Dendrimer-nanoparticle conjugates in nanomedicine.

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Dendrimer–nanoparticle conjugates in nanomedicine

Nanomedicine can take advantage of the recent developments in nanobiotechnology research areas for the creation of platforms with superior drug carrier capabilities, selective responsiveness to the environment, unique contrast enhancement profiles and improved accumulation at the disease site. Colloidal inorganic nanoparticles (NPs) have been attracting considerable interest in biomedicine, from drug and gene delivery to imaging, sensing and diagnostics. It is essential to modify the NPs surface to have enhanced biocompatibility and reach multifunctional systems for the in vitro and in vivo applications, especially in delivering drugs locally and recognizing overexpressed biomolecules. This paper describes the rational design for Dendrimer– nanoparticle conjugates elaboration and reviews their state-of-the-art uses as efficient nanomedicine tools. Keywords: dendrimers • dendrons • gold nanoparticles • MRI • optical imaging • quantum dots • superparamagnetic iron oxide • tumor targeting

As one of the newest areas of science, nanoscale science and technology are seen by many as the key technology of the 21st century, which of course raises the question as to what role this technology will play in medicine. An important area of nanoscale science is the development of nanostructured carriers for medical applications. Various colloidal inorganic nanoparticles (NPs) that exhibit unique inherent properties such as fluorescence properties (e.g., semiconductor quantum dots [QDs] [1] up-/down-conversion nanoparticles), [2] magnetic properties (e.g., metal oxide nanoparticles) [3] and plasmonic properties (e.g., noble metallic nanoparticles) [4] have been widely explored, particularly for biological and medical applications. For instance, various types of magnetic nanoparticles have a widespread range of applications such as in MRI [5] , magnetically guided drug/gene delivery [6] , magnetic hyperthermia (MH) and magnetic biosensors [7] ; up-/down-conversion nanoparticles and QDs and their niche in

10.2217/NNM.14.196 © 2015 Future Medicine Ltd

Audrey Parat1, Catalina Bordeianu1, Hanna Dib1, Antonio Garofalo1, Aurélie Walter1, Sylvie Bégin-Colin1 & Delphine Felder-Flesch*,1 Institut de Physique et de Chimie des Matériaux de Strasbourg IPCMS, UMR CNRS-UdS 7504, 23 rue du Loess, BP 43, 67034 STRASBOURG CEDEX 2, France *Author for correspondence: Tel.: +33 388 107 163 Fax: +33 388 107 246 Delphine.Felder@ ipcms.unistra.fr 1

biological and medical imaging [8] ; noble metallic nanoparticles can be used for photothermal therapy [9] and biosensing [10] . Designing nanoparticles for molecular diagnosis and targeted therapy is of the utmost importance. The wish list of such systems is long: they should selectively home in on the cells and organs of the body that are involved in the disease process, specifically targeting their potent healing effects on these cells and organs, while sparing cells not involved in the disease process. They should be completely nontoxic, biodegradable or capable of natural excretion, not be recognized or eliminated by the body’s own immune system before they have reached their target, and not induce any allergic reactions. Ideally, they are generic, in other words, they can be ‘programed’ to combat a wide variety of diseases by docking onto any target structures one chooses and being capable of carrying any medicines. A proper surface coating can stabilize particles and avoid agglomeration, which hence may increase the sensitivity of NPs-

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Review Bordeianu, Dib, Garofalo et al. based sensor. In addition, a proper surface coating enables the nanoparticles in response specifically to biological species and avoids nonspecific interactions with components in the complex matrix. Coating is also an effective manner of preventing the dissolution and release of core materials that may cause toxicity to biological system [11] . Furthermore, the steric hindrance of coating can affect the fate of NPs in biological system, such as cellular uptake and accumulation, circulation and clearance from body [12] . In addition, the surface can affect the maintenance of the intrinsic nanocrystal properties such as fluorescence and magnetic behavior. Moreover, appropriate surface functionality is the perquisite for conjugating biomolecules to NPs for biomedical applications. A dendritic approach as a coating strategy for the design of functional nanoparticles is particularly interesting in the field of cancer diagnostics (Figure 1) . The appeal of such strategy is due to the unique properties of the dendritic structures which can be chemically tuned to reach ideal biodistribution or highly and efficient targeting efficacies. Indeed, dendrimers are macromolecules consisting of multiple perfectly branched monomers and this architecture makes them versatile constructs for the simultaneous presentation of receptor binding ligands and other biologically relevant molecules. Additionally, dendrimers might serve as promising molecular scaffolds containing a number of ligands thereby inducing an apparent increase of ligand concentration and increasing the probability of statistical rebinding. Alternatively, dendrimers may align these ligands and induce multivalency when receptor clustering occurs or is initiated after initial mono

