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Dendrimers and their supramolecular nanostructures for biomedical applications
Accepted Manuscript Dendrimers and their supramolecular nanostructures for biomedical applications Markus Selin, Leena Peltonen, Jouni Hirvonen, Luis M. Bimbo
PII:
S1773-2247(16)30060-0
DOI:
10.1016/j.jddst.2016.02.008
Reference:
JDDST 172
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 31 December 2015 Revised Date:
26 February 2016
Accepted Date: 26 February 2016
Please cite this article as: M. Selin, L. Peltonen, J. Hirvonen, L.M. Bimbo, Dendrimers and their supramolecular nanostructures for biomedical applications, Journal of Drug Delivery Science and Technology (2016), doi: 10.1016/j.jddst.2016.02.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Dendrimers and their supramolecular nanostructures for biomedical applications Markus Selin*, Leena Peltonen, Jouni Hirvonen, Luis M. Bimbo
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Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, PO Box 56 (Viikinkaari 5E), FI-00014 University of Helsinki, Finland *Corresponding author: Markus Selin, [email protected], Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, PO Box 56 (Viikinkaari 5E), FI-00014 University of Helsinki, Finland
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ACCEPTED MANUSCRIPT Abstract
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Pharmaceutical nanotechnology is the field of materials science that focuses on techniques to process raw materials to produce nanostructures with desired properties for pharmaceutical applications. Nanostructures can be roughly divided into surface textures, tubular structures or particles, depending on how many of their dimensions are on the nanoscale. The structures may also be divided into hard and soft structures, depending on their physical properties. There has been lately a growing interest to study soft supramolecular structures. In this review, the main focus is to briefly introduce the reader to various aspects of dendrimers, including Janus dendrimers, and supramolecular structures and then focus on recent studies dealing with application of dendrimer-based supramolecular structures in the pharmaceutical field.
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Graphical Abstract
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Keywords: (Janus) dendrimers, dendrons, hydrogels, nanostructures, polymers, supramolecular structures
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ACCEPTED MANUSCRIPT 1. Introduction
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Polymers can be broadly divided into four classes based on their primary structure: 1) linear, 2) cross-linked with side-chains or side-functional groups, 3) branched, and 4) perfect dendrimers [1]. The primary structure of perfect dendrimers branches periodically and in a precise pattern. The name of an individual dendrimer is derived from the branching pattern and the generation of the dendrimer, i.e. an ordinal number stating the number of times the pattern is repeated. Generally, dendrimers display an open structure at low generations and become more globular and dense as the generations increase. Dendrimers without globular core-shell topology are sometimes termed dendrons to emphasize this detail. Dendrimers have intrinsic and unique features, e.g. high structural fidelity due to controlled iterative synthesis methods, and a high number of functionalities present in the intermediate and surface layers [2–4]. Especially dendrimers with globular core-shell topology can be viewed as nanoscale atom mimics [5] that are capable to aggregate and form nanoscale mimics of molecules via surface interactions [6].
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Structural effects of intermolecular interactions are studied in supramolecular chemistry. Among aspects covered are self-assembly, i.e. the hierarchical process where molecules spontaneously arrange into supramolecular assemblies that further self-organize into supramolecular structures and systems [7], and self-curing, i.e. ability of the supramolecular structures to reform after being damaged [8]. Supramolecular self-assembly processes may be driven by phase separation, a complex phenomenon where components of chemical mixtures or chemically distinct domains of molecules get spatially separated. One of the most ubiquitous self-assembly processes is the hierarchical organization of amphiphilic molecules into structures such as micelles, rods and vesicles [9]. Molecular recognition driven self-assembly processes differ from the phase separation driven ones since in this case a connection is formed when chemically different domains pair through interactions precisely complementing each other. A number of examples demonstrating supramolecular polymers formed via molecular recognition driven self-assembly processes and further phase separation driven self-organization of such polymers into complex structures, e.g. vesicles, may be found in a recent review [10]. As a specific example of a molecular recognition driven process, the hostquest chemistry between adamantane-modified hyperbranched polyglycerol and cationic β-cyclodextrin derivatives has been utilized to design self-assembling dendritic polymers for non-viral gene delivery [11].
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Supramolecular structures can be manifold, one example being supramolecular coatings. The coating molecules form monolayers upon adsorption to a solid support. An interesting coating study was made with amphiphilic peptide coatings incorporating a terminal L-lysine dendron and a terminal fatty acid on an oligopeptide [12]. Further, the dendrons were equipped with one or two cell adherence motifs. The coatings were then attached to the polymeric scaffold whereupon they adapted supramolecular β-sheet structures. In comparison to bare scaffolds and scaffolds coated with a linear peptide coating, the scaffolds coated with the L-lysine dendron bearing coatings showed improved colonization of the scaffold surface by human bladder smooth muscle cells (SMCs) and enhanced penetration of the cells into the scaffold. Thus, the dendron bearing coatings have potential to be used in bladder tissue regeneration applications. The results were speculated to be due to either improved accessibility of the adhesion sequences to the SMCs, or due to non-specific electrostatic attraction between the positively charged amines of the L-lysine residues and the SMCs. Dendrimers and dendrons have been shown to self-assemble into hierarchically organized structures [13,14], mediate chirality into structures [15,16], glue supramolecular structures [17], and facilitate the formation of crystalline complexes with viruses [18]. There are already reviews about dendrimers [19] and dendrimer-based supramolecular structures [20] in pharmaceutical and biomedical fields, as well as a thorough review about biomedical applications of supramolecular polymers [21]. Only very recently have dendrimers comprised of heterogeneous segments (Janus dendrimers) been designed for biomedical applications (Figure 1A-B). A brief overview of the reviewed studies dealing with dendrimers forming 3
ACCEPTED MANUSCRIPT supramolecular structures and dendrimers studied as drug carriers is provided (Table 1). In this review article various aspects of dendrimers and supramolecular structures are introduced the focus being on recent studies dealing with applications of dendrimer-based supramolecular structures in the pharmaceutical field. (Figure 1) (Table 1)
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2. Supramolecular hydrogels
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Hydrogels are viscous mixtures composed of a polymeric network and an aqueous phase that, by definition [22], expands the network throughout the volume of the gel. Most of the mass of a hydrogel comes from its aqueous phase. Covalent and physical hydrogel networks are formed from gelling agents connected through covalent bonds and physical interactions, respectively. Covalent hydrogels are usually prepared using a chemical reaction connecting the gelling agents through specially designed crosslinking molecules. Entanglement of polymer chains of cellulose derivatives, such as hydroxypropyl methylcellulose (HPMC) and carboxymethyl cellulose (CMC), suspended in an aqueous phase are typical examples of how physical hydrogel networks are formed. The supramolecular hydrogel networks demonstrate various levels structural hierarchy [23–26]. The amphiphilic molecules work as gelling agents comprising self-assembling supramolecular nanoscale tubules and fibrils that form hydrogel networks through physical and supramolecular crosslinks.
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If the dimensions of the expanded hydrogel network are within nanoscale, then the whole of the hydrogel should be viewed as a nanoparticle rather than a bulk gel. When the term nanogel is used to refer to such hydrogel particles, care should be taken to check that such particle fulfils the criteria of both being a hydrogel, i.e. having a network of crosslinked fibrils expanded by the aqueous phase [22], and being a nanoparticle, i.e. having dimensions in nanoscale [27]. Some hydrogels, micelles and vesicles may comply with the criteria, however, typical micelles do not since they lack a relevant number of water molecules inside them [28–30]. Vesicles have a membrane that is expanded by solvent in the inside cavity. The membrane of typical vesicles should not be viewed as a network of crosslinked fibrils since it is, in fact, a single continuous supramolecular structure. In this review, the studies dealing with supramolecular entities with all the dimensions on the nanoscale will be discussed under the subtitle ‘3. Supramolecular nanoparticles’.
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Hydrogels based on dendritic polymers can be classified into three classes: type I hydrogels, where dendrimers act as a cross-linkers, type II hydrogels, where hydrogels are formed via polymerization of dendrimer-based monomers, and type III hydrogels, where self-assembling dendritic compounds form hydrogel [31]. Type III supramolecular dendrimer hydrogels may be further divided based on their chemical and network structures (Figure 2). Early reports of amphiphilic dendrimer gels were presented by Newkome et al. [32], with later examples including multi-component [33] and chiral [34,35] gels tailored for high-end [36] and biomedical delivery applications [37]. Because self-assembling process is highly dependent on the environmental factors, the stability of those hydrogels may be affected by different environmental changes in vivo. (Figure 2) Dendrimer-based gels have been applied and studied for applications in drug release [24,38,39], as well as diagnostics, regenerative medicine and tissue engineering [40–45]. In dendritic gels, drug can be entrapped physically inside of the polymeric network structures. The rate of drug release from gels is usually controlled either by rate of drug diffusion or by hydrogel degradation [24,39]. The degradation of the 4
ACCEPTED MANUSCRIPT hydrogels becomes important especially if the drug molecules are covalently incorporated into or attached onto the hydrogel network. Some hydrogels respond to environmental factors, such as pH, metal ions and temperature [38,39,46,47]. Hydrogels may be equipped with adherence motifs, e.g. to promote either cell migration and proliferation [45] or competitive binding between dendrimer bound ligands and the assayed biomarkers [42]. 2.1 Polyethylene glycol (PEG) end-modified with dendrons
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Hydrogels were formed from PEG terminated with citric acid dendrons of various generations [48]. To prepare the G1 dendrimer, PEG bis(carboxymethyl) ether (Mn 600 g/mol) was esterified with citric acid using thionyl chloride in pyridine. The esterification reactions to produce the following dendritic generations succeeded using N,N' dicyclohexylcarbodiimide in pyridine. The gel formers were dissolved by heating, and the gels formed during subsequent cooling. 5-aminosalicylic acid, diclofenac, mefenamic acid, and pyridine were complexed with dendrimer generations 1-3 [38]. The molar ratio of complexed drug to dendrimer was increasing with the generations. When the complexes were hydrated they formed hydrogels that were studied in drug release experiments. Typically these drugs were released from the hydrogels at pH 7.4 and 1 within 6 to 8 hours. Hydrogel from G1 dendrimer was able to retain 70 % of the encapsulated 5-aminosalicylic acid, diclofenac and mefenamic acid at pH 7.4. 2.2 PAMAM dendrimers coated with polypeptides
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PAMAM dendrimers contain a lot of amino groups, which induces pH-sensitivity for these structures (Figure 1C). Experimentally the highest swelling with these hydrogels has been found to be around pH 4 [49]. The G4 PAMAM dendrimers coated with collagen model peptide (Pro-Pro-Gly)5 have gelatine mimicking characteristics [50,51]. Dendrimers were loaded with rose bengal (RB) in aqueous solution and subsequently excess of RB was removed from freeze dried complexes through precipitation in chloroform [52]. The release of RB was monitored at various temperatures and was slowest at 4 °C [50,51]. Dendrimer solution (8.4 mM) also formed a hydrogel when cooled to 4 °C. Subsequently, G4 PAMAM dendrimers were coated with (Pro-Pro-Gly)10 [53] and even later with (Pro-Hyp-Gly)10 [47]. The fore reported coating provided dendrimers that formed a gelatine mimicking hydrogel below 45 °C whereas the coating reported later provided dendrimers that formed a collagen mimicking hydrogel above 40 °C. The (Pro-Hyp-Gly)10 peptides on the G4 PAMAM dendrimer adapted a triple helical structure that was more thermostable than the helical structure of native collagen. The collagen mimicking hydrogel itself was stable for 1 hour at 80 °C. As an alternative way to make self-assembling supramolecular hydrogels from peptide coated PAMAM dendrimers, methodology for easy polymerization reaction to surface modify G2 and G4 amine terminated dendrimers with poly(γ-(2-(2-methoxyethoxy)ethyl) L-glutamate) has been published recently [54]. The dendrimer used as the core can be prepared easily without protection/activation steps using alternating aza-Michael addition and thiol-yne reactions [55]. 2.3 Asymmetric L-glutamic acid dendrons A L-glutamic acid dendron bound to a long chain fatty acid was characterized as a gel former [25]. When the gel former was dissolved in aqueous solution below pH 10, very thin helical nanoribbons formed and adapted further hierarchical morphologies, i.e. helical tubules 10 nm in diameter with 4 nm thick bilayers and superhelices of the tubules (Figure 2B). The asymmetric nature of the L-glutamic acid dendron might convey these structures their helical morphology. Fractal, needle-like fibrils were seen in solutions of the gel former at pH above 10. Supramolecular, metal-ion-shrinkable hydrogel was prepared from these gel formers [46]. The shrinking ratios and rates were studied over time in connection with various metal ions. Thiamine was entrapped in the hydrogel and the release was followed after addition of selected metal ions. The release of entrapped thiamine was controlled by the hydrogel shrinkage. A positively charged drug, pralidoxime, and phenol red were simultaneously entrapped in the hydrogel [39]. Their release was studied 5
ACCEPTED MANUSCRIPT in controlled pH conditions in the presence and absence of Mg2+ ions (Figure 3). In the absence of the ions in pH 3.2, the rates of release were almost the similar. In pH 3.2, the ions caused shrinkage of the hydrogel, increased the rate of phenol red release and halted the release of the drug below 30 % of the encapsulated amount. When the pH was subsequently adjusted to 6.2, the hydrogel degraded and the remaining drug was released. (Figure 3)
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2.4 Janus dendrimers Amphiphilic Janus dendrimers may be produced by combining polar and non-polar segments, which will be reflected in descriptors like molecular dipole moment and hydrophilic-lipophilic balance. Classifying individual cases of dendrimers as symmetric or Janus dendrimers is not always straightforward, e.g. the way of defining segments and cores influences the classification. In this review, we have considered simple fatty acid esters of dendrons as symmetric dendrons rather than as Janus dendrimers.
