential as slightly acidic (~6.3), which prevents its nonspecific binding to cell sur- therapeutics. Although viral vectors are generally effective for nucleic faces and proteins [33,34]. As a result, neutravidin can be coated on the acid delivery, virus-associated safety concerns make non-viral systems surface of quantum nanorods to stabilize them and prevent aggregation better candidates for therapeutic applications. Numerous non-viral [35]. In addition, neutravidin has been utilized as a bridge between platforms, especially nanoscale delivery systems including liposomes, 30 A. Jain, K. Cheng / Journal of Controlled Release 245 (2017) 27–40

Table 1 Physical-chemical properties of avidin and derivatives.

Properties Avidin Neutravidin Streptavidin Bradavidin II

Origin Chicken egg Derivative of avidin Streptomyces avidinii Bradyrhizobium japonicum Molecular weight (KDa) 67 60 53 58.4 Iso-electric point 10 6.5 5.3–6.5 9.6 Specificity ++ ++++ +++ +++ Biotin binding site 4 4 4 4 Applications Imaging, peptide delivery Imaging, pre-targeting, Immunological assays, nucleic Affinity purification mab-fusion protein delivery, sirna delivery, drug acid delivery, vaccine development, vaccine development, pre-targeting delivery, antigen detection mab-fusion protein delivery peptide nanocomplex, polymer-based nanoparticles, and inorganic In addition to their use as Trojan horses, viral proteins have also been nanoparticles, have been rigorously developed for nucleic acid delivery utilized to target nucleic acids to cells. A viral domain that specifically over the past two decades. targets a particular cell type is coupled to an avidin moiety via a covalent Nano-formulations for the delivery of siRNA face tremendous chal- bond, and the avidin molecule acts as a carrier for the delivery of the lenges, including aggregation, short systemic half-life and low cellular nucleic acids. Leisi et al. developed one such delivery system, in which uptake [52–54]. Protein-based delivery can efficiently counter such they utilized the VP1u domain of parovirus B19 conjugated to shortcomings. For the delivery of siRNA-like negatively charged mole- neutravidin to specifically deliver biotinylated DNA or fluorophores to cules, cationic proteins, peptides or polymers are recommended for the targeted erythroid cells that were undergoing differentiation use. These counter-ions produce a high likelihood of nanocomplex ag- around the proerythroblast stage. For this study, maleimide-activated gregation. In a related study, neutravidin was utilized deliver siRNA to neutravidin was used because it can attach to 5–6 VP1u in a manner podocytes by conjugation of a monovalent IgG (mIgG) to neutravidin that excludes the four biotin-binding sites. Neutravidin is very versatile through a sulfhydryl group (Fig. 2A). Here, instead of the biotinyation and retains its activity after minor chemical modifications. The results of the siRNA, protamine (a cationic protein) was linked to biotin that demonstrated that this VP1u-neutravidin nanocarrier is highly efficient was coupled to the neutravidin tag on the mIgG. Cationic protamine and has excellent therapeutic and diagnostic applications in a wide va- therefore condenses multiple siRNA molecules and deliver the complex riety of blood-associated conditions, including hematological ailments to the target podocytes. Within 30 min of incubation, the IgG with siRNA such as thalassemia or leukemia [56]. was detectable in the cytoplasm. This delivery system showed a tre- Similarly, streptavidin has been employed to deliver siRNA for the mendous reduction (greater than 80%) in the expression of the target treatment of liver fibrosis. The tetravalent structure of avidin makes protein (p57/Kip2) compared to the control [55]. this protein a very efficient tool for the delivery of biotinylated siRNA