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valent binding. To improve tumor-targeting efficacy and to obtain better in vivo imaging properties, several studies explored the multivalency effect of dendrimers or of a dendritic surface functionalization of nanomaterials [13] . Indeed, due to their conical-like architecture and focal points, dendritic structures are of particular interest as coatings of ultrasmall NPs with very high curvature. Indeed, such cone shapes are expected to improve steric resistance to macromolecules such as proteins while preventing better particle agglomeration by comparison with their linear counterparts [14] . This review provides a glimpse of how various dendrimer-based organic/inorganic hybrid NPs have been designed and used in nanomedicine, with a special emphasis on three families of hard NPs: iron oxides (IO); gold NPs; and quantum dots. Why dendrimers & dendrons? The use of dendrimers or dendritic compounds for biomedical applications is a flourishing area of research, mainly because of their precisely defined structure and composition, and also high tunable surface chemistry [15] . Dendrimers and their elementary unit ‘dendrons,’ possess excellent symmetry in geometry morphology and good controllability on molecular weight both of which profit from their special step-by-step synthetic pattern. Besides, there are various internal cavities inside the dendritic structure and abundant functional groups on the external surface. Moreover, in addition to a controlled multifunctionalization, dendrimers and dendron units allow a versatility of size (according to the generation [G]) and of physicochemical

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Figure 1. Dendronized (dendron-functionalized) nanoparticles as multimodal nanoplatforms.

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properties (hydrophilic and lipophilic) which can be precisely tuned (Figure 2) . The resulting effects on stability (dendrimer effect), pharmacokinetics and biodistribution can then clearly be identified. There is no doubt that dendrimer-based organic/inorganic hybrids represent highly advanced pharmaceutical tools, able to target a specific type of cell or organ, be tracked while doing it and deliver a specific drug in situ. Dendronized (dendron-functionalized) iron oxide nanoparticles Because of their unique magnetic properties [16] , nontoxicity and biodegradability [17] , iron oxide nanoparticles (IONPs) have been increasingly employed in MRI [18] , targeted drug delivery [19] , therapy [20] and cell separation [21] . In all these biological applications their colloidal stability is of utmost importance. It has been shown that the blood half-life, opsonization, biokinetics and biodistribution of IONPs is determined by both the surface chemical nature and the particle size [22] . To this end, dendrimers and dendrons could play a crucial role in the nontoxicity, biocompatibility, stability, reticulo-endothelial system (RES) escape and MRI properties of IONPs [23] . In the field of dendron grafting, various grafting procedures have been described involving bonding through catechol [24] , carboxylate [25] or stronger anchoring groups such as silane [26] or phosphonate [27] . IO NPs are also developed for MH. When exposed to alternating magnetic fields of appropriate amplitude and frequency, these NPs release heat locally (where they are concentrated), which reduces the cancer cells viability. Indeed, MH is shown to enhance the tumor cells sensitivity towards chemoor radio-therapy. Furthermore MH facilitates drug release or acts on cell membranes. However, one of


the main limitations of MH is the low heating power of currently used magnetic NPs, requiring a local injection of large quantities of NPs. There is thus a great challenge for optimizing the heating power of magnetic NPs. The amount of heat generated by NPs is highly dependent on the NPs structural and magnetic properties. First studies have shown that spherical IO NPs with a mean size around 20 nm are suitable for clinical MH. However the current progress in the NPs synthesis allows now the synthesis of NPs with variable and controlled shapes and core-shell structures [28] . Magnetic resonance imaging