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In favourable conditions, the intermolecular interactions cause amphiphilic Janus dendrimers in solution to aggregate and become directionally aligned. In effect, during this process the amphiphilic molecules form layers consisting of two separate phases. The intermolecular interactions and molecular properties may promote curvature in the layers and thereby drive formation of tubular structures or particles instead of planar surfaces. In a bilayer, two layers have aligned and merged their chemically similar phases. Cell membranes are a classical example of bilayers where the non-polar phases of two phospholipid layers constitute a merged interior phase, and the polar phases of the layers form two separate exterior phases. Properties of these layered surfaces, e.g. zeta-potential and surface energy, reflect the material behaviour.
(Figure 4)
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When two dodecanoxy substituted (azidomethyl)benzene derivatives were combined through a copper catalysed cycloaddition reaction with G3 dendritic 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) molecules built onto 2-propynol, amphiphilic dendrimers able to gelatinise aqueous solutions at low mass proportions were formed [24]. Self-assembling fibrils 4-6 nm in diameter formed supramolecular bundles with hexagonal cross-sections 15 nm in diameter, fibres less than 100 nm in diameter, and their crosslinked networks. Drug release from 5-(azidomethyl)benzene-1,2,3-triol-based dendrimer hydrogels was further studied (Figure 4). More than 80 % of encapsulated small molecule drug, nadolol, and more than 50 % of the encapsulated peptide drug, gonadorelin, was released over period of 3 hours. Also, horseradish peroxidase was entrapped into the hydrogel and its activity was confirmed after release.
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3. Supramolecular nanoparticles
In addition to hydrogels, dendrimers can also form supramolecular particles with nanoscale dimensions. The self-assembly principles also influence the formation of supramolecular particles. In the following chapters we have divided studies dealing with supramolecular particles to demonstrate 1) the phase separation as a driving force of self-assembly, and 2) the ways molecular recognition can be utilized in pharmaceutical applications of supramolecular structures. A molecule should contain chemically different domains to demonstrate supramolecular self-assembly via phase separation. In this case, similar domains are matched to construct the phases. Molecular recognition relies on interactions from chemically distinct functional groups precisely complementing each other. In the following examples, multiple pairs of interacting groups are utilized as the basis for supramolecular co-assembly and for coupling drug release from supramolecular structures to specific receptor binding events. 3.1 Phase separated supramolecular particles
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Supramolecular particles formed via phase separation processes can be spherical vesicles or micelles. For example, spherical particles 170-190 nm in diameter have been formed from PEG end-modified with G2 dendritic carbosilane molecules and micelles 32-92 nm in diameter were formed from PEG end-modified with dendritic benzyl ether molecules [56,57]. In aqueous environment, dendrimers formed by coupling PAMAM type dendron to a hydrophobic part bearing two alkyl chains self-assembled into vesicular structures termed dendrimersomes that are approx. 200 nm in diameter [58]. The dendrimersomes collapsed to clusters of small micelles 6-8 nm in diameter upon addition of siRNA. The micelles were used as vectors for siRNA delivery in vitro and in vivo. Tumour growth was slowed in PC-3 xenograft mice by the delivered siRNA (Figure 5). (Figure 5)
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Empty micelles and micelles encapsulating anticancer drug, doxorubicin, could also be formed from PAMAM type dendrimers via film dispersion method [59]. The doxorubicin encapsulating micelles were slightly larger than the empty micelles (10 nm in diameter). Less than 20 % and 35 % of the doxorubicin was released at 37 °C from the micelles in 24 hours at pH 7.4 and 5.0, respectively. The micelles enhanced the penetration of doxorubicin into tumour spheroids prepared by 3D culturing doxorubicin resistant breast cancer MCF-7R cells. When the effect was further studied, it was found out that the micelles were able to both deliver doxorubicin via macropinocytosis-dependent endocytosis thereby overcoming the doxorubicin resistance mediated by efflux pumps overexpressed by the cell line, and inhibit the efflux of internalized doxorubicin. The micelles loaded with a dye and injected intravascularly focused at tumour site in a mice model. Furthermore, doxorubicin loaded micelles enhanced antitumour effect of the doxorubicin and reduced its toxic off-target effects in vivo (Figure 6). It remains to be seen, if the therapeutic approaches based on siRNA and doxorubicin can be combined. (Figure 6)
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Janus dendrimers have also been found to self-assemble into supramolecular particles. Two series of G1-3 dendrons were prepared by building dendritic bis-MPA molecules onto either a single 2-propynol or a 6azidohexan-1-ol [60]. The alkyne containing dendrons were then esterified with stearic acid to get lipophilic dendrons. Next, the alkyne and azide bearing dendrons were modularly coupled via Cu(I)-catalysed 1,3dipolar cycloaddition to produce amphiphilic Janus dendrimers denoted as G1/G1-3, G2/G2-3 and G3/G3 using generations of their lipophilic and hydrophilic dendrons, respectively. When a G1/G2-3 and G2/G3 dendrimers aggregated in water, elongated micelles were formed. The G1/G1 dendrimer did not aggregate in water at concentrations below its solubility limit. The G2/G2 dendrimers formed well defined spherical structures 50 nm in diameter. The G3/G3 dendrimers produced vesicles where the thickness of the formed bilayers was 9-12 nm. The rest of the dendrimers produced elongated micelles. Lipophilic anticancer drug, plitidepsin, was complexed with G2/G2, G3/G3, and G2/G3 dendrimers. The process was less successful with G1/G3 and G1/G2 dendrimers, which was attributed to their less lipophilic nature. 3.2 Supramolecular particles demonstrating molecular recognition Molecular recognition helps silsesquioxane cored G2 poly(L-lysine) dendrimers to co-assemble with glutamic acid terminated poly(l-leucine) in aqueous environment into supramolecular nanoparticles 250300 nm in diameter [61]. Doxorubicin could be loaded into these nanoparticles and in 24 hours, approx. 80 % of the loaded doxorubicin was retained and released at pH 7.4 and pH 6.2, respectively [62]. The electrostatic interactions necessary for co-assembly dissipate at acidic conditions (Figure 7A). Human hepatocellular carcinoma cell line, HepG2, cells were incubated with doxorubicin, doxorubicin hydrochloride and nanoparticles loaded with doxorubicin. Confocal laser scanning microscopy (CSLM) of fluorescein isothiocyanate (FITC) labelled hydrophilic parts of the nanoparticles showed that initially spot7
ACCEPTED MANUSCRIPT like FITC signals spread after the 3 first hours of incubation (Figure 7B). Thereafter the FITC signal stayed associated with the cell membranes. Doxorubicin reached the nucleus within the first 3 hours, presumably after pH-dependent disassembly in response to a biological stimulus (figure 7C). The drug loaded nanoparticles enhanced internalization of doxorubicin by HepG2 cells compared to the free base form. However, comparable cytotoxic effects were seen in HepG2 samples incubated with doxorubicin loaded nanoparticles and doxorubicin hydrochloride.
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(Figure 7)
(Figure 8)
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Symmetric aromatic repeating and termination units were made amphiphilic using substituents [63]. These amphiphilic building blocks were then used to synthesize dendrimers that were able to form both micelles and inverted micelles incorporating model compounds as cargo. The substituents were later fixed as pentaethylene glycol monomethyl ether and 1-decanol to promote charge neutrality and temperature sensitivity in the micellar constructs [64]. To produce dendrimers that self-assemble to form micelles with molecular recognition properties, hydrophilic [65] and hydrophobic [66] ligands were attached onto these dendrimers by replacing one substituent per dendrimer. Attaching a hydrophilic biotin molecule onto G1-2 of these dendrimers yielded micelles that could be loaded with pyrene as model cargo [65]. The micelles dissembled and released the loaded pyrene when subjected to extravidin. Other proteins used did not result in such disassembly. Disassembly of the micelle bearing a hydrophobic ligand was studied using the interaction between dinitrophenyl (DNP) and rat anti-DNP immunoglobulin G antibody. Nile red loaded into the micelle was released and the measured size of the micelles diminished in the presence of the antibody [66]. Addition of proteins that do not work as receptors for dinitrophenyl did not release the loaded Nile red nor change the size of the micelles. This confirmed the hydrophobic ligands could also be used for archiving targeted disassembly. The mechanisms of ligand binding associated micellar disassembly processes are summarized in a figure (Figure 8).
4. Dendrimer based molecular drug carriers
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As stated above, supramolecular dendrimers have been shown to be promising delivery vehicles in biomedical applications. As molecular carriers, their branched and layered architectures display a high number of controlled terminal groups as well as cavities for physical entrapment. In the following chapters, novel approaches to utilize the potential of dendrimers as molecular drug carriers.
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4.1 PAMAM-type molecular drug carriers Covalent binding of doxorubicin to G4 PAMAM dendrimer first surface modified with glutamic acid and then coated with enzyme hydrolysed collagen peptide (2 kDa, Figure 9A) suppressed diffusion of doxorubicin from a collagen gel (Figure 9B) when compared to diffusion of free doxorubicin from the gel [67]. Doxorubicin attached to poly(glutamic acid) had negligible diffusion rate in a collagen gel [68]. The result was interpreted as entanglement of the polypeptide chain to the hydrogel network since almost similar molecular weight prodrug diffused in the gel. In the prodrug, doxorubicin was covalently attached to a globular dendrimer coated with acetylated glutamic acid. Replacing the acetyl groups with PEGs (2 kDa) did not change the diffusion rate. However, replacement of the acetyl groups with collagen peptides (either 2 kDa or 5kDa) slowed the diffusion. Matrix metalloproteinase (MMP) inhibitor reduced the toxicity of dendrimer conjugated doxorubicin embedded in a collagen gel against highly invasive MDA-MB-231 cells [67]. Thus, the gel offers an option to selectively target MMP secreting cells with the embedded prodrugs. The embedded collagen gel was able to inhibit metastasis of reporter gene bearing mouse breast cancer cell line cells implanted in mammary pad of BALC/c mice (Figure 9C). 8
ACCEPTED MANUSCRIPT (Figure 9) 4.2 Janus dendrimers
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Pan et al. designed and synthesized a range of amphiphilic Janus dendrimers to accomplish bone-targeted dendritic drug delivery [69]. These dendrimers carried acidic amino acids as targeting moieties simultaneously with multiple naproxen molecules. As a result, the dendrimers exhibited more than 80% binding rates to hydroxyapatite (HAP) in vitro and enhanced solubility of naproxen by the dendritic drug delivery system, while exhibiting no significant cytotoxicity against HEK293 cells. Another recent proof-ofconcept study utilized the Janus dendrimers for combination therapy. Acton et al. synthesized a series of novel Janus dendrimers with potential applications in combination therapy [70]. They reported the creation of different first and second generations of tertiary amine and PEG-based dendrons. Model compounds benzyl alcohol (BA) and 3-phenylpropionic acid (PPA) were covalently attached to the dendrons via carbonate and ester linkers, respectively. The four dendrons were coupled together via (3 + 2) cycloaddition chemistries to afford four Janus dendrimers, which contained varying amounts and different ratios of the BA and PPA to promote differential drug release in plasma (Figure 10). The Janus dendrimers provided sequential release of the two model compounds, with BA being released faster than PPA from all the dendrons. Haemolysis studies confirmed that these dendrimers are non-cytotoxic towards HUVEC cells and non-toxic toward red blood cells. (Figure 10) 5. Conclusions
6. Acknowledgements
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The recent studies showing promising results highlight the various aspects of dendrimer-based selfassembling structures and Janus dendrimers beneficial in developing biomedical applications. The fields with potential applications for these remarkable entities include regenerative medicine, targeted and controlled drug delivery, as well as combination therapy. Albeit the first in vivo results have been claimed from supramolecular dendrimer-based structures, still many details have to be clarified to realize the results of the research in clinical routines. Janus dendrimers can also be designed to work as intelligent drug carriers or pro-drugs, utilizing the multiple functionalities to graft drugs with desired properties.
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Financial support by the Academy of Finland (decisions No. 276377 for L.B.), Biocentrum Helsinki, The Finnish Cultural Foundation, Orion Research Foundation and the Jane and Aatos Erkko Foundation is gratefully acknowledged. REFERENCES [1]
D.A. Tomalia, Dendrons/dendrimers: quantized, nano-element like building blocks for soft-soft and soft-hard nano-compound synthesis, Soft Matter. 6 (2010) 456–474. doi:10.1039/b917370f.
[2]
S.M. Grayson, J.M.J. Fréchet, Convergent Dendrons and Dendrimers: from Synthesis to Applications, Chem. Rev. 101 (2001) 3819–3867. doi:10.1021/cr990116h.
[3]
A.W. Bosman, H.M. Janssen, E.W. Meijer, About Dendrimers : Structure, Physical Properties, and Applications, Chem. Rev. 99 (1999) 1665–1688. doi:10.1021/cr970069y.
[4]
S. Nummelin, M. Skrifvars, K. Rissanen, Polyester and Ester Functionalized Dendrimers, Top. Curr. Chem. 210 (2000) 1–67. doi:10.1007/3-540-46577-4_1.
[5]
D.A. Tomalia, Dendritic effects: dependency of dendritic nano-periodic property patterns on critical nanoscale design parameters (CNDPs), New J. Chem. 36 (2012) 264–281. doi:10.1039/c1nj20501c.