Protamine BCG HGN

A B C D

Chimeric mAvidin

siRNA Aβ1-40 Peptide mAB 125I Avidin or variants

Biotin Ligand

Small molecule drug E FGPolymer

Fig. 2. Various fabrication mechanism sof avidin-based nanoscale drug delivery systems. (A) Monovalent anti-podocyte anti body is coupled to protamine through the biotin-neutravidin linkage. Cationic protamine there for condenses multiple siRNA molecules and deliver the complex to the target podocytes.(B) Biotinylated siRNA and biotinylated ligand are anchored to the same streptavidin back bone.(C) Plasmonic hollow gold nanoshells (HGN) are decorated with siRNA molecules via thiol bond. The other end of the siRNA is biotinylated andcoupled to biotinylated TAT peptide through streptavidin. Upon exposure to near infrared (NIR) light (~800 nm), the siRNAs are released from the nanoshells due to gold-thioldissociation. (D) Bacillus Calmette-Guerin (BCG) bacteria are biotinylated and coupled to achimeric fusion protein composed of amonovalentavidin (mAvidin) and an antigen. The anti gencoated BCG is the ninoculatedin to animals to deliver antigens and subsequently induce specific T cell responses. (E) Radio labeled Aβ1–40 is biotinylated and coupled to the TfR-MAb-avidin fusion protein. This complexc and eliver the radio pharmaceutical across the blood brain barrier. (F) Liposome is functionalized with biotinylated PEG, which is coupled to abiotinylated antibody through streptavidin. (G) Neutravidin-biotin is used to link microhydrogel containing doxorubicin and biotinylated hyaluronicacid. A. Jain, K. Cheng / Journal of Controlled Release 245 (2017) 27–40 31 by producing stable nanocomplexes. These nanocomplexes have shown peptide mandated by the carrier [70]. The four biotin-binding sites on rapid uptake and silencing of the target gene. As shown in Fig. 2B, the bi- avidin can be exploited for the delivery of different peptide sequences otinylated siRNA and biotinylated cholesterol anchored to the for specific roles such as targeting a ligand, as depicted in Fig. 2B. In streptavidin backbone provide efficient silencing of the PCBP2 gene for one such study, a tumor-targeting peptide (bio-CREKA) and an argi- liver fibrosis treatment [57,58]. Streptavidin-biotin technology has nine-rich peptide (Bio-R8) were used simultaneously to deliver the also been utilized for the delivery of Kv1.3 siRNAs in the treatment of p53 tumor suppressor gene. Here, avidin allowed the use of the two bi- autoimmune disorders. Biotinylated polyethylene glycol and cholester- otinylated peptides, where the Bio-R8 moiety promoted the internaliza- ol were functionalized with biotinylated-CD45RO antibodies through tion of the nanocomplex and bio-CREKA provided for specificbindingto the use of streptavidin to provide highly specific targeting and uptake the receptors on MCF-7 cells [72]. of nanoparticles by memory T cells, which had the consequence of de- The internalization of a protein by a target cell involves overcoming creasing the calcium ion influx, thus producing a therapeutic effect a series of barriers. Investigators have developed fusion proteins that in- [59]. Several other simple and efficient strategies for nucleic acid deliv- clude avidin or streptavidin to enhance the uptake of small synthetic ery have recently been reported for the targeted delivery of nucleic molecules mediated by receptors. The presence of the streptavidin in acids. these fusion proteins triggers internalization and thus overcomes the Delivery of siRNA to normal cells is relatively easier compared to the delivery barriers [73]. The avidin modification not only provides a process in stem cells. Biocompatibility is a major concern because in- very useful carrier for peptide delivery but substantially promotes the compatibility could otherwise cause a fatal immunogenic response cellular internalization [74]. Several parameters including particle ag- [60,61]. Synthetic vectors pose various limitations for the transfection gregation, size and surface charge play vital roles in cellular internaliza- of undifferentiated human embryonic stem cells (hESC) with nucleic tion. Rational modification of the surfaces of the nanoparticles with acids. Huang and coworkers developed a light responsive siRNA deliv- avidin showed higher uptake than that of the unmodified nanoparticles. ery system using hollow gold nanoshells (HGN) in which the HIV-de- In contrast to the DSPE-PEG nanoparticles, Av-MPG-NPs showed a rived TAT peptide was utilized to deliver the siRNA into the hESCs higher uptake due to their surface charges, which resulted in a smaller under the influence of biocompatible infra-red radiation (~800 nm). size and a higher ligand density. These properties have a combined im- The direct conjugation of the TAT peptide to the siRNA resulted in aggre- pact on the internalization kinetics that resulted in the higher internal- gation due to surface charge. As depicted in Fig. 2C, the TAT and siRNA ization of the Av-MPG-NPs [75]. were coupled to different binding sites of the streptavidin molecule, Peptide sequences such as cell-penetrating peptides (CPP) hold which kept them separated. This structure prevents electrostatic con- considerable potential for the cellular internalization of molecules tact, which inhibits particle aggregation [62]. that are difficult to transfect. However, CPP-based delivery to colo- Another novel method of delivering nucleic acids is the avidin- rectal cancer (CRC) cell lines or metastatic cancer cells is even nucleic acid nanoassembly (ANANAS). Here, the nucleic acid acts as a more challenging. Streptavidin-like proteins have been used as a central unit onto which several avidin molecules are nucleated. This model protein carrier for the delivery of several CPP that can pro- assembly forms a toroidal structure, which is further complexed with mote the internalization of macromolecular drugs, such as DNA or biotinylated polyethylene glycol [63]. Due to the excellent pharmacoki- siRNA. Cell-penetrating sequences such as transportan-10 (TP-10) netic properties of avidin, after in vivo administration, fluorescently and transportan (TP) have recently been evaluated by Wierzbicki labeled ANANAS nanoparticles showed substantial subcellular internal- et al. Biotinylated analogues of TP and TP-10 have demonstrated an ization in the mucosal vasculature. This enabled the localization of endocytosis-independent and highly efficient delivery of siRNA nanoparticles at the target site, whereas no accumulation was observed when complexed with streptavidin in HCT116 (metastatic CRC in healthy tissues. ANANAS shows tremendously promising characteris- model) and HT29 (early stage CRC model) cell lines and resulted in tics, including easy preparation, no immunogenicity, and excellent a high silencing activity of SASH-1 mRNA [76]. It can clearly be in- pharmacokinetic properties, which make it an outstanding translational ferred that streptavidin provides a substantial advantage for cellular therapeutic and diagnostic tool [64,65]. and tissue internalization in addition to its use as a dynamic stoichio- metric support (back-bone) for nanocomplexes. 3.2. Protein and peptide delivery 3.3. Vaccine delivery Protein and peptide delivery has generated considerable interest in the past two decades on the basis of its potentially important applica- In recent years, vaccine development has generated much interest. tions in targeted therapy. Therapeutic peptides, enzymes and recombi- Rigorous efforts have been made to reduce the time between the dis- nant proteins are among the highest revenue-generating products covery of vaccine candidates and their clinical development. The among all the pharmaceutical products offered across the globe [66]. greatest challenge involves the production of antigens in an appropriate However, the macromolecular drugs face substantial delivery chal- quantity to induce an optimal immune response in body [77]. Avidin lenges including slow or low permeability across biological membranes also provides the advantage of varying and adjusting surface chimeric and low target-specific biodistribution [67]. The greatest hurdle in the proteins on bacteria to induce a higher immunogenic response toward delivery of peptides and proteins is their encapsulation. Special consid- the vaccine. Furthermore, biotinylation of the bacterial surface proteins eration must be given to the chemical and physical properties of the bi- has no effect on the phenotypic characteristics of the bacteria or the ex- ologics before contemplating the nanocarrier. Because proteins are ogenous properties of the protein, all of which make avidin a very appli- prone to structural distortion that may lead to the loss of biological ac- cable moiety for vaccine development [78].Inadditiontoalloftheother tivity [68], special care is needed in protein modification to minimize advantages, deglycosylated monovalent avidin substantially reduces the loss of the activity of these molecules. the chances that the vaccines will aggregate [78]. Due to the minimal modification required by the biotinylation of a The stability of vaccines is the most important aspect of vaccine de- protein