Recently, Neoh et al. prepared hyperbranched polyglycerol (HPG)-grafted IONPs (Supplementary Figure 1, available online at: www.futuremedicine.com/doi/full/ 10.2217/NNM.14.196) and evaluated their efficiency as MRI contrast agent in vitro [29] . They employed carboxylic function of 6-hydroxy caproïc acid as anchoring group on the surface of IONPs. The ‘grafting from’ strategy was put in use for this synthesis; the so obtained HPG-grafted magnetic NPs were superparamagnetic with a magnetization saturation (Ms) value of 30 emu g-1 and in vitro studies showed a very low macrophage uptake (Supplementary Figure 1A) . HPG-IONPs confirmed a high stability in both water and phosphate-buffered saline (PBS) under 1.5T which is highly advantageous for their intended applications as MRI contrast agent. The hyperbranched surface structure showing numerous hydroxyl end groups endowed these nano-objects with high protein resistance and flexibility for further modification. Muller et al. also developed a HPG-grafted IO NPs approach, but through a covalent linkage (silane groups) between the NP and the organic coating [30] .

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Review Bordeianu, Dib, Garofalo et al. In vitro relaxivity measurements showed negative MRI contrast enhancement and in vivo studies confirmed rapid renal excretion. Goodson et al. reported highly pH-sensitive MR responses induced by a series of superparamagnetic IONPs (SPIONs) functionalized by three generations (G) of melamine dendrons showing high molar relaxivities and excellent aqueous stability [31] . Cellular uptake in Henrietta Lacks (HeLa) cell cultures were performed (Supplementary Figure 2) and the high spin–spin relaxation rate (R 2*) values (Supplementary Figure 2E) observed were not only consistent with high cellular uptake but also reflected significant SPIONs clustering within the cells as a function of pH (Figure 3) . Indeed, molar relaxivities were found to exhibit great sensitivity to pH at physiologically relevant ionic strengths, with sharp inflections observed at pH values near the pKa of the melamine (polyamine) dendron. Finally, by comparison with other dendrons displaying alternative functionalities (derivatives of nitrilotriacetic acid or poly(1-vinylimidazole)), the authors showed that these pH-sensitive MR responses are highly dependent upon chemical composition of the surface species and thus amenable to modulation through rational design. Multimodal imaging

In 2011 S Begin-Colin, D Felder-Flesch and collaborators [32] investigated the synthesis of small-sized polyethyleneglycol (PEG) dendrons and their graft-

ing through monophosphonate anchor at the surface of IO NPs under optimal conditions according to the nature of the peripheral functional groups. The double objective of the study was to obtain a good colloidal stability in water and osmolar media (with a mean hydrodynamic diameter smaller than 100 nm) and to ensure the possibility of tuning the organic coating characteristics (e.g., morphology, functionalities, physicochemical properties, grafting of fluorescent or targeting molecules). In vitro and in vivo MRI and optical imaging were then demonstrated to be simultaneously possible using such versatile SPIONs covered by an optimized dendritic shell displaying either carboxylate or ammonium groups at their periphery. Indeed, very high enhancement contrast ratio (EHC) values obtained for these dendronized NPs confirmed their high contrast power even at high magnetic field (7T). For instance, on MR transverse relaxation time (T2) weighted image at 7T, EHC values were 15–75% higher than those obtained for Endorem™. Both positive and negative EHC values were observed after injection and could be explained by local differences of concentration: indeed, at lower concentration (in the kidneys and initially in the bladder), the relative hypersignal can be explained by a dominating longitudinal relaxation time (T1) effect, while at higher concentrations (liver, bladder and kidneys later in time), the T2 effect becomes very important and compensates widely for the T1 effect, at the origin of a decreased signal (Supplementary Figure 3A) . There was, however, a T2 saturation effect at very