9
ACCEPTED MANUSCRIPT D.A. Tomalia, H.M. Brothers II, L.T. Piehler, H.D. Durst, D.R. Swanson, Partial shell-filled core-shell tecto(dendrimers): A strategy to surface differentiated nano-clefts and cusps, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 5081–5087. doi:10.1073/pnas.062684999.
[7]
G.M. Whitesides, B. Grzybowski, Self-Assembly at All Scales, Science. 295 (2002) 2418–2421. doi:10.1126/science.1070821.
[8]
M. Zhang, D. Xu, X. Yan, J. Chen, S. Dong, B. Zheng, et al., Self-healing Supramolecular Gels Formed by Crown Ether Based Host-Guest Interactions, Angew. Chemie - Int. Ed. 51 (2012) 7011–7015. doi:10.1002/anie.201203063.
[9]
P. V Messina, J.M. Besada-Porto, J.M. Ruso, Self-Assembly Drugs: From Micelles to Nanomedicine, Curr. Top. Med. Chem. 14 (2014) 555–571. doi:10.2174/1568026614666140121112118.
[10]
R. Dong, Y. Zhou, X. Zhu, Supramolecular dendritic polymers: From synthesis to applications, Acc. Chem. Res. 47 (2014) 2006–2016. doi:10.1021/ar500057e.
[11]
R. Dong, L. Zhou, J. Wu, C. Tu, Y. Su, B. Zhu, et al., A supramolecular approach to the preparation of charge-tunable dendritic polycations for efficient gene delivery., Chem. Commun. (Camb). 47 (2011) 5473–5475. doi:10.1039/c1cc10934k.
[12]
D.A. Harrington, E.Y. Cheng, M.O. Guler, L.K. Lee, J.L. Donovan, R.C. Claussen, et al., Branched peptide-amphiphiles as self-assembling coatings for tissue engineering scaffolds, J. Biomed. Mater. Res. A. 78A (2006) 157–167. doi:10.1002/jbm.a.30718.
[13]
B.M. Rosen, C.J. Wilson, D.A. Wilson, M. Peterca, M.R. Imam, V. Percec, Dendron-Mediated SelfAssembly, Disassembly, and Self-Organization of Complex Systems, Chem. Rev. 109 (2009) 6275– 6540. doi:10.1021/cr900157q.
[14]
H.-J. Sun, S. Zhang, V. Percec, From structure to function via complex supramolecular dendrimer systems, Chem. Soc. Rev. 44 (2015) 3900–3923. doi:10.1039/c4cs00249k.
[15]
V. Percec, A.E. Dulcey, V.S.K. Balagurusamy, Y. Miura, J. Smidrkal, M. Peterca, et al., Self-assembly of amphiphilic dendritic dipeptides into helical pores., Nature. 430 (2004) 764–768. doi:10.1038/nature02770.
[16]
V. Percec, A.E. Dulcey, M. Peterca, M. Ilies, J. Ladislaw, B.M. Rosen, et al., The Internal Structure of Helical Pores Self-Assembled from Dendritic Dipeptides is Stereochemically Programmed and Allosterically Regulated, Angew. Chemie Int. Ed. 44 (2005) 6516–6521. doi:10.1002/anie.200501331.
[17]
A. Pantos, D. Tsiourvas, G. Nounesis, C.M. Paleos, Interaction of Functional Dendrimers with Multilamellar Liposomes: Design of a Model System for Studying Drug Delivery, Langmuir. 21 (2005) 7483–7490. doi:10.1021/la0510331.
[18]
J. Mikkilä, H. Rosilo, S. Nummelin, J. Seitsonen, J. Ruokolainen, M.A. Kostiainen, Janus-DendrimerMediated Formation of Crystalline Virus Assemblies, ACS Macro Lett. 2 (2013) 720–724. doi:10.1021/mz400307h.
[19]
Y. Kim, S.C. Zimmerman, Applications of dendrimers in bio-organic chemistry, Curr. Opin. Chem. Biol. 2 (1998) 733–742. doi:10.1016/S1367-5931(98)80111-7.
[20]
K.T. Al-Jamal, C. Ramaswamy, A.T. Florence, Supramolecular structures from dendrons and dendrimers, Adv. Drug Deliv. Rev. 57 (2005) 2238–2270. doi:10.1016/j.addr.2005.09.015.
[21]
R. Dong, Y. Zhou, X. Huang, X. Zhu, Y. Lu, J. Shen, Functional supramolecular polymers for biomedical applications, Adv. Mater. 27 (2015) 498–526. doi:10.1002/adma.201402975.
[22]
J. V. Alemán, A. V. Chadwick, J. He, M. Hess, K. Horie, R.G. Jones, et al., Definitions of terms relating to the structure and processing of sols, gels, networks, and inorganic-organic hybrid materials
AC C
EP
TE D
M AN U
SC
RI PT
[6]
10
ACCEPTED MANUSCRIPT (IUPAC Recommendations 2007), Pure Appl. Chem. 79 (2007) 1801–1829. doi:10.1351/pac200779101801. P.Y.W. Dankers, T.M. Hermans, T.W. Baughman, Y. Kamikawa, R.E. Kieltyka, M.M.C. Bastings, et al., Hierarchical Formation of Supramolecular Transient Networks in Water: A Modular Injectable Delivery System, Adv. Mater. 24 (2012) 2703–2709. doi:10.1002/adma.201104072.
[24]
S. Nummelin, V. Liljeström, E. Saarikoski, J. Ropponen, A. Nykänen, V. Linko, et al., Self-Assembly of Amphiphilic Janus Dendrimers into Mechanically Robust Supramolecular Hydrogels for Sustained Drug Release, Chem. - A Eur. J. 21 (2015) 14433–14439. doi:10.1002/chem.201501812.
[25]
P. Duan, L. Qin, X. Zhu, M. Liu, Hierarchical Self-Assembly of Amphiphilic Peptide Dendrons: Evolution of Diverse Chiral Nanostructures Through Hydrogel Formation Over a Wide pH Range, Chem. - A Eur. J. 17 (2011) 6389–6395. doi:10.1002/chem.201003049.
[26]
R. Dong, Y. Pang, Y. Su, X. Zhu, Supramolecular hydrogels: synthesis, properties and their biomedical applications, Biomater. Sci. 3 (2015) 937–954. doi:10.1039/C4BM00448E.
[27]
M. Vert, Y. Doi, K.-H. Hellwich, M. Hess, P. Hodge, P. Kubisa, et al., Terminology for biorelated polymers and applications (IUPAC Recommendations 2012), Pure Appl. Chem. 84 (2012) 337–410. doi:10.1351/PAC-REC-10-12-04.
[28]
F.M. Menger, J.M. Jerkunica, J.C. Johnston, The Water Content of a Micelle Interior. The Fjord vs. Reef Models, J. Am. Chem. Soc. 100 (1978) 4676–4678. doi:10.1021/ja00483a008.
[29]
A.D. MacKerell Jr., Molecular Dynamics Simulation Analysis of a Sodium Dodecyl Sulfate Micelle in Aqueous Solution : Decreased Fluidity of the Micelle Hydrocarbon Interior, J. Phys. Chem. 99 (1995) 1846–1855. doi:10.1021/j100007a011.
[30]
L. Nordstierna, P. V. Yushmanov, I. Furó, Solute-Solvent Contact by Intermolecular Cross-Relaxation. 2. The Water-Micelle Interface and the Micellar Interior, J. Phys. Chem. B. 110 (2006) 25775–25781. doi:10.1021/jp0647885.
[31]
D.K. Smith, Dendritic Gels - Many Arms Make Light Work, Adv. Mater. 18 (2006) 2773–2778. doi:10.1002/adma.200600474.
[32]
G.R. Newkome, G.R. Baker, S. Arai, M.J. Saunders, P.S. Russo, K.J. Theriot, et al., Cascade molecules. Part 6. Synthesis and Characterization of Two-Directional Cascade Molecules and Formation of Aqueous Gels, J. Am. Chem. Soc. 112 (1990) 8458–8465. doi:10.1021/ja00179a034.
[33]
A.R. Hirst, D.K. Smith, M.C. Feiters, H.P.M. Geurts, Two-Component Dendritic Gel: Effect of Stereochemistry on the Supramolecular Chiral Assembly, Chem. - A Eur. J. 10 (2004) 5901–5910. doi:10.1002/chem.200400502.
[34]
J.J.D. de Jong, N. Lucas, Linda, R.M. Kellogg, J.H. van Esch, B.L. Feringa, Reversible Optical Transcription of Supramolecular Chirality into Molecular Chirality, Science. 304 (2004) 278–281. doi:10.1126/science.1095353.
[35]
D.K. Smith, Lost in translation? Chirality effects in the self-assembly of nanostructured gel-phase materials, Chem. Soc. Rev. 38 (2009) 684–694. doi:10.1039/b800409a.
[36]
A.R. Hirst, B. Escuder, J.F. Miravet, D.K. Smith, High-Tech Applications of Self-Assembling Supramolecular Nanostructured Gel-Phase Materials: From Regenerative Medicine to Electronic Devices, Angew. Chemie Int. Ed. 47 (2008) 8002–8018. doi:10.1002/anie.200800022.
[37]
J. Boekhoven, M. Koot, T.A. Wezendonk, R. Eelkema, J.H. van Esch, A Self-Assembled Delivery Platform with Post-production Tunable Release Rate, J. Am. Chem. Soc. 134 (2012) 12908–12911. doi:10.1021/ja3051876.
AC C
EP
TE D
M AN U
SC
RI PT
[23]
11
ACCEPTED MANUSCRIPT H. Namazi, M. Adeli, Dendrimers of citric acid and poly (ethylene glycol) as the new drug-delivery agents, Biomaterials. 26 (2005) 1175–1183. doi:10.1016/j.biomaterials.2004.04.014.
[39]
L. Qin, F. Xie, P. Duan, M. Liu, A Peptide Dendron-Based Shrinkable Metallo-Hydrogel for Charged Species Separation and Stepwise Release of Drugs., Chem. - A Eur. J. 20 (2014) 15419–15425. doi:10.1002/chem.201404035.
[40]
I.L.M. Isa, A. Srivastava, D. Tiernan, P. Owens, P. Rooney, P. Dockery, et al., Hyaluronic Acid Based Hydrogels Attenuate Inflammatory Receptors and Neurotrophins in Interleukin-1β Induced Inflammation Model of Nucleus Pulposus Cells, Biomacromolecules. 16 (2015) 1714–1725. doi:10.1021/acs.biomac.5b00168.
[41]
L. Degoricija, P.N. Bansal, S.H.M. Söntjens, N.S. Joshi, M. Takahashi, B. Snyder, et al., Hydrogels for Osteochondral Repair Based on Photocrosslinkable Carbamate Dendrimers, Biomacromolecules. 9 (2008) 2863–2872. doi:10.1021/bm800658x.
[42]
B.L. Ibey, H.T. Beier, R.M. Rounds, G.L. Coté, V.K. Yadavalli, M. V. Pishko, Competitive Binding Assay for Glucose Based on Glycodendrimer-Fluorophore Conjugates, Anal. Chem. 77 (2005) 7039–7046. doi:10.1021/ac0507901.
[43]
A. Weeks, D. Luensmann, A. Boone, L. Jones, H. Sheardown, Hyaluronic acid as an internal wetting agent in model DMAA/TRIS contact lenses, J. Biomater. Appl. 27 (2012) 423–432. doi:10.1177/0885328211410999.
[44]
S.H.M. Söntjens, D.L. Nettles, M.A. Carnahan, L.A. Setton, M.W. Grinstaff, Biodendrimer-Based Hydrogel Scaffolds for Cartilage Tissue Repair, Biomacromolecules. 7 (2006) 310–316. doi:10.1021/bm050663e.
[45]
Y. Wang, Q. Zhao, H. Zhang, S. Yang, X. Jia, A Novel Poly(amido amine)-Dendrimer-Based Hydrogel as a Mimic for the Extracellular Matrix, Adv. Mater. 26 (2014) 4163–4167. doi:10.1002/adma.201400323.
[46]
L. Qin, P. Duan, F. Xie, L. Zhang, M. Liu, A metal ion triggered shrinkable supramolecular hydrogel and controlled release by an amphiphilic peptide dendron, Chem. Commun. 49 (2013) 10823– 10825. doi:10.1039/c3cc47004k.
[47]
C. Kojima, T. Suehiro, T. Tada, Y. Sakamoto, T. Waku, N. Tanaka, Preparation of heat-induced artificial collagen gels based on collagen-mimetic dendrimers, Soft Matter. 7 (2011) 8991–8997. doi:10.1039/c1sm06157g.
[48]
H. Namazi, M. Adeli, Novel linear-globular thermoreversible hydrogel ABA type copolymers from dendritic citric acid as the A blocks and poly(ethyleneglycol) as the B block, Eur. Polym. J. 39 (2003) 1491–1500. doi:10.1016/S0014-3057(02)00385-3.
[49]
B. Unal, R.C. Hedden, pH-dependent swelling of hydrogels containing highly branched polyamine macromonomers, Polymer (Guildf). 50 (2009) 905–912. doi:10.1016/j.polymer.2008.11.049.
[50]
C. Kojima, S. Tsumura, A. Harada, K. Kono, A Collagen-Mimic Dendrimer Capable of Controlled Release, J. Am. Chem. Soc. 131 (2009) 6052–6053. doi:10.1021/ja809639c.