molecule, avidins have been employed by several researchers velopment. Much effort is being directed toward improving the stability as a carrier for peptide delivery, such as cell penetrating peptides and efficiency of the development of vaccines for a range of conditions, (CPP) [69,70] and TAT peptide [71]. The biotinylated peptide sequences from HIV to dengue. One issue is that the addition of protein molecules are complexed with avidin to reduce the chance of aggregation. Addi- that may help to stabilize a vaccine may compromise its activity. Solu- tionally, covalent modifications of peptides may induce conformational tions have recently been devised to express an avidin fusion protein changes that can interfere with their ability to be translocated. Howev- along with the target protein for stable and efficient antigen expression. er, biotinylation acts as a spacer that reduces the constraints on a In one such study, shown in Fig. 2D, Bacillus Calmette-Guerin (BCG) 32 A. Jain, K. Cheng / Journal of Controlled Release 245 (2017) 27–40 bacteria were biotinylated, and the surface of the bacteria was decorat- 3.4. Monoclonal antibody (MAb) delivery ed with a monovalent avidin fusion protein. This technique produced BCG vaccines that are more responsive to T cells, and which were repro- Avidin-biotin technology has made it possible for a monoclonal an- ducible and highly stable for an extended period of time after freeze tibody to carry a payload to the vital target sites. Avidin-coupling or fu- drying [78]. Another research group developed a technique to employ sion proteins not only make it easier to formulate the delivery carrier avidin in the development of a Lassa fever vaccine that undergoes but help to increase the uptake at a target site. Avidin provides a greater self-assembly. Here, MtbHSP70-avidin proteins were coupled to bio- advantage in the delivery of drugs via monoclonal antibodies. Avidin-fu- tinylated peptides from the Lassa GP1 and GP2 proteins, which are nat- sion proteins coupled to monoclonal antibodies by genetic engineering urally immunogenic. This self-assembling vaccine showed tremendous have shown excellent target specificity. Molecular Trojan horse and av- stability and optimal immunogenicity [79]. idin-biotin technologies have been recently exploited to delivery bio- The vaccine research and development process also faces the chal- logics across the blood brain barrier (BBB) via transferrin receptors lenge of the extended time required to reach an effective in vivo immu- (TfR). Specifically, TfR-MAb functions as a ferry to transport the bio- nogen concentration. Investigators have reported efforts to minimize logics into the brain through the BBB TfR [90]. Chemically cross-linking the long time required for the B cell-specific antigen detection and the avidin with TfR-MAb is one strategy that has been applied to deliver a slow onset of the immune response. This method involves the sequen- peptide across the BBB [91]. Unfortunately, the specificity of monoclonal tial staining of biotinylated antigenic gonadotropin-releasing hormone antibodies is compromised when they are chemically modified. Howev- (GnRH)-like peptides with streptavidin or neutravidin. This process in- er, an IgG-Avidin fusion protein expressed in Chinese hamster ovary creases the ligand avidity, and the ‘single epitope multiple staining’ (CHO) cells retained the specificity of the MAb. Similarly, avidin-chime- principle permits the rapid detection of the B cells after immunization ric-TfR-MAb fusion protein was also expressed and engineered in bio- [80]. tin-depleted CHO cells [92]. Using the same methodology, the amyloid In addition to binding to the biotinylated antigen, streptavidin is also plaques that accumulate in the brain and result in Alzheimer's disease used as a potent immunostimulant in cases of less immunogenic anti- have also been targeted for imaging and diagnosis using an TfR-MAb- gen-based cancer vaccines. Currently, cancer vaccine research utilizes avidin fusion protein. As depicted in Fig. 2E, radiolabeled Aβ1–40 was at- “self” tumor antigens, which are only weakly immunogenic. These anti- tached to the TfR-MAb-avidin fusion protein. This complete conjugate gens are not much different from other cells in patient's body, hence, the was used to ferry the radio-pharmaceutical across the BBB [93]. patient becomes tolerant very rapidly. To circumvent such problems, Antibodies are also extensively used in radioimmunotherapy, which immunostimulants of bacterial origin, such as streptavidin, have been requires a highly specific interacting molecule for the binding of the ef- used. Moreover, streptavidin has also been demonstrated to have a fector to the pre-targeting molecule [94]. To achieve a high specificity of high affinity for surface-bound tumor proteins. The RYDS sequence of the effector for the pre-targeting molecule, avidin-biotin technology has streptavidin assists in cell adhesion through the RGD cell adhesion do- been recently implemented. In contrast, a fusion protein approach using main [81]. These two qualities have led to the use of streptavidin for the avidin-biotin system is very effective and produces a superior ho- the delivery of cancer vaccines. The vaccine-streptavidin combination mogeneity. However, this approach is highly time-consuming and re- produced 6 times higher reactivity than the vaccines alone. Streptavidin quires extensive efforts and optimization [95]. A pretargeted 211At- bound to biotinylated soluble tumor proteins have successfully pro- radioimmunotherapy developed by Frost et al. consists of an avidin- duced tumor reduction and remission in a 9 L glioma rat model and in monoclonal antibody that has a high affinity toward radiolabeled and canine patients [82]. biotinylated poly-L-lysine conjugates. This radioimmunotherapy deliv- Vaccine development requires the vaccine immunogen to be pre- ery system is highly efficient, less time-consuming and demonstrates sented at a high density with a uniform orientation through which it excellent tumor specificity [96]. Compared with conventional can attain ideal epitope spacing and mimic the multivalent epitope radioimmunotherapy, the avidin-conjugated monoclonal antibody pro- of a virus [83,84]. Controlling the orientation and density of the duced a higher uptake of radiopharmaceuticals [97]. coupled antigen by chemical conjugation poses a tremendous chal- Patrick Ng et al. also developed an avidin-antibody fusion protein lenge. Additionally, the incorporation of complex antigens onto (Av-anti-rat TfR IgG3) that is capable of efficient delivery of various virus-like particles (VLPs) compromises their assembly and thus molecules into cancer cells. In this approach, the CH3 region of a the activity. Thrane et al. designed a genetically modified HPV16 L1 human lgG3 (rat transferrin receptor-specific) is genetically fused VLP (human papilloma virus 16 L1 virus-like particle) to overcome with avidin. Generally, the fusion protein approach requires the chemi- these challenges. The insertion of AviTagTM (Avidin tag) in the L1 cal conjugation of specific protein components with various applica- coding sequence of HPV16 L1 VLP permits it to be biotinylated specif- tions. This results in decreases in the activities of the respective ically. This, in turn, can be utilized to fuse the antigen with monova- components, and its development is very difficult. In contrast, the avi- lent streptavidin without disrupting the assembly of VLPs and their din-Mab construct is a universal construct that eliminates the need to activity [85]. use a specific protein for a particular application. Ng and coworkers The use of avidin in recombinant vectors has increased the possibil- demonstrated that the Av-anti-rat TfR IgG3 possesses an ability to deliv- ity of delivering various classes of biotinylated antigens. Vectors that er various biotinylated molecules and has a strong pro-apoptotic activ- target dendritic cells using a single chain antibody (scFv) fused with a ity against the T cell lymphoma cell line Y3-Ag1.2.3 as well as the rat C58 streptavidin core is an example of this approach. Dendritic cells play (NT) D.1.G.OVAR (malignant cancer cells), while a non-recombinant an essential function in the management of antigen-specific immunoge- anti-rat TfR IgG3 did not show any activity [98]. They further showed nicity. Antigenic vaccines that can specifically target the dendritic cells that this recombinant avidin system may induce a high antitumor activ- to activate the required immunogenic response are being developed ity in vivo by delivering the biotinylated agents into the cancer cells and tested clinically [86,87]. However, the optimal immune response [98]. was not observed following either in vivo or ex vivo stimulation. A re- Molecular Trojan horses are also a type of genetically engineered ceptor-targeting approach has been developed to use dendritic cells re- MAb that has recently become the most promising tool for the delivery ceptor-specific ligands that can efficiently deliver antigens to the of macro-molecules across biological membranes such as the BBB. The dendritic cells [88]. Investigators