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high concentrations, for example in the liver, especially at the initial time. No significant liver (fluorescent intensity being equivalent to control), skin and spleen uptake was observed (no RES uptake). A very fast urinary fluorescence indicating urinary elimination was observed (no kidney signal). In contrast, a very fast (no persistent liver uptake at 20 min) and very high fluorescent signal was detected in intestinal tract, indicating hepatobiliary elimination (main elimination pathway; Supplementary Figure 3B ). Observation of fluorescence in feces confirmed this hypothesis. Another PEG-dendronized IONPs system has been developed by Felder-Flesch et al. for magnetooptical detection [33] . A Patent Blue dye (PB VF) was covalently connected to the dendritic coating. In perspectives, such system could be dedicated to sentinel node detection in breast cancer. Lately, Felder-Flesch et al. proposed a new method for the design of biocompatible and bifunctional dendrons for IO NPs [34] : a biphosphonic acid tweezers was used as coupling agent allowing a stronger binding to NPs than the monophosphonate previously reported [35,36] and connected to a PEGylated G0.5 poly(amido)amine (PAMAM) displaying carboxylic acid end groups. Relaxivity measurements at 7T and room temperature presented a high longitudinal relaxivity (135 Mm-1s-1) with an interesting r2 /r1 of 69.5. In vivo a large contrast enhancement was observed after intravenous (IV) injection, reaching -60% in the liver and kidneys excretory cavity. This was confirmed by in vivo optical imaging (Figure 4) . Tumor targeting & drug release

A system combining tumor targeting to pH-responsive properties was developed by Li and coworkers [37] . Targeted anticancer drug delivery was based on folic acid (FA) conjugated to PEG-modified PAMAM dendrimers with Doxorubicin (DOX) and SPIONs (FA-PEG-PAMAM-DOX@SPIONs; Supplementary Figure 4 ). In vitro drug release studies indicated that DOX was mainly dissociated from the conjugates within the tumor cells nuclei or regions nearby, since FA is an efficient targeting molecule. In vivo experiments showed great potential for application in both MRI detection and cancer therapy by virtue of their targeting function, which allowed the anticancer drug to be released directly to the target sites. In another study reporting on IONPs that can specifically target cancer cells overexpressing FA receptors, Baker and coworkers demonstrated a unique functionalization approach which combines a layer-by-layer (LbL) self-assembly with dendrimer chemistry [38] .


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Using an electrostatic LbL technique, IONPs were modified with a bilayer composed of a polyelectrolyte and a G5 PAMAM dendrimer prefunctionalized with FA and fluorescein isothiocyanate (FITC) moieties. The stability of the system has to be improved and both in vitro and in vivo tests to be accomplished. Nevertheless, this approach opens a new avenue for numerous biological sensing and therapeutic applications of IONPs. Indeed, various PAMAM dendrimer-IO NPs conjugates were also employed as nanocarriers of DOX [39] and Streptavidin [40] for delivery applications. Gene delivery

Gene therapy is a technique using foreign nucleic acid (DNA & siRNA) as medicine to repair defective genes which are responsible for genetic disorders. Development of highly efficient nonviral gene-delivery vectors remains a great challenge. Dendronized magnetic IONPs can be employed as magnetoplexes for magnetofection, which is a powerful technology of gene delivery. In magnetofection, nucleic acid drugs are associated with SPIONs to form magnetoplexes which can rapidly concentrate on the target cells with the help of an additional magnetic field. As a result, high-level transgene expression can be achieved with a relatively short incubation time, low DNA dose and a small amount of magnetic NPs (Figure 5) [41] . Prussian blue staining and cellular uptake of Cytochrome 3 (Cy-3) labeled DNA demonstrated that an applied magnetic field could quickly gather G6-PA M A M-SPIONs /DNA /Polyethyleneimine (PEI) magnetoplexes to the surface of target cells and consequently enhanced their cellular uptake (Figure 6) [41] . Recently, Huang et al. prepared covalently linked PAMAM-IONPs conjugates of different PAMAM generation and used them to bind antisense survivin oligonucleotides (asODN) to inhibit tumor cells growth [42] . The investigation was focused on gene transfection efficacy, uptake mechanism and biological effects of the nano-objects (Supplementary Figure 5) with breast (MCF-7 and MDA-MB-435) or liver (HepG2) cancer cell lines. Marked downregulation of the survivin gene and protein together with inhibited cell growth in dose- and time-dependent means were highlighted. Gold dendriplexes Gold nanoparticle (AuNP) also called gold colloid or gold nanocluster are extensively investigated in various fields of research but are of special interest in biological applications thanks to their favorable chemical stability, attractive optical properties and ease of functionalization which provide a versatile platform