[51]
C. Kojima, S. Tsumura, A. Harada, K. Kono, A collagen-mimic dendrimer capable of controlled release, J. Am. Chem. Soc. 131 (2009) 10334–10334. doi:10.1021/ja904242j.
[52]
C. Kojima, Y. Toi, A. Harada, K. Kono, Preparation of Poly(ethylene glycol)-Attached Dendrimers Encapsulating Photosensitizers for Application to Photodynamic Therapy, Bioconjug. Chem. 18 (2007) 663–670. doi:10.1021/bc060244u.
[53]
T. Suehiro, T. Tada, T. Waku, N. Tanaka, C. Hongo, S. Yamamoto, et al., Temperature-Dependent Higher Order Structures of the (Pro-Pro-Gly)₁₀-Modified Dendrimer, Biopolymers. 95 (2011) 270–
AC C
EP
TE D
M AN U
SC
RI PT
[38]
12
ACCEPTED MANUSCRIPT 277. doi:10.1002/bip.21576. Y. Shen, S. Zhang, Y. Wan, W. Fu, Z. Li, Hydrogels assembled from star-shaped polypeptides with a dendrimer as the core, Soft Matter. 11 (2015) 2945–2951. doi:10.1039/c5sm00083a.
[55]
Y. Shen, Y. Ma, Z. Li, Facile Synthesis of Dendrimers Combining aza-Michael Addition with Thiol-yne Click Chemistry, J. Polym. Sci. Part A Polym. Chem. 51 (2013) 708–715. doi:10.1002/pola.26429.
[56]
Y. Chang, C. Kim, Synthesis and Photophysical Characterization of Amphiphilic Dendritic–Linear– Dendritic Block Copolymers, J. Polym. Sci. Part A Polym. Chem. 39 (2001) 918–926. doi:10.1002/1099-0518(20010315)39:6<918::AID-POLA1066>3.0.CO;2-P.
[57]
I. Gitsov, K.R. Lambrych, V.A. Remnant, R. Pracitto, Micelles with Highly Branched Nanoporous Interior: Solution Properties and Binding Capabilities of Amphiphilic Copolymers with Linear Dendritic Architecture, J. Polym. Sci. Part A Polym. Chem. 38 (2000) 2711–2727. doi:10.1002/10990518(20000801)38:15<2711::AID-POLA110>3.0.CO;2-1.
[58]
X. Liu, J. Zhou, T. Yu, C. Chen, Q. Cheng, K. Sengupta, et al., Adaptive Amphiphilic Dendrimer-Based Nanoassemblies as Robust and Versatile siRNA Delivery Systems, Angew. Chemie - Int. Ed. 53 (2014) 11822–11827. doi:10.1002/anie.201406764.
[59]
T. Wei, C. Chen, J. Liu, C. Liu, P. Posocco, X. Liu, et al., Anticancer drug nanomicelles formed by selfassembling amphiphilic dendrimer to combat cancer drug resistance, Proc. Natl. Acad. Sci. U. S. A. 112 (2015) 2978–2983. doi:10.1073/pnas.1418494112.
[60]
E. Fedeli, A. Lancelot, J.L. Serrano, P. Calvo, T. Sierra, Self-assembling amphiphilic Janus dendrimers: mesomorphic properties and aggregation in water, New J. Chem. 39 (2015) 1960–1967. doi:10.1039/c4nj02071e.
[61]
X. Xu, H. Yuan, J. Chang, B. He, Z. Gu, Cooperative Hierarchical Self-Assembly of Peptide Dendrimers and Linear Polypeptides into Nanoarchitectures Mimicking Viral Capsids, Angew. Chemie - Int. Ed. 51 (2012) 3130–3133. doi:10.1002/anie.201106080.
[62]
X. Xu, Y. Li, H. Li, R. Liu, M. Sheng, B. He, et al., Smart Nanovehicles Based on pH-Triggered Disassembly of Supramolecular Peptide-Amphiphiles for Efficient Intracellular Drug Delivery, Small. 10 (2014) 1133–1140. doi:10.1002/smll.201301885.
[63]
D.R. Vutukuri, S. Basu, S. Thayumanavan, Dendrimers with Both Polar and Apolar Nanocontainer Characteristics, J. Am. Chem. Soc. 126 (2004) 15636–15637. doi:10.1021/ja0449628.
[64]
S. V. Aathimanikandan, E.N. Savariar, S. Thayumanavan, Temperature-Sensitive Dendritic Micelles, J. Am. Chem. Soc. 127 (2005) 14922–14929. doi:10.1021/ja054542y.
[65]
M.A. Azagarsamy, V. Yesilyurt, S. Thayumanavan, Disassembly of Dendritic Micellar Containers Due to Protein Binding, J. Am. Chem. Soc. 132 (2010) 4550–4551. doi:10.1021/ja100746d.
[66]
V. Yesilyurt, R. Ramireddy, M.A. Azagarsamy, S. Thayumanavan, Accessing Lipophilic Ligands in Dendrimer-Based Amphiphilic Supramolecular Assemblies for Protein-Induced Disassembly, Chem. A Eur. J. 18 (2012) 223–229. doi:10.1002/chem.201102727.
[67]
C. Kojima, T. Suehiro, K. Watanabe, M. Ogawa, A. Fukuhara, E. Nishisaka, et al., Doxorubicinconjugated dendrimer/collagen hybrid gels for metastasis-associated drug delivery systems, Acta Biomater. 9 (2013) 5673–5680. doi:10.1016/j.actbio.2012.11.013.
[68]
C. Kojima, E. Nishisaka, T. Suehiro, K. Watanabe, A. Harada, T. Goto, et al., The synthesis and evaluation of polymer prodrug/collagen hybrid gels for delivery into metastatic cancer cells, Nanomedicine Nanotechnology, Biol. Med. 9 (2013) 767–775. doi:10.1016/j.nano.2013.01.004.
[69]
J. Pan, M. Wen, D. Yin, B. Jiang, D. He, L. Guo, Design and synthesis of novel amphiphilic Janus
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ACCEPTED MANUSCRIPT Figure captions Figure 1: Examples of the chemical structures of dendrimers. A) Generations 1-3 of Janus dendrimers with a triazole in the core [60], B) generations 1-2 of Janus dendrimers with aspartic acid as targeting moiety [69], and C) generations 1-3 of PAMAM dendrimers.
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Figure 2: Illustrations showcasing supramolecular structures. A) A structure formed through entanglement of peptide coated G4 poly(amidoamine) (PAMAM) dendrimers. Adapted from Ref [47] with permission of The Royal Society of Chemistry. (http://dx.doi.org/10.1039/C1SM06157G). B) A structure formed through phase separation of esterified amino acid dendrons. Adapted from Ref [25] with permission of John Wiley & Sons; © 2011 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim. (http://dx.doi.org/10.1002/chem.201003049).
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Figure 3: A) A pH dependent release profiles of pralidoxime iodide (PI) and phenol red (PR) from a hydrogel in the presence and absence of metal ions (Mg2+), and B) schematic illustration of the release processes. Reprinted from Ref [39] with permission of John Wiley & Sons; © 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. (http://dx.doi.org/10.1002/chem.201404035).
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Figure 4: A) Drug (and enzyme) release profiles from Janus dendrimer-based hydrogel, and B) activity of the enzyme was maintained even after release from the gel. Reprinted from Ref [24] with permission of John Wiley & Sons; © 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. (http://dx.doi.org/10.1002/chem.201501812). Figure 5: Tumour growth in PC-3 xenograft mice after various treatments: combination of micelles and Hsp27 siRNA slowed tumour growth. Reprinted from Ref [58] with permission of John Wiley & Sons; © 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. (http://dx.doi.org/10.1002/anie.201406764).
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Figure 6: Doxorubicin encapsulated in supramolecular micelles A) enhanced drug effect on tumour volume, B) abolished body weight changes due to doxorubicin, C) increased mice survival during the experiment, D) reduced tumour proliferation, and E) reduced heart toxicity. T. Wei, C. Chen, J. Liu, C. Liu, P. Posocco, X. Liu, Q. Cheng, S. Huo, Z. Liang, M. Fermeglia, S. Pricl, XJ. Liang, P. Rocchi and L. Peng, Anticancer drug nanomicelles formed by self-assembling amphiphilic dendrimer to combat cancer drug resistance, Proc. Natl. Acad. Sci. U. S. A. 112: 2978–83, 2015. Reprinted with permission of PNAS. (http://dx.doi.org/10.1073/pnas.1418494112).
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Figure 7: A) Molecular recognition guided self-assembly and pH-dependent disassembly, and B) confocal laser scanning microscopy tracking of the drug delivery system components, and C) schematic illustration of supramolecular micelle internalization leading to pH-guided disassembly and in-situ drug release within a cell. Reprinted from Ref [62] with permission of John Wiley & Sons; © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. (http://dx.doi.org/10.1002/anie.201106080). Figure 8: Schematic illustration of drug release from supramolecular micelles upon receptor binding of a covalently attached A) hydrophilic and B) hydrophobic ligand. Reprinted from Ref [66] with permission of John Wiley & Sons; © 2012 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim. (http://dx.doi.org/10.1002/chem.201102727). Figure 9: A) Chemical structure of the dendrimer-prodrug, B) comparison of the release profiles of the dendrimer-prodrug and free drug (in gel), and C) in vivo experimental result showing inhibition of metastasis (i) compared to controls (ii-iv). Reprinted from Acta Biomaterialia, Vol 9 (3), C. Kojima, T. Suehiro, K. Watanabe, M. Ogawa, A. Fukuhara, E. Nishisaka, A. Harada, K. Kono, T. Inui and Y. Magata, Doxorubicin-conjugated dendrimer/collagen hybrid gels for metastasis-associated drug delivery systems,
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ACCEPTED MANUSCRIPT Pages No. 5673–5680, Copyright 2012, with permission from Elsevier. (http://dx.doi.org/10.1016/j.actbio.2012.11.013).
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Figure 10: Ratio of drugs released into plasma during in vitro drug release experiment. Adapted with permission from A.L. Acton, C. Fante, B. Flatley, S. Burattini, I.W. Hamley, Z. Wang, F. Greco and W. Hayes, Janus PEG-based dendrimers for use in combination therapy: Controlled multi-drug loading and sequential release, Biomacromolecules. 14(2): 564–574, 2013. Copyright 2013 American Chemical Society. (http://dx.doi.org/10.1021/bm301881h).
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ACCEPTED MANUSCRIPT Table 1: Summary of recent biomedical applications of supramolecular dendrimer-based structures.
Glutamic acid end-capped G4 PAMAM dendrimer covered with 2 kDa collagen peptide Coupling of G1-3 bis-MPA dendrons with and without esterification
Effect Released over period of 3 hours
Reference [24]
Hydrogel formed upon hydration
Released in 6-8 hours, release from G1 was pH dependent
[38,48]
Helical tubules forming metal ion shrinkable hydrogel degrading at neutral pH
Negatively charged drugs released during shrinkage and positive drugs during degradation of the gel
[25,39,46]
Collagen gel embedding the dendrimer-prodrugs
Injection to tumour site inhibits cancer cell line metastasis in mice
[67]
Plitidepsin (encapsulated)
Spherical aggregates using oil-in-water method: particle with 50 nm diameter, vesicles with 9-12 nm bilayers, and elongated micelles Micellar aggregates complexing siRNA; micelles encapsulating doxorubicin
PAMAM type dendrimers coupled to a hydrophobic part bearing two alkyl chains
siRNA; doxorubicin
Silsesquioxane cored G2 poly(L-lysine) dendrimers co-assembling with glutamic acid terminated poly(l-leucine)
Doxorubicin (encapsulated)
Dendrimers with substituted biaryl as the dendritic repeating unit
Pyrene / Nile red as model cargo
A range of amphiphilic Janus dendrimers from coupling of dendrons modified with peripheral acidic amino acids as targeting moieties and dendrons ester bond to naproxen Generations (first and second) of PEGbased dendrons were first coupled with 2 model compounds to get four dendrons, and then the dendrons were coupled to get four dendrimers carrying both drugs
Naproxen
Molecular drug carrier for targeted delivery
Benzyl alcohol (BA) and 3-phenylpropionic acid (PPA) attached to the dendrimers via 2 different linkers (carbonate and ester)
Molecular drug carrier for combinational therapy
Varied concentrations of the drug in solutions confirmed using HPLC and gravimetry
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Supramolecular nanoparticles 250-300 nm in diameter in aqueous environment Biotin / dinitrophenyl bearing micelles
[60]
Delivery of siRNA slowing tumour growth in PC-3 xenograft mice; doxorubicin resistance mediated by efflux pumps overcome Enhanced internalization of doxorubicin; comparable cytotoxic effects with doxorubicin hydrochloride Release of cargo at binding onto extravidin / rat anti-DNP immunoglobulin G antibody Hydroxyapatite targeted drug carrier enhanced naproxen solubility and was non-cytotoxic for HEK293 cell line.