have developed a single chain anti- only caveat of this fusion technology is that the protein cannot be body (scFv) fused with a streptavidin core that targets the DEC-205 re- fused with oligonucleotides. However, the avidin-biotin technology en- ceptor of dendritic cells. This streptavidin core and biotinylated antigen ables the targeting of siRNA and peptides in vivo in association with fu- subsequently form a complex and delivers the antigens to the target sion protein technology [99]. Merging the Trojan horse technology and dendritic cells [89]. the avidin-biotin technology has also allowed a highly specific receptor- A. Jain, K. Cheng / Journal of Controlled Release 245 (2017) 27–40 33 mediated means to deliver siRNA using Mab, which have extensive sta- In another approach (Fig. 2G), hyaluronic acid (HA)-based micro bility in vivo. In a similar study, human insulin receptors (HIR) were hydrogels were prepared by exploiting the avidin-biotin technology. targeted using HIRMAb that were conjugated with streptavidin Here, neutravidin-biotin was used to link micro-hydrogel containing (HIRMAb-SA) and subsequently bound to a 3′-biotinyl-siRNA. The de- doxorubicin to the HA molecule to function as a switch. Avidin-biotin livery of luciferase siRNA with HIR-MAb-SA caused 90% silencing of has also been exploited by several researchers for the hydrogel grafting the luciferase gene. In comparison, avidin, unconjugated streptavidin, [112,113]. These HA-biotin- neutravidin micro-hydrogels show fine or the HIR-MAb alone showed negligible effect [100]. porosity in conjunction with micro-beads. Upon addition of excess bio- Non-convalent avidin-biotin interactions are very helpful for the tin, the HA microhydrogel-encapsulated drug disassembles to rapidly surface decoration of nanoparticles with antibodies. The use of avidin- release additional doxorubicin. In this application, biotin acts as the biotin to conjugate the MAbs to the nano-carrier surface helps to retain triggering agent or a switch to release the drug at the target site [114]. the MAb's function. Hajdu et al. biotinylated antibodies to CD45RO, a We have described the advantage of avidin in the tumor-specificde- cell surface marker for memory T cells. These biotinylated MAbs were livery of drug molecules. Delivery across the BBB and other complex bi- used to functionalize the surfaces of nanoparticles made from ological barriers is even more challenging than metastasized malignant pegylated/biotinylated phosphoethanolamine and cholesterol through tumor cells. To address this issue, investigators have devised several ap- the use of streptavidin. The resulting CD45RO-functionalized nanoparti- proaches including the use of apolipoproteins in the design of nanopar- cles showed highly efficient targeting to memory T cells for potential ticles that mimic natural particles [115]. Low-density lipoprotein therapy for autoimmunity (Fig. 2F) [59]. receptor-specific apolipoprotein E (Apo-E) target the endothelial cells of the BBB very efficiently. However, chemical conjugation and other modifications of Apo-E reduce its function substantially. Avidin-biotin 3.5. Small molecule delivery technology serves as a convenient tool to attach these Apo-E molecules to a drug delivery carrier without compromising their activity. Here, Nanotechnology has provided many ways to efficiently deliver che- Apo-E was biotinylated and used to functionalize avidin-conjugated motherapeutic drugs to the site of action. Efficient delivery of small mol- solid lipid nanoparticles. The avidin-biotin system is thus useful for ecules requires high specificity to the target site which can only be the strategic functionalization of nanocarriers for highly efficient deliv- achieved by the attachment of receptor-specific ligands [101]. Avidin ery of therapeutic small molecule drugs [116]. provides an excellent bridge for diagnostically relevant ligands that tar- Stimulus-responsive technology for drug development, controlled get specificreceptors[75]. These ligands may or may not be functionally release and targeted therapy has been extensively harnessed by re- modified with stimulus-responsive cleavable linkage for delivery of search groups across the globe. In this context, the switchable release these small molecules. Mesoporous silica nanoparticles capped with av- of avidin from biotin is one of the most simplistic, intriguing and inno- idin functionalized with an MMP9 (matrix metalloproteinase 9) specific vative strategies. Imino-biotin is a type of biotin which has avidin-bind- cleavable linker is one such example [102]. Because MMP9 is over- ing properties similar to those of natural biotin. Imino-biotin is coupled expressed in the areas of lung tumors that are beginning to metastasize, to the nano-carrier, where it retains its structure and its noncovalent it allows the controlled release of the drug at the site of metastasis [103]. bond with avidin at physiological pH. Upon protonation under lower The mesoporous silica nanoparticles mentioned above are constituted physiological pH conditions, its affinity for avidin is decreased, and it re- with tunable pores and volume for higher drug loading. The outer sur- leases the drug attached to it. This switch-release technology using a faces of these mesoporous silica nanoparticles can be functionalized modified biotin is highly efficient in comparison to the disulfide linkages with certain molecules that do not interfere with the pore morphology [117,118]. and integrity. In one such example, the mesoporous silica nanoparticles were tightly capped with avidin molecules, and the pores were blocked 4. Applications of nanoscale avidin systems in diagnosis by biotin, which prevented the release of the payload (cisplatin) from and biotechnology the core [102]. Thus, the avidin-biotin served as a guard on the surface of the nanoparticles to regulate the release of drug. 4.1. Surface antigen detection The avidin-biotin system has also been widely used as a pretargeting strategy [104,105]. Additionally, biotin and avidin have been reported to The interaction of avidin with biotin provides an excellent platform target and accumulate, respectively, in tumors [106–109]. These prop- for the development of various assay systems. Although antibody- erties make them an excellent choice for a tumor-targeting formulation based assay systems are highly specific, chemical conjugation with fluo- strategy. High avidin accumulation in the liver after treatment, as well rescent dyes or chemiluminescent compounds may have some impacts as the properties mentioned above, have been exploited to target hepat- on the antibody specificity. Quantum dots have proven to be a highly ef- ic carcinoma. Recently, chitosan nanoparticles modified with biotin and ficient tool compared to the traditional antibody-fluorescent dye-based avidin were designed in which the mannose sugar and acetyl glucos- assays. Quantum dots provide several advantages such as high signal amine of avidin were used to target the liver. Moreover, avidin allows strength, high photo stability and high quantum yield for the detection the addition of multiple ligands on the surface to achieve highly specific of proteins with an enhanced signal amplification [119]. Protein quanti- targeting. These authors demonstrated that the avidin bound to the bi- fication using quantum dots in elemental mass spectrometry (ICP-MS) otin on the surface of the nanoparticles was responsible for the higher based immunoassays has also proven to be very effective and efficient. liver accumulation of nanoparticles and thus enhanced the anticancer Quantum dots possess outstanding photoluminescent properties, activity [110]. which makes them very important and novel as an antigen-detection Biotin labeling of the surface of the carrier dramatically increases the system. Quantum dots have a tremendous capacity to improve possibility of surface functionalization through avidin-modified moie- bioanalytical applications that use bio-labeling and bioimaging ties. This technique has been applied to couple liposomes to the surface methods. To fully exploit the functions of quantum dots, it is necessary of microbubbles. Microbubble-liposome therapeutic carriers for which to establish strict control for their synthesis and surface modification. ultrasound is used to target the therapy toward breast cancer has re- The noncovalent interaction between biotin and streptavidin serves as cently a field of extensive research. Yan et al. proposed this novel form the best tool for the surface modification of the quantum dots and per- of therapy in which they used avidin for the surface conjugation of pac- mits the development of selective assay systems without compromising litaxel-loaded liposomes on the microbubbles. Ultrasound exposure their target-binding specificity. In a similar development, an immunoas- caused the drug payload to be efficiently delivered to the target site say platform based on streptavidin-conjugated quantum dots, which [111]. are used to bind to a biotinylated antibody and can be detected using 34 A. Jain, K. Cheng / Journal of Controlled Release 245 (2017) 27–40