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Figure 4. Bioelimination of dendronized iron oxide nanoparticles followed by optical imaging. (A1, B1 & B2) In vivo whole body and (A2 and B3) ex vivo organs optical images of PEGylated PAMAM dendronized IONPs with Alexa 647 performed at (A1) 7 min, (B1) 20 min, (A2) 30 min and (B2 & B3) 24 h postintravenous injection. The yellow square includes ex vivo organ images of control mouse imaged with the same parameters (1 = lungs, 2 = urines, 3 = heart, 4 = spleen, 5 = kidney, 6 = liver, 7 = brain, 8 = digestive tract, 9 = bone sample, 10 = skin sample, 11 = blood sample, 12 = muscle sample). Fine arrows indicate bladder signal, large arrow indicates liver signal. Reproduced with permission from [34] .

for nanobiological assemblies [43] . AuNPs have demonstrated potential in drug delivery [44,45] , diagnostics and imaging [46,47] as sensors [48] or therapeutics [49] . The synthesis of Au NPs has been well-developed over the last century. While a number of strategies were reported for their preparation, the emphasis has been shifted to control the size and shape of these useful materials. As a kind of a template for their preparation, dendrimers could satisfy the requirements of the controllability due to their hyperbranched 3D structure, good monodispersity and nanosized (even smaller) internal cavities. The activity and efficiency of AuNPs in biological applications are primarily influenced by their shape, size, and size distribution, which are strongly dependent on the synthesis methods and preparation procedures [50] . Various macromolecules were used as stabilizers in order to disperse Au NPs in solution among which dendrimers have been increasingly

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used as templates for their preparation since the end of the last century. The unique structural features of dendrimers make them ideal candidates for templatebased Au NPs synthesis due to multiple end groups at the molecular surface of dendrimer and their interior cavities [51,52] . Many examples of dendrimers entrapped (encapsulated) AuNPs (DENPs) and their use in biological applications have been reported to date. Balogh et al. was the first to report the use of PAMAM DENPs as DNA carrier [53] . They also investigated the behavior of gold/PAMAM nanocomposites under various biological conditions. Shi et al. reported a new approach to improve the biocompatibility of Au DENPs by functionalization of the surface [54] . Computed tomography imaging

As one of the important molecular imaging (MI) technologies, CT affords better spatial and density resolu-



tion than other imaging modalities. These advantages become particularly apparent when CT is used to diagnose diseases in the thoracic region, such as lung cancer. To achieve sensitive CT imaging capability, development of suitable CT MI probes is necessary. Shi et al. have shown that Au DENPs have a great potential to be used for CT imaging of cancer cells [55–57] when covalently linked to targeting and imaging ligands such as FA and FITC. In vitro studies showed that FA- and FITC-modified Au DENPs bind specifically to KB (HeLa derivative) cells that overexpress high-affinity folate receptors and are internalized predominantly into lysosomes of target cells within 2 h. A new usage of FA-modified Au DENPs was reported by Shi et al. in 2013 as nanoprobes for in vitro and in vivo targeted CT imaging of human lung adenocarcinoma [58] . The tumor sections were stained with a silver enhancer kit and observed using an optical microscope to verify that the Au DENPs-FA are able to be delivered to the tumor site for targeted CT imaging. No black spots O

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associated with the Au DENPs-FA were observed in the negative control sample (Supplementary Figure 6A) . In contrast, in the sections of the tumors injected with Au DENPs-FA either IP or IV, there were numerous black spots clearly localized in the cytoplasm of the cells, indicating the presence of the Au DENPs-FA at 6 h postinjection (Supplementary Figure 6B & C) . Such targeting efficiency toward FA receptors was also reached with PEGylated Au DENPs [59,60] while in another in vitro study, PAMAM dendrimers functionalized with FITC and RGD peptide Au DENPs showed improved targeting efficacy towards cells overexpressing integrin receptor [61] . Lactobionic acid (LA)-modified Au DENPs have also been reported as efficient probes for targeted CT imaging of human hepatocellular carcinoma (HepG2 cells) through the receptor-mediated active targeting pathway (Supplementary Figure 7) [62] . Finally, a promising approach combining two radiodense elements, gold and iodine, within one single O

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Figure 5. Modification of superparamagnetic iron oxide nanoparticles with poly(amido)amine dendrons for magnetofection. Adapted with permission from [41] .