[58,59]
Sequential release, BA being released faster than PPA from all the dendrons
[70]
[61,62]
[63–66]
[69]
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Esterified L-glutamic acid dendron
Structure Supramolecular hydrogels upon ethanol injection
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PEG with G1-3 Citric acid dendrons
Compounds Nadolol; gonadorelin; horseradish peroxidase (entrapped) 5-aminosalicylic acid, diclofenac, mefenamic acid, and pyridine complexed with dendrimers Thiamine; pralidoxime and phenol red simultaneously (entrapped with heating) Doxorubicin (covalently attached)
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Dendrimer Three G3 dendritic bis-MPA and (azidomethyl)benzene-based dendrimers
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PII:
S1773-2247(16)30060-0
DOI:
10.1016/j.jddst.2016.02.008
Reference:
JDDST 172
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 31 December 2015 Revised Date:
26 February 2016
Accepted Date: 26 February 2016
Please cite this article as: M. Selin, L. Peltonen, J. Hirvonen, L.M. Bimbo, Dendrimers and their supramolecular nanostructures for biomedical applications, Journal of Drug Delivery Science and Technology (2016), doi: 10.1016/j.jddst.2016.02.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT Dendrimers and their supramolecular nanostructures for biomedical applications Markus Selin*, Leena Peltonen, Jouni Hirvonen, Luis M. Bimbo
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Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, PO Box 56 (Viikinkaari 5E), FI-00014 University of Helsinki, Finland *Corresponding author: Markus Selin, [email protected], Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, PO Box 56 (Viikinkaari 5E), FI-00014 University of Helsinki, Finland
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ACCEPTED MANUSCRIPT Abstract
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Pharmaceutical nanotechnology is the field of materials science that focuses on techniques to process raw materials to produce nanostructures with desired properties for pharmaceutical applications. Nanostructures can be roughly divided into surface textures, tubular structures or particles, depending on how many of their dimensions are on the nanoscale. The structures may also be divided into hard and soft structures, depending on their physical properties. There has been lately a growing interest to study soft supramolecular structures. In this review, the main focus is to briefly introduce the reader to various aspects of dendrimers, including Janus dendrimers, and supramolecular structures and then focus on recent studies dealing with application of dendrimer-based supramolecular structures in the pharmaceutical field.
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Graphical Abstract
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Keywords: (Janus) dendrimers, dendrons, hydrogels, nanostructures, polymers, supramolecular structures
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ACCEPTED MANUSCRIPT 1. Introduction
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Polymers can be broadly divided into four classes based on their primary structure: 1) linear, 2) cross-linked with side-chains or side-functional groups, 3) branched, and 4) perfect dendrimers [1]. The primary structure of perfect dendrimers branches periodically and in a precise pattern. The name of an individual dendrimer is derived from the branching pattern and the generation of the dendrimer, i.e. an ordinal number stating the number of times the pattern is repeated. Generally, dendrimers display an open structure at low generations and become more globular and dense as the generations increase. Dendrimers without globular core-shell topology are sometimes termed dendrons to emphasize this detail. Dendrimers have intrinsic and unique features, e.g. high structural fidelity due to controlled iterative synthesis methods, and a high number of functionalities present in the intermediate and surface layers [2–4]. Especially dendrimers with globular core-shell topology can be viewed as nanoscale atom mimics [5] that are capable to aggregate and form nanoscale mimics of molecules via surface interactions [6].
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Structural effects of intermolecular interactions are studied in supramolecular chemistry. Among aspects covered are self-assembly, i.e. the hierarchical process where molecules spontaneously arrange into supramolecular assemblies that further self-organize into supramolecular structures and systems [7], and self-curing, i.e. ability of the supramolecular structures to reform after being damaged [8]. Supramolecular self-assembly processes may be driven by phase separation, a complex phenomenon where components of chemical mixtures or chemically distinct domains of molecules get spatially separated. One of the most ubiquitous self-assembly processes is the hierarchical organization of amphiphilic molecules into structures such as micelles, rods and vesicles [9]. Molecular recognition driven self-assembly processes differ from the phase separation driven ones since in this case a connection is formed when chemically different domains pair through interactions precisely complementing each other. A number of examples demonstrating supramolecular polymers formed via molecular recognition driven self-assembly processes and further phase separation driven self-organization of such polymers into complex structures, e.g. vesicles, may be found in a recent review [10]. As a specific example of a molecular recognition driven process, the hostquest chemistry between adamantane-modified hyperbranched polyglycerol and cationic β-cyclodextrin derivatives has been utilized to design self-assembling dendritic polymers for non-viral gene delivery [11].
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Supramolecular structures can be manifold, one example being supramolecular coatings. The coating molecules form monolayers upon adsorption to a solid support. An interesting coating study was made with amphiphilic peptide coatings incorporating a terminal L-lysine dendron and a terminal fatty acid on an oligopeptide [12]. Further, the dendrons were equipped with one or two cell adherence motifs. The coatings were then attached to the polymeric scaffold whereupon they adapted supramolecular β-sheet structures. In comparison to bare scaffolds and scaffolds coated with a linear peptide coating, the scaffolds coated with the L-lysine dendron bearing coatings showed improved colonization of the scaffold surface by human bladder smooth muscle cells (SMCs) and enhanced penetration of the cells into the scaffold. Thus, the dendron bearing coatings have potential to be used in bladder tissue regeneration applications. The results were speculated to be due to either improved accessibility of the adhesion sequences to the SMCs, or due to non-specific electrostatic attraction between the positively charged amines of the L-lysine residues and the SMCs. Dendrimers and dendrons have been shown to self-assemble into hierarchically organized structures [13,14], mediate chirality into structures [15,16], glue supramolecular structures [17], and facilitate the formation of crystalline complexes with viruses [18]. There are already reviews about dendrimers [19] and dendrimer-based supramolecular structures [20] in pharmaceutical and biomedical fields, as well as a thorough review about biomedical applications of supramolecular polymers [21]. Only very recently have dendrimers comprised of heterogeneous segments (Janus dendrimers) been designed for biomedical applications (Figure 1A-B). A brief overview of the reviewed studies dealing with dendrimers forming 3
ACCEPTED MANUSCRIPT supramolecular structures and dendrimers studied as drug carriers is provided (Table 1). In this review article various aspects of dendrimers and supramolecular structures are introduced the focus being on recent studies dealing with applications of dendrimer-based supramolecular structures in the pharmaceutical field. (Figure 1) (Table 1)
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2. Supramolecular hydrogels
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Hydrogels are viscous mixtures composed of a polymeric network and an aqueous phase that, by definition [22], expands the network throughout the volume of the gel. Most of the mass of a hydrogel comes from its aqueous phase. Covalent and physical hydrogel networks are formed from gelling agents connected through covalent bonds and physical interactions, respectively. Covalent hydrogels are usually prepared using a chemical reaction connecting the gelling agents through specially designed crosslinking molecules. Entanglement of polymer chains of cellulose derivatives, such as hydroxypropyl methylcellulose (HPMC) and carboxymethyl cellulose (CMC), suspended in an aqueous phase are typical examples of how physical hydrogel networks are formed. The supramolecular hydrogel networks demonstrate various levels structural hierarchy [23–26]. The amphiphilic molecules work as gelling agents comprising self-assembling supramolecular nanoscale tubules and fibrils that form hydrogel networks through physical and supramolecular crosslinks.
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If the dimensions of the expanded hydrogel network are within nanoscale, then the whole of the hydrogel should be viewed as a nanoparticle rather than a bulk gel. When the term nanogel is used to refer to such hydrogel particles, care should be taken to check that such particle fulfils the criteria of both being a hydrogel, i.e. having a network of crosslinked fibrils expanded by the aqueous phase [22], and being a nanoparticle, i.e. having dimensions in nanoscale [27]. Some hydrogels, micelles and vesicles may comply with the criteria, however, typical micelles do not since they lack a relevant number of water molecules inside them [28–30]. Vesicles have a membrane that is expanded by solvent in the inside cavity. The membrane of typical vesicles should not be viewed as a network of crosslinked fibrils since it is, in fact, a single continuous supramolecular structure. In this review, the studies dealing with supramolecular entities with all the dimensions on the nanoscale will be discussed under the subtitle ‘3. Supramolecular nanoparticles’.
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Hydrogels based on dendritic polymers can be classified into three classes: type I hydrogels, where dendrimers act as a cross-linkers, type II hydrogels, where hydrogels are formed via polymerization of dendrimer-based monomers, and type III hydrogels, where self-assembling dendritic compounds form hydrogel [31]. Type III supramolecular dendrimer hydrogels may be further divided based on their chemical and network structures (Figure 2). Early reports of amphiphilic dendrimer gels were presented by Newkome et al. [32], with later examples including multi-component [33] and chiral [34,35] gels tailored for high-end [36] and biomedical delivery applications [37]. Because self-assembling process is highly dependent on the environmental factors, the stability of those hydrogels may be affected by different environmental changes in vivo. (Figure 2) Dendrimer-based gels have been applied and studied for applications in drug release [24,38,39], as well as diagnostics, regenerative medicine and tissue engineering [40–45]. In dendritic gels, drug can be entrapped physically inside of the polymeric network structures. The rate of drug release from gels is usually controlled either by rate of drug diffusion or by hydrogel degradation [24,39]. The degradation of the 4
ACCEPTED MANUSCRIPT hydrogels becomes important especially if the drug molecules are covalently incorporated into or attached onto the hydrogel network. Some hydrogels respond to environmental factors, such as pH, metal ions and temperature [38,39,46,47]. Hydrogels may be equipped with adherence motifs, e.g. to promote either cell migration and proliferation [45] or competitive binding between dendrimer bound ligands and the assayed biomarkers [42]. 2.1 Polyethylene glycol (PEG) end-modified with dendrons
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Hydrogels were formed from PEG terminated with citric acid dendrons of various generations [48]. To prepare the G1 dendrimer, PEG bis(carboxymethyl) ether (Mn 600 g/mol) was esterified with citric acid using thionyl chloride in pyridine. The esterification reactions to produce the following dendritic generations succeeded using N,N' dicyclohexylcarbodiimide in pyridine. The gel formers were dissolved by heating, and the gels formed during subsequent cooling. 5-aminosalicylic acid, diclofenac, mefenamic acid, and pyridine were complexed with dendrimer generations 1-3 [38]. The molar ratio of complexed drug to dendrimer was increasing with the generations. When the complexes were hydrated they formed hydrogels that were studied in drug release experiments. Typically these drugs were released from the hydrogels at pH 7.4 and 1 within 6 to 8 hours. Hydrogel from G1 dendrimer was able to retain 70 % of the encapsulated 5-aminosalicylic acid, diclofenac and mefenamic acid at pH 7.4. 2.2 PAMAM dendrimers coated with polypeptides
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PAMAM dendrimers contain a lot of amino groups, which induces pH-sensitivity for these structures (Figure 1C). Experimentally the highest swelling with these hydrogels has been found to be around pH 4 [49]. The G4 PAMAM dendrimers coated with collagen model peptide (Pro-Pro-Gly)5 have gelatine mimicking characteristics [50,51]. Dendrimers were loaded with rose bengal (RB) in aqueous solution and subsequently excess of RB was removed from freeze dried complexes through precipitation in chloroform [52]. The release of RB was monitored at various temperatures and was slowest at 4 °C [50,51]. Dendrimer solution (8.4 mM) also formed a hydrogel when cooled to 4 °C. Subsequently, G4 PAMAM dendrimers were coated with (Pro-Pro-Gly)10 [53] and even later with (Pro-Hyp-Gly)10 [47]. The fore reported coating provided dendrimers that formed a gelatine mimicking hydrogel below 45 °C whereas the coating reported later provided dendrimers that formed a collagen mimicking hydrogel above 40 °C. The (Pro-Hyp-Gly)10 peptides on the G4 PAMAM dendrimer adapted a triple helical structure that was more thermostable than the helical structure of native collagen. The collagen mimicking hydrogel itself was stable for 1 hour at 80 °C. As an alternative way to make self-assembling supramolecular hydrogels from peptide coated PAMAM dendrimers, methodology for easy polymerization reaction to surface modify G2 and G4 amine terminated dendrimers with poly(γ-(2-(2-methoxyethoxy)ethyl) L-glutamate) has been published recently [54]. The dendrimer used as the core can be prepared easily without protection/activation steps using alternating aza-Michael addition and thiol-yne reactions [55]. 2.3 Asymmetric L-glutamic acid dendrons A L-glutamic acid dendron bound to a long chain fatty acid was characterized as a gel former [25]. When the gel former was dissolved in aqueous solution below pH 10, very thin helical nanoribbons formed and adapted further hierarchical morphologies, i.e. helical tubules 10 nm in diameter with 4 nm thick bilayers and superhelices of the tubules (Figure 2B). The asymmetric nature of the L-glutamic acid dendron might convey these structures their helical morphology. Fractal, needle-like fibrils were seen in solutions of the gel former at pH above 10. Supramolecular, metal-ion-shrinkable hydrogel was prepared from these gel formers [46]. The shrinking ratios and rates were studied over time in connection with various metal ions. Thiamine was entrapped in the hydrogel and the release was followed after addition of selected metal ions. The release of entrapped thiamine was controlled by the hydrogel shrinkage. A positively charged drug, pralidoxime, and phenol red were simultaneously entrapped in the hydrogel [39]. Their release was studied 5
ACCEPTED MANUSCRIPT in controlled pH conditions in the presence and absence of Mg2+ ions (Figure 3). In the absence of the ions in pH 3.2, the rates of release were almost the similar. In pH 3.2, the ions caused shrinkage of the hydrogel, increased the rate of phenol red release and halted the release of the drug below 30 % of the encapsulated amount. When the pH was subsequently adjusted to 6.2, the hydrogel degraded and the remaining drug was released. (Figure 3)
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2.4 Janus dendrimers Amphiphilic Janus dendrimers may be produced by combining polar and non-polar segments, which will be reflected in descriptors like molecular dipole moment and hydrophilic-lipophilic balance. Classifying individual cases of dendrimers as symmetric or Janus dendrimers is not always straightforward, e.g. the way of defining segments and cores influences the classification. In this review, we have considered simple fatty acid esters of dendrons as symmetric dendrons rather than as Janus dendrimers.