ICP-MS, has been designed for amplified protein quantitation. This sys- such as challenging preparation, purification, and a low DNA-to-anti- tem enables the quantitation of protein in samples with concentrations body ratio that results in a low sensitivity and high non-specificback- as low as 50 ng/mL [120]. Neutravidin bioconjugated to highly lumines- ground signals. To address these issues, as depicted in Fig. 4, modified cent quantum dots has also been developed for the detection of the ty- liposomes with an encapsulated reporter DNA have been designed. rosine kinase B (Trk-B) receptors present on the neurons of the The surface of the liposomes is conjugated to a biotin-PEG (polyethyl- hippocampus. Here, neutravidin was specifically used instead of avidin ene glycol) phospholipid that acts as a detection agent. Microplate or streptavidin because the presence of the lysine allows the amide to wells on which the target antigen specific antibody is immobilized bind to the carboxylic groups on the quantum dots. The quantum dot- capture the antigen. A biotin-labeled secondary antibody with a neutravidin bound to the biotinylated anti-TrkB antibody specifically neutravidin bridge binds to the antigen. Biotin-coated liposomes bind detected the distribution of the TrkB receptors. The quantum dot- to the neutravidin-bridge and anchor the reporter DNA-loaded lipo- neutravidin conjugated to the biotin-TrkB serve as an excellent tool somes. Upon binding to the target antigen, the liposome bursts to re- for long-term observations of the trafficking of the fluorescence signals lease the reporter DNA, which permits the target protein to be during live imaging of neurons [121]. Quantum dots have also been quantified using real-time PCR [129]. employed for detecting and tracking enzymes in vivo. As demonstrated Although sandwich ELISA-based assays are well known and widely in Fig. 3, several hydrolase enzymes have been conjugated to quantum applied [130,131], a new assay approach that harnesses the dots through the avidin-biotin interaction. It was found that the catalyt- streptavidin-biotin interaction and exhibits a 10-fold higher sensitivity ic activity was retained by the quantum dot-conjugated enzymes com- has been recently developed [132]. In addition, this approach reduces pared to the free biotinylated enzymes. The avidin-biotin interaction in the cost by 20-fold in comparison to RT-PCR. In this assay, named this application was critical because it is a robust interaction with a high NLFOA (nuclease-linked fluorescence oligonucleotide assay), nanopar- resistance toward wide range of pH. The avidin–biotin system has ubiq- ticles coupled with several streptavidin molecules interacts specifically uitously been proven to be the best method to functionalize the lumi- with the biotinylated TurboNuclease for detection of the HIV-1 p24 an- nescent quantum dots-enzyme conjugates compared with the tigen. The NLFOA provides a highly sensitive assay system for the clini- traditional approaches [122]. cal diagnosis of HIV or other infectious diseases [132]. Non-invasive bio-medical imaging techniques such as magnetic res- onance imaging (MRI), single-photon emission computed tomography 4.2. Imaging and diagnosis (SPECT) and positron emission tomography (PET) can evaluate and di- agnose the progression of numerous diseases through the use of surface Imaging technology has been extensively evolved in recent years. Its antigen detection. Among these techniques, MRI is considered to be the biggest application is the diagnostics and detection of biological technique of choice for imaging soft tissues [123]. Iron oxide nanoparti- markers for specific diseases, such as cancer. Several radiolabeled anti- cles are widely utilized as the MRI contrast agent. However, iron oxide bodies are being extensively studied or are now undergoing clinical tri- nanoparticles need to be stabilized with low-molecular-weight high-af- als for the imaging and diagnosis of cancer [133,134]. However, this finity dispersants [124] and polymers, such as poly ethylene glycol, to method poses several disadvantages that prevents its broad clinical ap- stabilize them against oxidization. In one such study, iron oxide nano- plication. These include slower tumor diffusion and delayed clearance particles were stabilized with poly-ethylene glycol coated with kinetics that lead to a decrease in the tumor-to-nontumor ratio. To ad- neutravidin for the immobilization of the biotinylated VCAM-1 (vascu- dress these shortcomings, the concept of pretargeting has been pro- lar cell adhesion molecule 1) antibodies. VCAM-1 bearing neutravidin- posed. For pretargeting, avidin and streptavidin can be conjugated to coated nanoparticles showed outstanding affinity and specificity to- either monoclonal antibody or radioisotope. The extremely high bind- ward the VCAM chimeras. Neutravidin also provides flexibility in vary- ing affinity, rapid blood clearance and high tumor uptake of biotin and ing the ligand density on the nanoparticles, which makes it an excellent agent for the detection of antigens in-vivo [125,126]. Liposome containing Quantification of a specific antigen at extremely low levels and over reporter DNA a wide range is a very important capability in the field of life science [127,128]. For dynamic sensitivity over a wide range of antigen concertation, immuno-PCR (IPCR) is a system that utilizes antibodies and DNA conjugates. However, this method has several disadvantages,