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Figure 6. Cellular uptake of magnetoplexes. Monkey kidney fibroblast (COS-7) cells transfected with superparamagnetic iron oxide nanoparticles-G6/DNA/PEI ternary magnetoplexes at optimal mass ratio of 2.0 for superparamagnetic iron oxide nanoparticles-G6/DNA and optimal N/P ratio of 10 for polyethyleneimine/DNA in (B & D) the absence or (A & C) presence of a magnetic field. (A & B) Prussian blue staining experiments detect the presence of iron inside the cells. (C & D) Confocal laser scanning microscopy images of Cy3-labeled magnetoplexes (Luciferase as the reporter gene). Reproduced with permission from [41] .

dendrimer-based nanodevice showed impressive x-ray attenuation properties [63,64] . Polymerase chain reaction amplification

PCR has been recognized as a basic technique in modern biology research and clinical medicine. This gene amplification technique can increase the number of copies of target genes by six orders of magnitude. However, it lacks from specificity and efficiency. To overcome this problem, an addition of Au DENPs to the PCR mixture can significantly increase the specificity and efficiency of PCR at their optimal concentrations in the error-prone two-round PCR system [65,66] . It is generally accepted that there are three different mechanisms in the PCR optimization with NPs: the NPs can somehow mimic the function of single-stranded (ss)DNA binding protein which selectively binds to ssDNA rather than double-stranded (ds)DNA and then minimizes the mispairing between the primers

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and the templates in the PCR system; the NPs are able to strongly interact or bind with the DNA template or DNA polymerase, significantly increasing the local concentration of the polymerase or DNA template and leading to improved specificity and efficiency of PCR; NPs with high thermal conductivity in the PCR mixture help increasing the specific annealing of primers with templates, lowering the chances of forming nonspecific or smear products. All three mechanisms may occur simultaneously or individually. Although the related molecular mechanism of the enhancing effect is currently unclear, it can be hypothesized that in some cases, the surface charge-mediated interaction between NPs and PCR components may play an important role in optimizing PCR. To be more specific, the interaction between dendrimers and PCR components is expected to be different after the interiors of dendrimers are occupied by metal NPs because the 3D morphology of dendrimers upon interaction with PCR components can thus be well maintained by opposition to that of dendrimers without metal NPs entrapped. In addition, the entrapped metal NPs may offer additional thermal conductivity to the PCR system, allowing for the involvement of different PCR-enhancing mechanisms. Therefore, utilization of Au DENPs may be much more beneficial than the use of dendrimers without metal NPs or metal NPs in the absence of dendrimers in terms of PCR-enhancing effect. Gene delivery

Au DENPs as nonviral gene delivery vectors were demonstrated to be able to effectively compact plasmid DNA (pDNA) to form polyplexes with a smaller size when compared with dendrimers without AuNPs entrapped [67] . Therewith, Au DENPs enabled enhanced gene delivery with gene transfection efficiency more than 100-times higher than that obtained with the dendrimer without Au NPs entrapped. It is believed that the entrapment of Au NPs within dendrimer templates preserves the dendrimers 3D spherical shape thus enabling high DNA compaction to form smaller particles, and consequently resulting in enhanced gene delivery. FA-modified Au DENPs were also used as nonviral vector for the delivery of pDNA into cancer model cell line (HeLa cells) overexpressing high-affinity FA receptors [68] . In contrast to the control cells that did not show any green fluorescence signals, both Au DENPs and Au DENPs-FA vectors generated green fluorescence signals within the cells. However, Au DENPs-FA vector-transfected HeLa cells show more prominent green fluorescence features thus highlighting the targeting role of the FA grafting onto the Au DENPs surfaces (Supplementary Figure 8) .