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In favourable conditions, the intermolecular interactions cause amphiphilic Janus dendrimers in solution to aggregate and become directionally aligned. In effect, during this process the amphiphilic molecules form layers consisting of two separate phases. The intermolecular interactions and molecular properties may promote curvature in the layers and thereby drive formation of tubular structures or particles instead of planar surfaces. In a bilayer, two layers have aligned and merged their chemically similar phases. Cell membranes are a classical example of bilayers where the non-polar phases of two phospholipid layers constitute a merged interior phase, and the polar phases of the layers form two separate exterior phases. Properties of these layered surfaces, e.g. zeta-potential and surface energy, reflect the material behaviour.
(Figure 4)
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When two dodecanoxy substituted (azidomethyl)benzene derivatives were combined through a copper catalysed cycloaddition reaction with G3 dendritic 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) molecules built onto 2-propynol, amphiphilic dendrimers able to gelatinise aqueous solutions at low mass proportions were formed [24]. Self-assembling fibrils 4-6 nm in diameter formed supramolecular bundles with hexagonal cross-sections 15 nm in diameter, fibres less than 100 nm in diameter, and their crosslinked networks. Drug release from 5-(azidomethyl)benzene-1,2,3-triol-based dendrimer hydrogels was further studied (Figure 4). More than 80 % of encapsulated small molecule drug, nadolol, and more than 50 % of the encapsulated peptide drug, gonadorelin, was released over period of 3 hours. Also, horseradish peroxidase was entrapped into the hydrogel and its activity was confirmed after release.
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3. Supramolecular nanoparticles
In addition to hydrogels, dendrimers can also form supramolecular particles with nanoscale dimensions. The self-assembly principles also influence the formation of supramolecular particles. In the following chapters we have divided studies dealing with supramolecular particles to demonstrate 1) the phase separation as a driving force of self-assembly, and 2) the ways molecular recognition can be utilized in pharmaceutical applications of supramolecular structures. A molecule should contain chemically different domains to demonstrate supramolecular self-assembly via phase separation. In this case, similar domains are matched to construct the phases. Molecular recognition relies on interactions from chemically distinct functional groups precisely complementing each other. In the following examples, multiple pairs of interacting groups are utilized as the basis for supramolecular co-assembly and for coupling drug release from supramolecular structures to specific receptor binding events. 3.1 Phase separated supramolecular particles
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Supramolecular particles formed via phase separation processes can be spherical vesicles or micelles. For example, spherical particles 170-190 nm in diameter have been formed from PEG end-modified with G2 dendritic carbosilane molecules and micelles 32-92 nm in diameter were formed from PEG end-modified with dendritic benzyl ether molecules [56,57]. In aqueous environment, dendrimers formed by coupling PAMAM type dendron to a hydrophobic part bearing two alkyl chains self-assembled into vesicular structures termed dendrimersomes that are approx. 200 nm in diameter [58]. The dendrimersomes collapsed to clusters of small micelles 6-8 nm in diameter upon addition of siRNA. The micelles were used as vectors for siRNA delivery in vitro and in vivo. Tumour growth was slowed in PC-3 xenograft mice by the delivered siRNA (Figure 5). (Figure 5)
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Empty micelles and micelles encapsulating anticancer drug, doxorubicin, could also be formed from PAMAM type dendrimers via film dispersion method [59]. The doxorubicin encapsulating micelles were slightly larger than the empty micelles (10 nm in diameter). Less than 20 % and 35 % of the doxorubicin was released at 37 °C from the micelles in 24 hours at pH 7.4 and 5.0, respectively. The micelles enhanced the penetration of doxorubicin into tumour spheroids prepared by 3D culturing doxorubicin resistant breast cancer MCF-7R cells. When the effect was further studied, it was found out that the micelles were able to both deliver doxorubicin via macropinocytosis-dependent endocytosis thereby overcoming the doxorubicin resistance mediated by efflux pumps overexpressed by the cell line, and inhibit the efflux of internalized doxorubicin. The micelles loaded with a dye and injected intravascularly focused at tumour site in a mice model. Furthermore, doxorubicin loaded micelles enhanced antitumour effect of the doxorubicin and reduced its toxic off-target effects in vivo (Figure 6). It remains to be seen, if the therapeutic approaches based on siRNA and doxorubicin can be combined. (Figure 6)
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Janus dendrimers have also been found to self-assemble into supramolecular particles. Two series of G1-3 dendrons were prepared by building dendritic bis-MPA molecules onto either a single 2-propynol or a 6azidohexan-1-ol [60]. The alkyne containing dendrons were then esterified with stearic acid to get lipophilic dendrons. Next, the alkyne and azide bearing dendrons were modularly coupled via Cu(I)-catalysed 1,3dipolar cycloaddition to produce amphiphilic Janus dendrimers denoted as G1/G1-3, G2/G2-3 and G3/G3 using generations of their lipophilic and hydrophilic dendrons, respectively. When a G1/G2-3 and G2/G3 dendrimers aggregated in water, elongated micelles were formed. The G1/G1 dendrimer did not aggregate in water at concentrations below its solubility limit. The G2/G2 dendrimers formed well defined spherical structures 50 nm in diameter. The G3/G3 dendrimers produced vesicles where the thickness of the formed bilayers was 9-12 nm. The rest of the dendrimers produced elongated micelles. Lipophilic anticancer drug, plitidepsin, was complexed with G2/G2, G3/G3, and G2/G3 dendrimers. The process was less successful with G1/G3 and G1/G2 dendrimers, which was attributed to their less lipophilic nature. 3.2 Supramolecular particles demonstrating molecular recognition Molecular recognition helps silsesquioxane cored G2 poly(L-lysine) dendrimers to co-assemble with glutamic acid terminated poly(l-leucine) in aqueous environment into supramolecular nanoparticles 250300 nm in diameter [61]. Doxorubicin could be loaded into these nanoparticles and in 24 hours, approx. 80 % of the loaded doxorubicin was retained and released at pH 7.4 and pH 6.2, respectively [62]. The electrostatic interactions necessary for co-assembly dissipate at acidic conditions (Figure 7A). Human hepatocellular carcinoma cell line, HepG2, cells were incubated with doxorubicin, doxorubicin hydrochloride and nanoparticles loaded with doxorubicin. Confocal laser scanning microscopy (CSLM) of fluorescein isothiocyanate (FITC) labelled hydrophilic parts of the nanoparticles showed that initially spot7
ACCEPTED MANUSCRIPT like FITC signals spread after the 3 first hours of incubation (Figure 7B). Thereafter the FITC signal stayed associated with the cell membranes. Doxorubicin reached the nucleus within the first 3 hours, presumably after pH-dependent disassembly in response to a biological stimulus (figure 7C). The drug loaded nanoparticles enhanced internalization of doxorubicin by HepG2 cells compared to the free base form. However, comparable cytotoxic effects were seen in HepG2 samples incubated with doxorubicin loaded nanoparticles and doxorubicin hydrochloride.
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(Figure 7)
(Figure 8)
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Symmetric aromatic repeating and termination units were made amphiphilic using substituents [63]. These amphiphilic building blocks were then used to synthesize dendrimers that were able to form both micelles and inverted micelles incorporating model compounds as cargo. The substituents were later fixed as pentaethylene glycol monomethyl ether and 1-decanol to promote charge neutrality and temperature sensitivity in the micellar constructs [64]. To produce dendrimers that self-assemble to form micelles with molecular recognition properties, hydrophilic [65] and hydrophobic [66] ligands were attached onto these dendrimers by replacing one substituent per dendrimer. Attaching a hydrophilic biotin molecule onto G1-2 of these dendrimers yielded micelles that could be loaded with pyrene as model cargo [65]. The micelles dissembled and released the loaded pyrene when subjected to extravidin. Other proteins used did not result in such disassembly. Disassembly of the micelle bearing a hydrophobic ligand was studied using the interaction between dinitrophenyl (DNP) and rat anti-DNP immunoglobulin G antibody. Nile red loaded into the micelle was released and the measured size of the micelles diminished in the presence of the antibody [66]. Addition of proteins that do not work as receptors for dinitrophenyl did not release the loaded Nile red nor change the size of the micelles. This confirmed the hydrophobic ligands could also be used for archiving targeted disassembly. The mechanisms of ligand binding associated micellar disassembly processes are summarized in a figure (Figure 8).
4. Dendrimer based molecular drug carriers
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As stated above, supramolecular dendrimers have been shown to be promising delivery vehicles in biomedical applications. As molecular carriers, their branched and layered architectures display a high number of controlled terminal groups as well as cavities for physical entrapment. In the following chapters, novel approaches to utilize the potential of dendrimers as molecular drug carriers.
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4.1 PAMAM-type molecular drug carriers Covalent binding of doxorubicin to G4 PAMAM dendrimer first surface modified with glutamic acid and then coated with enzyme hydrolysed collagen peptide (2 kDa, Figure 9A) suppressed diffusion of doxorubicin from a collagen gel (Figure 9B) when compared to diffusion of free doxorubicin from the gel [67]. Doxorubicin attached to poly(glutamic acid) had negligible diffusion rate in a collagen gel [68]. The result was interpreted as entanglement of the polypeptide chain to the hydrogel network since almost similar molecular weight prodrug diffused in the gel. In the prodrug, doxorubicin was covalently attached to a globular dendrimer coated with acetylated glutamic acid. Replacing the acetyl groups with PEGs (2 kDa) did not change the diffusion rate. However, replacement of the acetyl groups with collagen peptides (either 2 kDa or 5kDa) slowed the diffusion. Matrix metalloproteinase (MMP) inhibitor reduced the toxicity of dendrimer conjugated doxorubicin embedded in a collagen gel against highly invasive MDA-MB-231 cells [67]. Thus, the gel offers an option to selectively target MMP secreting cells with the embedded prodrugs. The embedded collagen gel was able to inhibit metastasis of reporter gene bearing mouse breast cancer cell line cells implanted in mammary pad of BALC/c mice (Figure 9C). 8
ACCEPTED MANUSCRIPT (Figure 9) 4.2 Janus dendrimers
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Pan et al. designed and synthesized a range of amphiphilic Janus dendrimers to accomplish bone-targeted dendritic drug delivery [69]. These dendrimers carried acidic amino acids as targeting moieties simultaneously with multiple naproxen molecules. As a result, the dendrimers exhibited more than 80% binding rates to hydroxyapatite (HAP) in vitro and enhanced solubility of naproxen by the dendritic drug delivery system, while exhibiting no significant cytotoxicity against HEK293 cells. Another recent proof-ofconcept study utilized the Janus dendrimers for combination therapy. Acton et al. synthesized a series of novel Janus dendrimers with potential applications in combination therapy [70]. They reported the creation of different first and second generations of tertiary amine and PEG-based dendrons. Model compounds benzyl alcohol (BA) and 3-phenylpropionic acid (PPA) were covalently attached to the dendrons via carbonate and ester linkers, respectively. The four dendrons were coupled together via (3 + 2) cycloaddition chemistries to afford four Janus dendrimers, which contained varying amounts and different ratios of the BA and PPA to promote differential drug release in plasma (Figure 10). The Janus dendrimers provided sequential release of the two model compounds, with BA being released faster than PPA from all the dendrons. Haemolysis studies confirmed that these dendrimers are non-cytotoxic towards HUVEC cells and non-toxic toward red blood cells. (Figure 10) 5. Conclusions
6. Acknowledgements
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The recent studies showing promising results highlight the various aspects of dendrimer-based selfassembling structures and Janus dendrimers beneficial in developing biomedical applications. The fields with potential applications for these remarkable entities include regenerative medicine, targeted and controlled drug delivery, as well as combination therapy. Albeit the first in vivo results have been claimed from supramolecular dendrimer-based structures, still many details have to be clarified to realize the results of the research in clinical routines. Janus dendrimers can also be designed to work as intelligent drug carriers or pro-drugs, utilizing the multiple functionalities to graft drugs with desired properties.
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Financial support by the Academy of Finland (decisions No. 276377 for L.B.), Biocentrum Helsinki, The Finnish Cultural Foundation, Orion Research Foundation and the Jane and Aatos Erkko Foundation is gratefully acknowledged. REFERENCES [1]
D.A. Tomalia, Dendrons/dendrimers: quantized, nano-element like building blocks for soft-soft and soft-hard nano-compound synthesis, Soft Matter. 6 (2010) 456–474. doi:10.1039/b917370f.
[2]
S.M. Grayson, J.M.J. Fréchet, Convergent Dendrons and Dendrimers: from Synthesis to Applications, Chem. Rev. 101 (2001) 3819–3867. doi:10.1021/cr990116h.
[3]
A.W. Bosman, H.M. Janssen, E.W. Meijer, About Dendrimers : Structure, Physical Properties, and Applications, Chem. Rev. 99 (1999) 1665–1688. doi:10.1021/cr970069y.
[4]
S. Nummelin, M. Skrifvars, K. Rissanen, Polyester and Ester Functionalized Dendrimers, Top. Curr. Chem. 210 (2000) 1–67. doi:10.1007/3-540-46577-4_1.
[5]
D.A. Tomalia, Dendritic effects: dependency of dendritic nano-periodic property patterns on critical nanoscale design parameters (CNDPs), New J. Chem. 36 (2012) 264–281. doi:10.1039/c1nj20501c.