Enzyme

Antigen Polymer

Capture Antibody

Fig. 4. Avidin-based immunoliposome-PCR. Liposomes encapsulating reporter DNA are conjugated to abiotin-PEG (polyethyleneglycol) phospholipid that acts as a detection Fig. 3. Streptavidin coated quantum dots modified with biotinylated enzyme. Streptavidin agent. A biotin-labeled secondary antibody with a neutravidin bridge binds to the is linked to the carboxylic groups on the surface of the quantum dots. The streptavidin antigen. Biotin-coated liposomes bind to the neutravidin-bridge and anchor the reporter conjugated quantum dots are coupled to biotinylated enzyme via the avidin-biotin DNA-loaded liposomes. Upon binding to the target antigen, the liposome bursts to interaction for in-vivo tracking of the enzyme activity in vivo tracking of antigens like release the reporter DNA, which permits the target protein to be quantified using real- enzyme. time PCR. A. Jain, K. Cheng / Journal of Controlled Release 245 (2017) 27–40 35 avidin make them highly reliable and essentially the best candidate for technology. Streptavidin-coupled antibodies are highly selective for pretargeting imaging technology [94]. Generally, avidin is used to clear the tumor tissues or malignant cells. Once the streptavidin-coupled an- the circulating biotinylated antibodies from the systemic circulation, but tibodies accumulate at the target site, a biotinylated radioactive small for the pretargeting strategy streptavidin or neutravidin are extensively molecule is administered that specifically binds to the streptavidin used. Interestingly, it has been found that the fluorescently labeled or that has accumulated in the malignant cells or tumor site. Green et al. radiolabeled streptavidin showed a higher uptake in the tumors com- developed such a streptavidin-biotin pretargeted radioimmunotherapy pared to the normal tissues than that of the radiolabeled antibody system that targets the CD38 antigen for the delivery of a radionuclide alone [135–137]. for the eradication of multiple myeloma. Here, anti-CD38 was conjugat- For diagnostic purposes, the expression of a specific antigen on a ed to streptavidin, and 90Yttrium was bound to biotin for pretargetting particular cell type can be harnessed and subsequently detected with fa- [144,145].The90Y–DOTA–biotin treatment produced the maximum vorable techniques [138,139]. Similarly, the types of antigens or survival rate in treated mice [145]. markers present on cancer cells reveal the degrees of malignancy, inva- Another diagnostic modality that has received significant interest sion, neovascularization, and metastasis. Fluorescently tagged antibod- due to its unique properties and characteristics (Fig. 6)isnanoparticle ies specific for these cell-specificmarkersareusedforefficient clusters. However, their structurally and morphologically controlled de- detection via imaging or other diagnostic tools. Because avidin is very velopment is highly challenging. For successful size-controlled con- stable to the chemical modification, it serves as a good candidate for struction of nanoparticle clusters, Ryu et al. proposed a method using conjugation purposes. Maleimide activated avidin was utilized to mod- a DNA-binding zinc finger protein that utilizes the avidin-biotin system. ify a MAb which is specific for the embryonic cancer delta-like protein Direct chemical conjugation of metal ions (Zn) to DNA limits their fur- (Dlk-1). A fluorescent bioluminescent protein (FBP) was designed by ther ability to derivatize the nanoparticle surfaces. To circumvent the conjugation of biotinylated Cypridina luciferase (CLu) to a far-red these challenges, DNA was conjugated to biotinylated zinc finger pro- derivative of fluorescent indocyanine (Fig. 5). The FBP was subsequently tein and subsequently incubated with neutravidin-conjugated nanopar- coupled to the Dlk-1-specific MAb to identify the embryonic cancer an- ticles. These nanoparticle clusters showed not only the successful tigen Dlk-1 via the avidin-biotin interaction [140]. development of a size-controlled construct but also increased spin– Theranostic modalities that can simultaneously diagnose and treat a spin relaxivity three-fold compared to conventional contrast agents condition have driven the development of calcium phosphosilicate such as Feridex [146]. nanoparticles (CPNP). CPNP is another type of vehicle that is used for PET scanning is also a highly sensitive and extensively used tech- various imaging and therapeutic applications in biological systems nique for the quantitative imaging of tumor specific markers. Hypoxic [141,142]. These particles function by accumulating in the solid tumors inducible factor 1 (HIF-1) plays a key role in the progression of malig- via enhanced permeation and retention (EPR) effect. Conjugating these nant tumor and radiotherapy resistance. The oxygen-dependent degra- nanoparticles to fluorescent probes has proven to be highly effective. dation domain (ODD) of HIF-1α has therefore been used for the Barth et al. utilized avidin to engineer a novel CPNP that was conjugated development of tumor imaging and therapeutic agents. HIF-1-positive to biotinylated diferric transferrin (human holotransferrin), biotinylat- tumor cells are one type of the targets that have been most quantitative- ed anti-CD71 antibody (transferrin receptor specific), and biotinylated ly diagnosed with the help of noninvasive imaging techniques such as pentagastrin using an avidin-biotin coupling. These avidin-CPNP- PET [147]. A protein transduction domain (PTD) coupled to the oxy- based theranostic materials were shown to specifically target, diagnose gen-dependent degradation domain and to monovalent streptavidin and treat breast cancer and other rapidly dividing and transferrin-ex- was used to produce the fusion protein PTD-ODD-streptavidin (POS) pressing cells [143]. because of the outstanding in-vivo stability of biotin-streptavidin. Another widely accepted theranostic application is Streptavidin modification reduces the degradation of POS. The POS con- radioimmunotherapy, which employs streptavidin for the coupling jugated with radioactive 123I–IBB specifically target the HIF-1 exclusive strategy. Direct radionuclide labeling of MAb has been conventionally regions in tumors [148]. Another streptavidin fusion protein was devel- used for disease control. However, complete eradication is still a chal- oped by the same group to deliver the radiolabeled biotin derivative lenge because the tumor to normal cell localization ratio is extremely synthesized as 4-18F-fluorobenzoyl-norbiotinamide (18F–FBB). It was low. Malignant plasma cells are highly radiosensitive, and the manage- ment of plasmacytomas and multiple myeloma is possible with the help of radioimmunotherapy combined with streptavidin and biotin NP