Virus inhibition with dendronized gold NPs

petition of fluorescent DiD (1,1’ -Dioctadecyl-3,3,3’,3’ -tetramethylindodicarbocyanine perchlorate)-labeled virus to AuNPs-SO4Na and RBC. RBCs were chosen for this assay because they bind VSV but as they lack endocytic activity are unable to internalize viruses. Miscellaneous

Highly stable positively charged dendron-encapsulated Au NPs (PCD-AuNPs) have been reported by Hackley et al. with the study of their stability in biological media and a wide range of pH values. They have also used the human lung epithelial cell line 665 nm A549 and a monkey kidney Vero cell line to evaluate the dose dependent cell viability [73] . The appearance of large black agglomerates on the cells was noted after 48 h. Gentle pipetting action resulted in the agglomerates waving in the flow current suggesting that they are partially anchored to the surface of the cells. A reduction in the intensity of ruby red color of

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Besides the use of Au DENPs, few publications reported the use of dendronized Au NPs for biomedical applications. Hussain et al. reported the synthesis of poly-l-ysine dendron-DNA AuNPs and studied the release profiles of the surface-tethered DNA in water and cell culture media, together with the effect of ionic strength on dendron-Au NPs and DNA-dendron-Au NP complexes [69,70] . Haag et al. reported the preparation of Au NPs functionalized by sialicacid-coated polyglycerol dendrons [71] and their use in the inhibition of influenza virus infection. More recently, Haag et al. reported a virus inhibition induced by dendronized AuNPs-SO4Na of different sizes (Figure 7) [72] . For the quantification of the size-dependent vesicular stomatitis virus (VSV)-cell binding inhibition by AuNPs-SO4Na, a binding assay was established as shown in Figure 7. The assay was based on a binding com-

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Figure 7. Quantification of the virus–cell binding. 1,1’ -Dioctadecyl-3,3,3’,3’ - tetramethylindodicarbocyanine perchlorate (DiD)-labeled vesicular stomatitis virus (VSV) were preincubated with AuNPs–SO 4Na before human erythrocytes were added. (A) Smaller sized AuNPs–SO 4Na weakly inhibit VSV–erythrocyte binding, resulting in less remaining DiD signal in the supernatant. (B) Larger sized AuNPs–SO 4Na efficiently inhibit the virus–erythrocyte binding, resulting in a high DiD signal due to a larger virus fraction in the supernatant. Adapted with permission from [72] .



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Review Bordeianu, Dib, Garofalo et al. the culture media was also observed, suggesting that a large fraction of the NPs precipitated when cells were present. Quantum dots dendritic nanoboxes Semiconductor nanocrystals, typically referred to as ‘quantum dots’ (QDs) have shown promising applications as nanosensors, light-emitting diodes and bioprobes, especially in cellular imaging and drug delivery [74] . QDs have narrow, symmetric and sizetunable emission spectra, broad absorption spectra, high level of brightness and photostability, large Stokes shift and long fluorescence lifetime [75] . Aqueous stability is the common problem for all types of QDs when they are employed in biological researches, such as in vitro and in vivo imaging. To circumvent this problem, ligand exchange and polymer coating are proven to be effective, besides synthesizing QDs in aqueous solutions directly. Toxicity is another big concern especially for in vivo studies. Ligand protection and core/shell structure can partly solve this problem. Along with various surfactants used as stabilizing molecules in QD syntheses, an assortment of polymers has been employed as well. They include diblock ionomers, double-hydrophilic block copolymers, and block copolymer micelles [76] . Dendrimers developed for semiconductor nanocrystals have been based on thiol as the anchoring groups to the surface cations. In 2002, Wang and coworkers reported the synthesis of core-shell QDs with high relaxivity and photoluminescence [77] . Dendron ligands of different generations (G1–G3), displaying a thiol function at the focal point, are used for stabilizing CdSe and Au nanocrystals. The outer terminal groups of the dendron ligands are amides, carboxylic acids, alcohols, or esters which resemble that of a hydrophilic protein or a sugar. Photochemical, thermal, and chemical stability of CdSe and Au dendron-nanocrystals are exceptionally good in comparison to those of the nanocrystals coated by single-chain thiol ligands. The thin, about 1–2 nm, but closely packed and tangled ligand shell provides sufficient stability for the ‘dendron-protected nanocrystals’ to withstand the rigors of the coupling chemistry and the standard separation/purification techniques. Guo and coworkers described the design of semiconductor box nanocrystals prepared via dendrimers bridging [78,79] . The globally cross-linking of the dendron ligands on the surface of the nanocrystals practically sealed each nanocrystal inside a dendron box, which resulted in very stable box-nanocrystals. The chemistry presented by these authors has been applied for the development of a new generation of biomedical labeling reagents.