9
ACCEPTED MANUSCRIPT D.A. Tomalia, H.M. Brothers II, L.T. Piehler, H.D. Durst, D.R. Swanson, Partial shell-filled core-shell tecto(dendrimers): A strategy to surface differentiated nano-clefts and cusps, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 5081–5087. doi:10.1073/pnas.062684999.
[7]
G.M. Whitesides, B. Grzybowski, Self-Assembly at All Scales, Science. 295 (2002) 2418–2421. doi:10.1126/science.1070821.
[8]
M. Zhang, D. Xu, X. Yan, J. Chen, S. Dong, B. Zheng, et al., Self-healing Supramolecular Gels Formed by Crown Ether Based Host-Guest Interactions, Angew. Chemie - Int. Ed. 51 (2012) 7011–7015. doi:10.1002/anie.201203063.
[9]
P. V Messina, J.M. Besada-Porto, J.M. Ruso, Self-Assembly Drugs: From Micelles to Nanomedicine, Curr. Top. Med. Chem. 14 (2014) 555–571. doi:10.2174/1568026614666140121112118.
[10]
R. Dong, Y. Zhou, X. Zhu, Supramolecular dendritic polymers: From synthesis to applications, Acc. Chem. Res. 47 (2014) 2006–2016. doi:10.1021/ar500057e.
[11]
R. Dong, L. Zhou, J. Wu, C. Tu, Y. Su, B. Zhu, et al., A supramolecular approach to the preparation of charge-tunable dendritic polycations for efficient gene delivery., Chem. Commun. (Camb). 47 (2011) 5473–5475. doi:10.1039/c1cc10934k.
[12]
D.A. Harrington, E.Y. Cheng, M.O. Guler, L.K. Lee, J.L. Donovan, R.C. Claussen, et al., Branched peptide-amphiphiles as self-assembling coatings for tissue engineering scaffolds, J. Biomed. Mater. Res. A. 78A (2006) 157–167. doi:10.1002/jbm.a.30718.
[13]
B.M. Rosen, C.J. Wilson, D.A. Wilson, M. Peterca, M.R. Imam, V. Percec, Dendron-Mediated SelfAssembly, Disassembly, and Self-Organization of Complex Systems, Chem. Rev. 109 (2009) 6275– 6540. doi:10.1021/cr900157q.
[14]
H.-J. Sun, S. Zhang, V. Percec, From structure to function via complex supramolecular dendrimer systems, Chem. Soc. Rev. 44 (2015) 3900–3923. doi:10.1039/c4cs00249k.
[15]
V. Percec, A.E. Dulcey, V.S.K. Balagurusamy, Y. Miura, J. Smidrkal, M. Peterca, et al., Self-assembly of amphiphilic dendritic dipeptides into helical pores., Nature. 430 (2004) 764–768. doi:10.1038/nature02770.
[16]
V. Percec, A.E. Dulcey, M. Peterca, M. Ilies, J. Ladislaw, B.M. Rosen, et al., The Internal Structure of Helical Pores Self-Assembled from Dendritic Dipeptides is Stereochemically Programmed and Allosterically Regulated, Angew. Chemie Int. Ed. 44 (2005) 6516–6521. doi:10.1002/anie.200501331.
[17]
A. Pantos, D. Tsiourvas, G. Nounesis, C.M. Paleos, Interaction of Functional Dendrimers with Multilamellar Liposomes: Design of a Model System for Studying Drug Delivery, Langmuir. 21 (2005) 7483–7490. doi:10.1021/la0510331.
[18]
J. Mikkilä, H. Rosilo, S. Nummelin, J. Seitsonen, J. Ruokolainen, M.A. Kostiainen, Janus-DendrimerMediated Formation of Crystalline Virus Assemblies, ACS Macro Lett. 2 (2013) 720–724. doi:10.1021/mz400307h.
[19]
Y. Kim, S.C. Zimmerman, Applications of dendrimers in bio-organic chemistry, Curr. Opin. Chem. Biol. 2 (1998) 733–742. doi:10.1016/S1367-5931(98)80111-7.
[20]
K.T. Al-Jamal, C. Ramaswamy, A.T. Florence, Supramolecular structures from dendrons and dendrimers, Adv. Drug Deliv. Rev. 57 (2005) 2238–2270. doi:10.1016/j.addr.2005.09.015.
[21]
R. Dong, Y. Zhou, X. Huang, X. Zhu, Y. Lu, J. Shen, Functional supramolecular polymers for biomedical applications, Adv. Mater. 27 (2015) 498–526. doi:10.1002/adma.201402975.
[22]
J. V. Alemán, A. V. Chadwick, J. He, M. Hess, K. Horie, R.G. Jones, et al., Definitions of terms relating to the structure and processing of sols, gels, networks, and inorganic-organic hybrid materials
AC C
EP
TE D
M AN U
SC
RI PT
[6]
10
ACCEPTED MANUSCRIPT (IUPAC Recommendations 2007), Pure Appl. Chem. 79 (2007) 1801–1829. doi:10.1351/pac200779101801. P.Y.W. Dankers, T.M. Hermans, T.W. Baughman, Y. Kamikawa, R.E. Kieltyka, M.M.C. Bastings, et al., Hierarchical Formation of Supramolecular Transient Networks in Water: A Modular Injectable Delivery System, Adv. Mater. 24 (2012) 2703–2709. doi:10.1002/adma.201104072.
[24]
S. Nummelin, V. Liljeström, E. Saarikoski, J. Ropponen, A. Nykänen, V. Linko, et al., Self-Assembly of Amphiphilic Janus Dendrimers into Mechanically Robust Supramolecular Hydrogels for Sustained Drug Release, Chem. - A Eur. J. 21 (2015) 14433–14439. doi:10.1002/chem.201501812.
[25]
P. Duan, L. Qin, X. Zhu, M. Liu, Hierarchical Self-Assembly of Amphiphilic Peptide Dendrons: Evolution of Diverse Chiral Nanostructures Through Hydrogel Formation Over a Wide pH Range, Chem. - A Eur. J. 17 (2011) 6389–6395. doi:10.1002/chem.201003049.
[26]
R. Dong, Y. Pang, Y. Su, X. Zhu, Supramolecular hydrogels: synthesis, properties and their biomedical applications, Biomater. Sci. 3 (2015) 937–954. doi:10.1039/C4BM00448E.
[27]
M. Vert, Y. Doi, K.-H. Hellwich, M. Hess, P. Hodge, P. Kubisa, et al., Terminology for biorelated polymers and applications (IUPAC Recommendations 2012), Pure Appl. Chem. 84 (2012) 337–410. doi:10.1351/PAC-REC-10-12-04.
[28]
F.M. Menger, J.M. Jerkunica, J.C. Johnston, The Water Content of a Micelle Interior. The Fjord vs. Reef Models, J. Am. Chem. Soc. 100 (1978) 4676–4678. doi:10.1021/ja00483a008.
[29]
A.D. MacKerell Jr., Molecular Dynamics Simulation Analysis of a Sodium Dodecyl Sulfate Micelle in Aqueous Solution : Decreased Fluidity of the Micelle Hydrocarbon Interior, J. Phys. Chem. 99 (1995) 1846–1855. doi:10.1021/j100007a011.
[30]
L. Nordstierna, P. V. Yushmanov, I. Furó, Solute-Solvent Contact by Intermolecular Cross-Relaxation. 2. The Water-Micelle Interface and the Micellar Interior, J. Phys. Chem. B. 110 (2006) 25775–25781. doi:10.1021/jp0647885.
[31]
D.K. Smith, Dendritic Gels - Many Arms Make Light Work, Adv. Mater. 18 (2006) 2773–2778. doi:10.1002/adma.200600474.
[32]
G.R. Newkome, G.R. Baker, S. Arai, M.J. Saunders, P.S. Russo, K.J. Theriot, et al., Cascade molecules. Part 6. Synthesis and Characterization of Two-Directional Cascade Molecules and Formation of Aqueous Gels, J. Am. Chem. Soc. 112 (1990) 8458–8465. doi:10.1021/ja00179a034.
[33]
A.R. Hirst, D.K. Smith, M.C. Feiters, H.P.M. Geurts, Two-Component Dendritic Gel: Effect of Stereochemistry on the Supramolecular Chiral Assembly, Chem. - A Eur. J. 10 (2004) 5901–5910. doi:10.1002/chem.200400502.
[34]
J.J.D. de Jong, N. Lucas, Linda, R.M. Kellogg, J.H. van Esch, B.L. Feringa, Reversible Optical Transcription of Supramolecular Chirality into Molecular Chirality, Science. 304 (2004) 278–281. doi:10.1126/science.1095353.
[35]
D.K. Smith, Lost in translation? Chirality effects in the self-assembly of nanostructured gel-phase materials, Chem. Soc. Rev. 38 (2009) 684–694. doi:10.1039/b800409a.
[36]
A.R. Hirst, B. Escuder, J.F. Miravet, D.K. Smith, High-Tech Applications of Self-Assembling Supramolecular Nanostructured Gel-Phase Materials: From Regenerative Medicine to Electronic Devices, Angew. Chemie Int. Ed. 47 (2008) 8002–8018. doi:10.1002/anie.200800022.
[37]
J. Boekhoven, M. Koot, T.A. Wezendonk, R. Eelkema, J.H. van Esch, A Self-Assembled Delivery Platform with Post-production Tunable Release Rate, J. Am. Chem. Soc. 134 (2012) 12908–12911. doi:10.1021/ja3051876.
AC C
EP
TE D
M AN U
SC
RI PT
[23]
11
ACCEPTED MANUSCRIPT H. Namazi, M. Adeli, Dendrimers of citric acid and poly (ethylene glycol) as the new drug-delivery agents, Biomaterials. 26 (2005) 1175–1183. doi:10.1016/j.biomaterials.2004.04.014.
[39]
L. Qin, F. Xie, P. Duan, M. Liu, A Peptide Dendron-Based Shrinkable Metallo-Hydrogel for Charged Species Separation and Stepwise Release of Drugs., Chem. - A Eur. J. 20 (2014) 15419–15425. doi:10.1002/chem.201404035.
[40]
I.L.M. Isa, A. Srivastava, D. Tiernan, P. Owens, P. Rooney, P. Dockery, et al., Hyaluronic Acid Based Hydrogels Attenuate Inflammatory Receptors and Neurotrophins in Interleukin-1β Induced Inflammation Model of Nucleus Pulposus Cells, Biomacromolecules. 16 (2015) 1714–1725. doi:10.1021/acs.biomac.5b00168.
[41]
L. Degoricija, P.N. Bansal, S.H.M. Söntjens, N.S. Joshi, M. Takahashi, B. Snyder, et al., Hydrogels for Osteochondral Repair Based on Photocrosslinkable Carbamate Dendrimers, Biomacromolecules. 9 (2008) 2863–2872. doi:10.1021/bm800658x.
[42]
B.L. Ibey, H.T. Beier, R.M. Rounds, G.L. Coté, V.K. Yadavalli, M. V. Pishko, Competitive Binding Assay for Glucose Based on Glycodendrimer-Fluorophore Conjugates, Anal. Chem. 77 (2005) 7039–7046. doi:10.1021/ac0507901.
[43]
A. Weeks, D. Luensmann, A. Boone, L. Jones, H. Sheardown, Hyaluronic acid as an internal wetting agent in model DMAA/TRIS contact lenses, J. Biomater. Appl. 27 (2012) 423–432. doi:10.1177/0885328211410999.
[44]
S.H.M. Söntjens, D.L. Nettles, M.A. Carnahan, L.A. Setton, M.W. Grinstaff, Biodendrimer-Based Hydrogel Scaffolds for Cartilage Tissue Repair, Biomacromolecules. 7 (2006) 310–316. doi:10.1021/bm050663e.
[45]
Y. Wang, Q. Zhao, H. Zhang, S. Yang, X. Jia, A Novel Poly(amido amine)-Dendrimer-Based Hydrogel as a Mimic for the Extracellular Matrix, Adv. Mater. 26 (2014) 4163–4167. doi:10.1002/adma.201400323.
[46]
L. Qin, P. Duan, F. Xie, L. Zhang, M. Liu, A metal ion triggered shrinkable supramolecular hydrogel and controlled release by an amphiphilic peptide dendron, Chem. Commun. 49 (2013) 10823– 10825. doi:10.1039/c3cc47004k.
[47]
C. Kojima, T. Suehiro, T. Tada, Y. Sakamoto, T. Waku, N. Tanaka, Preparation of heat-induced artificial collagen gels based on collagen-mimetic dendrimers, Soft Matter. 7 (2011) 8991–8997. doi:10.1039/c1sm06157g.
[48]
H. Namazi, M. Adeli, Novel linear-globular thermoreversible hydrogel ABA type copolymers from dendritic citric acid as the A blocks and poly(ethyleneglycol) as the B block, Eur. Polym. J. 39 (2003) 1491–1500. doi:10.1016/S0014-3057(02)00385-3.
[49]
B. Unal, R.C. Hedden, pH-dependent swelling of hydrogels containing highly branched polyamine macromonomers, Polymer (Guildf). 50 (2009) 905–912. doi:10.1016/j.polymer.2008.11.049.
[50]
C. Kojima, S. Tsumura, A. Harada, K. Kono, A Collagen-Mimic Dendrimer Capable of Controlled Release, J. Am. Chem. Soc. 131 (2009) 6052–6053. doi:10.1021/ja809639c.