F1 Luciferin F2 F1, F2 and F3 O2 Cypridina Luciferase CLuc F3 Zinc Fingers

CO2 NP Indocyanine dye Oxyluciferin

NP Nanoparticle Dlk-1 mAb NP F1

F2

F3 Dlk-1 Receptor NP Cancer Cell

Fig. 5. Avidin-based fluorescent bioluminescent protein probe for cancer imaging. A Fig. 6. Neutravidin-based nanoparticle clusters for diagnosis and imaging. Biotinylated fluorescent bioluminescent protein (FBP) is designed by the conjugation of biotinylated zinc finger protein is coupled to avidin conjugated nanoparticles. The zinc finger Cypridina luciferase (CLu) to a far-red derivative of fluorescent indocyanine. These FBP proteins (F1, F2, F3) bind to the DNA template. These zinc fingers and DNA templates is subsequently coupled to the Dlk-1-specific MAb to identify the embryonic cancer are highly sequence specific which helps in the size-controlled construction of NPC for antigen Dlk-1 via the avidin-biotin interaction. application in diagnosis and imaging. 36 A. Jain, K. Cheng / Journal of Controlled Release 245 (2017) 27–40 concluded that the areas showing 18F–FBB localization corresponds to biotinylated and incubated with streptavidin to immobilize grafting the HIF-1α positive areas [149]. materials. The streptavidin-coupled HUVEC showed significant spread- Streptavidin-based imaging modalities have negligible effect on the ing in comparison to uncoupled HUVEC [157]. In a similar study, ABBS pharmacokinetics of their parent drug, which makes them ideal for the was utilized in the calcium phosphate glass scaffold for bone tissue ad- application in diagnosis. A streptavidin-based nanoparticle has been de- hesion. The scaffold immobilized with avidin showed substantially veloped as multimodal imaging agents for the fluorescence and nuclear higher cell attachment. ABBS has a major advantage over other scaffolds imaging and detection of tumors in a mouse model. Liang et al. designed because it does not inhibit cell proliferation. Scaffold functionalized with a biotin-binding streptavidin-based nanocomplex that was coupled to a ABBS can improve its adhesion with osteoblast-like cells. It was con- biotinylated anti-Her2 antibody. The tumor targeting was achieved cluded that the ABBS scaffold helps in proliferation after efficient attach- using a biotinylated anti-Her2 (Herceptin antibody), and diagnostic im- ment of the osteoblast like cells [12]. aging was performed using a biotinylated DOTA-chelator labeled with In addition to a prominent scaffold for cell adhesion, the biggest pre- 111ln and a biotinylated Cy5.5 fluorophore. Both radiolabeled and requisite for tissue engineering is to imitate the local bio-microenviron- fluorophore-labeled streptavidin nanoparticles complexes have proved ment and bio-ceramic matrix to provide a 3D support that enables the to be very efficient and promising tools for Her2-positive tumor imaging growth of bone tissue [158]. Biologically active molecule, such as pro- [150]. tein and peptide, decorated on the surface of these bioceramics can im- prove bone regeneration [159]. However, there are still few challenges 4.3. Tissue engineering for tissue engineering: (i) a strong interaction between proteins and the surface of scaffold; (ii) no change in the native structure and biolog- Skin grafts are considered the most common method used for the ical activity of the proteins; and (iii) site-directed immobilization. Baeza clinical repair of skin defects [151]. However, contraction of the grafts, et al. utilized the avidin-biotin technology to address these challenges in rejection due to immunity and graft dysfunction are a few shortcomings tissue engineering [160]. Another research group developed biotinylat- of the grafting technique. Tissue engineering is one promising alterna- ed nanofibrous hydrogels for efficient cell adhesion in a 3D matrix of C2 tive to overcome the deficiencies of the graft techniques This alternative based gelators made from 1, 4-benzyldacarboxamide. As shown in strategy involves the fabrication of a scaffold that mimics the natural ex- Fig. 7, avidin modified cells were used to adhere on the biotinylated tracellular matrix (ECM) of the target [152]. Avidin is immobilized on gelator-based 3D matrix. This system has shown to produce a universal biomaterial surfaces, and the cell membranes are conjugated with bio- 3D matrix system where avidin modified cells freely proliferate without tin. The extraordinary affinity of these two molecules mediates the effi- any sign of denaturation. Moreover, the density of the cells in the matrix cient attachment of the cells to the biomaterials. Natural cellular can be manipulated by varying the biotin-gelator amount in the matrix adhesion occurs by the formation of integrin-mediated bonds between [161]. integrin in the cell membrane and adhesion proteins on matrix [153]. 3D matrices used in tissue regeneration are required to be biode- Investigators have demonstrated that that avidin-biotin binding system gradable. Repair and regeneration of a bone defect with the help of a was found to be superior to the integrin-serum protein system with re- biodegradable polymer-based scaffolds are often used along with spect to the cell adhesion strength [154]. Recently, the avidin-biotin bone-inducing factors and/or osteogenic cells. In addition to the bio- binding system (ABBS) was used to fabricate PLCL/Pluronic nanofiber compatibility and appropriate mechanical strength for weight bearing, matrix for skin care application [155]. scaffold for bone regeneration must have interconnected porous struc- Silk biomaterials with attached bioactive molecules are extensively ture for tissue in-growth which can support the regeneration [162– used for drug delivery and tissue engineering. Covalent coupling has a 164]. BMP-2 and BMP-7 (bone morphogenetic protein) are two main very large negative impact on the bioactivity of the biomolecules due osteo-inductive factors which have the potential to induce differentia- to the amine group reactivity. Non-covalent methods of coupling such tion of osteocytes, followed by mineralization and bone regeneration as avidin-biotin are therefore recommended. The versatility and [165,166]. Encapsulation of these growth factors on to polymeric scaf- simplicity of the avidin-biotin system made it possible to immobilize folds using various solvents and electric field for fabrication may result various moieties, such as growth factors and gelators for tissue regener- in the loss of biological activity [167]. To overcome such challenges, ation [156]. streptavidin was employed for binding biotin-BMP2 and biotin-SAP New bone ingrowth at a bone defect site is also the most important (self-assembling peptides). Furthermore, with the help of streptavidin, alternative to simple bone replacement, which prevents the possibility the degree of BMP2 and biotinylated peptide can be controlled. Intra of bone rejection by immune system. Investigators have reported the scaffold retention of BMP2 was also increased by tethering of BMP2 application of avidin and biotin-based systems to attach cells to the sur- which further prolongs its half-life as well [168]. faces of nonporous 2D and 3D biodegradable scaffolds. A group of scientists also focused on the localized 3D differentiation Streptavidin has been used with grafting materials, such as Mylar™ of BM-MSCs (bone marrow-derived mesenchymal stem cells) by or- and Teflon-AF™, for adhesion and proliferation of endothelial cells. In thogonal matrix-immobilization of BMP-2. A recombinant Glutamine- one such study, human umbilical vein endothelial cells (HUVEC) were streptavidin linker peptide, a bio-mimetic scaffold, was genetically