[51]
C. Kojima, S. Tsumura, A. Harada, K. Kono, A collagen-mimic dendrimer capable of controlled release, J. Am. Chem. Soc. 131 (2009) 10334–10334. doi:10.1021/ja904242j.
[52]
C. Kojima, Y. Toi, A. Harada, K. Kono, Preparation of Poly(ethylene glycol)-Attached Dendrimers Encapsulating Photosensitizers for Application to Photodynamic Therapy, Bioconjug. Chem. 18 (2007) 663–670. doi:10.1021/bc060244u.
[53]
T. Suehiro, T. Tada, T. Waku, N. Tanaka, C. Hongo, S. Yamamoto, et al., Temperature-Dependent Higher Order Structures of the (Pro-Pro-Gly)₁₀-Modified Dendrimer, Biopolymers. 95 (2011) 270–
AC C
EP
TE D
M AN U
SC
RI PT
[38]
12
ACCEPTED MANUSCRIPT 277. doi:10.1002/bip.21576. Y. Shen, S. Zhang, Y. Wan, W. Fu, Z. Li, Hydrogels assembled from star-shaped polypeptides with a dendrimer as the core, Soft Matter. 11 (2015) 2945–2951. doi:10.1039/c5sm00083a.
[55]
Y. Shen, Y. Ma, Z. Li, Facile Synthesis of Dendrimers Combining aza-Michael Addition with Thiol-yne Click Chemistry, J. Polym. Sci. Part A Polym. Chem. 51 (2013) 708–715. doi:10.1002/pola.26429.
[56]
Y. Chang, C. Kim, Synthesis and Photophysical Characterization of Amphiphilic Dendritic–Linear– Dendritic Block Copolymers, J. Polym. Sci. Part A Polym. Chem. 39 (2001) 918–926. doi:10.1002/1099-0518(20010315)39:6<918::AID-POLA1066>3.0.CO;2-P.
[57]
I. Gitsov, K.R. Lambrych, V.A. Remnant, R. Pracitto, Micelles with Highly Branched Nanoporous Interior: Solution Properties and Binding Capabilities of Amphiphilic Copolymers with Linear Dendritic Architecture, J. Polym. Sci. Part A Polym. Chem. 38 (2000) 2711–2727. doi:10.1002/10990518(20000801)38:15<2711::AID-POLA110>3.0.CO;2-1.
[58]
X. Liu, J. Zhou, T. Yu, C. Chen, Q. Cheng, K. Sengupta, et al., Adaptive Amphiphilic Dendrimer-Based Nanoassemblies as Robust and Versatile siRNA Delivery Systems, Angew. Chemie - Int. Ed. 53 (2014) 11822–11827. doi:10.1002/anie.201406764.
[59]
T. Wei, C. Chen, J. Liu, C. Liu, P. Posocco, X. Liu, et al., Anticancer drug nanomicelles formed by selfassembling amphiphilic dendrimer to combat cancer drug resistance, Proc. Natl. Acad. Sci. U. S. A. 112 (2015) 2978–2983. doi:10.1073/pnas.1418494112.
[60]
E. Fedeli, A. Lancelot, J.L. Serrano, P. Calvo, T. Sierra, Self-assembling amphiphilic Janus dendrimers: mesomorphic properties and aggregation in water, New J. Chem. 39 (2015) 1960–1967. doi:10.1039/c4nj02071e.
[61]
X. Xu, H. Yuan, J. Chang, B. He, Z. Gu, Cooperative Hierarchical Self-Assembly of Peptide Dendrimers and Linear Polypeptides into Nanoarchitectures Mimicking Viral Capsids, Angew. Chemie - Int. Ed. 51 (2012) 3130–3133. doi:10.1002/anie.201106080.
[62]
X. Xu, Y. Li, H. Li, R. Liu, M. Sheng, B. He, et al., Smart Nanovehicles Based on pH-Triggered Disassembly of Supramolecular Peptide-Amphiphiles for Efficient Intracellular Drug Delivery, Small. 10 (2014) 1133–1140. doi:10.1002/smll.201301885.
[63]
D.R. Vutukuri, S. Basu, S. Thayumanavan, Dendrimers with Both Polar and Apolar Nanocontainer Characteristics, J. Am. Chem. Soc. 126 (2004) 15636–15637. doi:10.1021/ja0449628.
[64]
S. V. Aathimanikandan, E.N. Savariar, S. Thayumanavan, Temperature-Sensitive Dendritic Micelles, J. Am. Chem. Soc. 127 (2005) 14922–14929. doi:10.1021/ja054542y.
[65]
M.A. Azagarsamy, V. Yesilyurt, S. Thayumanavan, Disassembly of Dendritic Micellar Containers Due to Protein Binding, J. Am. Chem. Soc. 132 (2010) 4550–4551. doi:10.1021/ja100746d.
[66]
V. Yesilyurt, R. Ramireddy, M.A. Azagarsamy, S. Thayumanavan, Accessing Lipophilic Ligands in Dendrimer-Based Amphiphilic Supramolecular Assemblies for Protein-Induced Disassembly, Chem. A Eur. J. 18 (2012) 223–229. doi:10.1002/chem.201102727.
[67]
C. Kojima, T. Suehiro, K. Watanabe, M. Ogawa, A. Fukuhara, E. Nishisaka, et al., Doxorubicinconjugated dendrimer/collagen hybrid gels for metastasis-associated drug delivery systems, Acta Biomater. 9 (2013) 5673–5680. doi:10.1016/j.actbio.2012.11.013.
[68]
C. Kojima, E. Nishisaka, T. Suehiro, K. Watanabe, A. Harada, T. Goto, et al., The synthesis and evaluation of polymer prodrug/collagen hybrid gels for delivery into metastatic cancer cells, Nanomedicine Nanotechnology, Biol. Med. 9 (2013) 767–775. doi:10.1016/j.nano.2013.01.004.
[69]
J. Pan, M. Wen, D. Yin, B. Jiang, D. He, L. Guo, Design and synthesis of novel amphiphilic Janus
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[54]
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ACCEPTED MANUSCRIPT Figure captions Figure 1: Examples of the chemical structures of dendrimers. A) Generations 1-3 of Janus dendrimers with a triazole in the core [60], B) generations 1-2 of Janus dendrimers with aspartic acid as targeting moiety [69], and C) generations 1-3 of PAMAM dendrimers.
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Figure 2: Illustrations showcasing supramolecular structures. A) A structure formed through entanglement of peptide coated G4 poly(amidoamine) (PAMAM) dendrimers. Adapted from Ref [47] with permission of The Royal Society of Chemistry. (http://dx.doi.org/10.1039/C1SM06157G). B) A structure formed through phase separation of esterified amino acid dendrons. Adapted from Ref [25] with permission of John Wiley & Sons; © 2011 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim. (http://dx.doi.org/10.1002/chem.201003049).
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Figure 3: A) A pH dependent release profiles of pralidoxime iodide (PI) and phenol red (PR) from a hydrogel in the presence and absence of metal ions (Mg2+), and B) schematic illustration of the release processes. Reprinted from Ref [39] with permission of John Wiley & Sons; © 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. (http://dx.doi.org/10.1002/chem.201404035).
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Figure 4: A) Drug (and enzyme) release profiles from Janus dendrimer-based hydrogel, and B) activity of the enzyme was maintained even after release from the gel. Reprinted from Ref [24] with permission of John Wiley & Sons; © 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. (http://dx.doi.org/10.1002/chem.201501812). Figure 5: Tumour growth in PC-3 xenograft mice after various treatments: combination of micelles and Hsp27 siRNA slowed tumour growth. Reprinted from Ref [58] with permission of John Wiley & Sons; © 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. (http://dx.doi.org/10.1002/anie.201406764).
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Figure 6: Doxorubicin encapsulated in supramolecular micelles A) enhanced drug effect on tumour volume, B) abolished body weight changes due to doxorubicin, C) increased mice survival during the experiment, D) reduced tumour proliferation, and E) reduced heart toxicity. T. Wei, C. Chen, J. Liu, C. Liu, P. Posocco, X. Liu, Q. Cheng, S. Huo, Z. Liang, M. Fermeglia, S. Pricl, XJ. Liang, P. Rocchi and L. Peng, Anticancer drug nanomicelles formed by self-assembling amphiphilic dendrimer to combat cancer drug resistance, Proc. Natl. Acad. Sci. U. S. A. 112: 2978–83, 2015. Reprinted with permission of PNAS. (http://dx.doi.org/10.1073/pnas.1418494112).
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Figure 7: A) Molecular recognition guided self-assembly and pH-dependent disassembly, and B) confocal laser scanning microscopy tracking of the drug delivery system components, and C) schematic illustration of supramolecular micelle internalization leading to pH-guided disassembly and in-situ drug release within a cell. Reprinted from Ref [62] with permission of John Wiley & Sons; © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. (http://dx.doi.org/10.1002/anie.201106080). Figure 8: Schematic illustration of drug release from supramolecular micelles upon receptor binding of a covalently attached A) hydrophilic and B) hydrophobic ligand. Reprinted from Ref [66] with permission of John Wiley & Sons; © 2012 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim. (http://dx.doi.org/10.1002/chem.201102727). Figure 9: A) Chemical structure of the dendrimer-prodrug, B) comparison of the release profiles of the dendrimer-prodrug and free drug (in gel), and C) in vivo experimental result showing inhibition of metastasis (i) compared to controls (ii-iv). Reprinted from Acta Biomaterialia, Vol 9 (3), C. Kojima, T. Suehiro, K. Watanabe, M. Ogawa, A. Fukuhara, E. Nishisaka, A. Harada, K. Kono, T. Inui and Y. Magata, Doxorubicin-conjugated dendrimer/collagen hybrid gels for metastasis-associated drug delivery systems,
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ACCEPTED MANUSCRIPT Pages No. 5673–5680, Copyright 2012, with permission from Elsevier. (http://dx.doi.org/10.1016/j.actbio.2012.11.013).
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Figure 10: Ratio of drugs released into plasma during in vitro drug release experiment. Adapted with permission from A.L. Acton, C. Fante, B. Flatley, S. Burattini, I.W. Hamley, Z. Wang, F. Greco and W. Hayes, Janus PEG-based dendrimers for use in combination therapy: Controlled multi-drug loading and sequential release, Biomacromolecules. 14(2): 564–574, 2013. Copyright 2013 American Chemical Society. (http://dx.doi.org/10.1021/bm301881h).
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ACCEPTED MANUSCRIPT Table 1: Summary of recent biomedical applications of supramolecular dendrimer-based structures.
Glutamic acid end-capped G4 PAMAM dendrimer covered with 2 kDa collagen peptide Coupling of G1-3 bis-MPA dendrons with and without esterification
Effect Released over period of 3 hours
Reference [24]
Hydrogel formed upon hydration
Released in 6-8 hours, release from G1 was pH dependent
[38,48]
Helical tubules forming metal ion shrinkable hydrogel degrading at neutral pH
Negatively charged drugs released during shrinkage and positive drugs during degradation of the gel
[25,39,46]
Collagen gel embedding the dendrimer-prodrugs
Injection to tumour site inhibits cancer cell line metastasis in mice
[67]
Plitidepsin (encapsulated)
Spherical aggregates using oil-in-water method: particle with 50 nm diameter, vesicles with 9-12 nm bilayers, and elongated micelles Micellar aggregates complexing siRNA; micelles encapsulating doxorubicin
PAMAM type dendrimers coupled to a hydrophobic part bearing two alkyl chains
siRNA; doxorubicin
Silsesquioxane cored G2 poly(L-lysine) dendrimers co-assembling with glutamic acid terminated poly(l-leucine)
Doxorubicin (encapsulated)
Dendrimers with substituted biaryl as the dendritic repeating unit
Pyrene / Nile red as model cargo
A range of amphiphilic Janus dendrimers from coupling of dendrons modified with peripheral acidic amino acids as targeting moieties and dendrons ester bond to naproxen Generations (first and second) of PEGbased dendrons were first coupled with 2 model compounds to get four dendrons, and then the dendrons were coupled to get four dendrimers carrying both drugs
Naproxen
Molecular drug carrier for targeted delivery
Benzyl alcohol (BA) and 3-phenylpropionic acid (PPA) attached to the dendrimers via 2 different linkers (carbonate and ester)
Molecular drug carrier for combinational therapy
Varied concentrations of the drug in solutions confirmed using HPLC and gravimetry
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Supramolecular nanoparticles 250-300 nm in diameter in aqueous environment Biotin / dinitrophenyl bearing micelles
[60]
Delivery of siRNA slowing tumour growth in PC-3 xenograft mice; doxorubicin resistance mediated by efflux pumps overcome Enhanced internalization of doxorubicin; comparable cytotoxic effects with doxorubicin hydrochloride Release of cargo at binding onto extravidin / rat anti-DNP immunoglobulin G antibody Hydroxyapatite targeted drug carrier enhanced naproxen solubility and was non-cytotoxic for HEK293 cell line.
[58,59]
Sequential release, BA being released faster than PPA from all the dendrons
[70]
[61,62]
[63–66]
[69]
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Esterified L-glutamic acid dendron
Structure Supramolecular hydrogels upon ethanol injection
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PEG with G1-3 Citric acid dendrons
Compounds Nadolol; gonadorelin; horseradish peroxidase (entrapped) 5-aminosalicylic acid, diclofenac, mefenamic acid, and pyridine complexed with dendrimers Thiamine; pralidoxime and phenol red simultaneously (entrapped with heating) Doxorubicin (covalently attached)
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Dendrimer Three G3 dendritic bis-MPA and (azidomethyl)benzene-based dendrimers
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