Avidin modified cells

Biotinylated Polymer (C2 based Gelators)

Fig. 7. Avidin-based 3D nanofibrous hydrogels for tissue regeneration. MC3T3 osteoblastic, EAhy926 human endothelial, and SMMC-7721 human hepatoma cells were biotinylated withan EZ Links Sulfo-NHS-LC-LC-Biotin. Using avidin as a bridge, the modified cells were in situ encapsulated in a 3D hydrogel system made of biotinylated and nonbiotinylated 1,4- benzyldicarboxamide (C2) based supramolecular gelator. A. Jain, K. Cheng / Journal of Controlled Release 245 (2017) 27–40 37 engineered. It was covalently coupled to the TG–PEG (Trans-glutamase) [3] M.n. González, C.E. Argaraña, G.D. Fidelio, Extremely high thermal stability of streptavidin and avidin upon biotin binding, Biomol. Eng. 16 (1–4) (1999) 67–72. hydrogels. Controlled presentation of recombinant rhBMP-2 with [4] J.N. Rybak, et al., Purification of biotinylated proteins on streptavidin resin: a pro- streptavidin coupled PEG hydrogels provides the consequent 3D-local- tocol for quantitative elution, Proteomics 4 (8) (2004) 2296–2299. ized osteogenic differentiation of BM-MSCs [169]. [5] D. Ellison, et al., Limited proteolysis of native proteins: the interaction between av- idin and proteinase K. Protein science: a publication of the protein society, 4 (7) (1995) 1337–1345. 5. Conclusion [6] G. Elia, Biotinylation reagents for the study of cell surface proteins, Proteomics 8 (19) (2008) 4012–4024. [7] O. Shimoni, et al., Macromolecule functionalization of disulfide-bonded polymer As one of the strongest non-covalent interactions in the nature, the hydrogel capsules and cancer cell targeting, ACS Nano 6 (2) (2012) 1463–1472. avidin-biotin interaction has evolved to become a very versatile plat- [8] J. Gao, et al., Biofunctionalization of polyelectrolyte microcapsules with biotinylat- ed polyethylene glycol-grafted liposomes, Macromol. Biosci. 11 (8) (2011) form for a great variety of applications in biotechnology and nanotech- 1079–1087. nology. At a constant pace, new analogues of avidin are being developed [9] Q. Hu, et al., RGD-targeted ultrasound contrast agent for longitudinal assessment of with better efficiency and physical-chemical properties to serve specific Hep-2 tumor angiogenesis in vivo, PLoS One 11 (2) (2016), e0149075. purposes. As simplistic this system is in its fundamental principle, avi- [10] J. Ren, et al., Imaging rhodopsin degeneration in vivo in a new model of ocular is- chemia in living mice, FASEB J. 30 (2) (2016) 612–623. din-biotin system is as strong and applicable [23]. Extensive use of this [11] M. Omichi, et al., Fabrication of enzyme-degradable and size-controlled protein system in a wide variety of fields, such as drug delivery, antigen nanowires using single particle nano-fabrication technique, Nat. Commun. 5 targeting, diagnostics, and tissue engineering is the proof of its unparal- (2014) 3718. [12] M.C. Kim, et al., Bone tissue engineering by using calcium phosphate glass scaffolds leled advantages. and the avidin-biotin binding system, Ann. Biomed. Eng. 43 (12) (2015) Noncovalent interactions including hydrogen bonding, hydrophobic 3004–3014. interaction, van der Waals interaction, and ionic interaction have also [13] Z. Yao, et al., Avidin targeting of intraperitoneal tumor xenografts, J. Natl. Cancer Inst. 90 (1) (1998) 25–29. been utilized for nanoscale drug delivery systems. These interactions [14] C. Rosano, P. Arosio, M. Bolognesi, The X-ray three-dimensional structure of avidin, are stronger when present collectively but are very weak individually. Biomol. 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