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Cell recognition & cellular assays

In 2008, Chen et al. reported galactoside-capped gallamide dendritic anchor via click chemistry with a thiol anchoring group [80–82] . The highly hydrophilic dendron was efficiently anchored onto CdSe/ ZnS core/shell nanoparticles in a covalent fashion and with intact core fluorescent property. Confocal microscopic analyses showed the QD-nanohybrid could be smoothly and selectively uptaken by lung cancer cells enriched with membrane-bound asialoprotein receptors in 2–3 h (Supplementary Figure 9) . The newly designed multivalent nanoprobe is potentially useful for studying the multimeric carbohydrate interactions, understanding endocytic process as well as cell adhesion and recognition. Algarra and coworkers developed a thiol polypropylenimine diaminobutyl (DAB)-dendrimer of G5 coated with fluorescent ZnSe QDs for selective recognition of C-reactive protein (CRP) [83] . The main goal was to obtain a quenching of fluorescence caused by CRP in order to provide a rapid diagnosis of inflammation and infections in patients. By modification of the pH media, the fluorescence emission bands positions changed proving recognition of CRP by the 30 nm QDs sensor. Liu and coworkers reported on hydrophilic dendronized CdSe/ZnS core/shell nanocrystals conjugated to different antibodies [84] . The so obtained bioconjugates were sensitive for the detection of their corresponding targets, Escherichia coli for bacteria and hepatitis B (HBsAg) for viruses. A biosensor system based on a flowing chamber equipped with a microporous immunofilter and the CdSe/ZnS dendron nanocrystals as fluorescent labels was developed (Supplementary Figure 10). The detection limit of this new biosensor system was 500-fold lower than other published biosensor. Zinc oxide QDs are promising alternatives for diagnosis and imaging but their aqueous instability has markedly limited their use. Generations 1 (G1), 2 (G2) and 3 (G3) PAMAM dendrons bearing a siloxane group at the focal point were grafted onto ZnO QDs of 5 nm in size [85] . After dispersion in water, the absorption spectra of ZnO@G1 and ZnO@G2 QDs showed a strong green-yellow emission centered around 550 nm (2.25 eV) after excitation at 365 nm. Luminescent ZnO QDs were successfully used for imaging Gram-positive bacteria Staphylococcus aureus (Supplementary Figure 11) . The ZnO@G2-PAMAM QDs labeling of S. aureus cells varies per individual (shown as a histogram) and was found to have a bell-shaped distribution which may result from physiological variability, and therefore different labeling susceptibility. Upon incubation, positively charged ZnO@G2.



PAMAM QDs may self-assemble on the negatively charged bacteria surfaces. Throughout the observation time (ca. 1 h), only negligible fluorescence fading was observed, thus showing the photostability of the ZnO QDs under prolonged and repeated imaging conditions. Compared to Cd-based QDs, cytotoxicity experiments of ZnO@G2-PAMAM showed that these QDs could be used with concentrations up to 1 mM without altering cell growth. The biocompatibility of these QDs is markedly improved compared with their Cd-based counterparts. Therefore, these QDs should play an important role in a variety of nanocrystal-based biomedical applications in a near future. Molecular imaging

In the last years, the use QDs in biomedical research has grown, however successful examples of clinical applications are still absent due to many clinical concerns. Gao et al. reported on stable and biocompatible dendron-coated InP/ZnS core/shell QD as a clinically translatable nanoprobe for molecular imaging applications [86] . The InP/ZnS core/shell nanocrystals can emit from about 450–750 nm, with a quantum yield as high as 40%. The bright core/shell nanocrystals are stable in air and can be dispersed in water after being coated with dendritic and PEGylated RGD dimers (Supplementary Figure 12) [86] . In vivo and ex vivo fluorescence imaging indicated that the QD-dendron-RGD2 nanoprobe clearly imaged integrin αv β3 -positive tumors (human ovary cancer cells SKOV3) with high specificity (active targeting), while QD-dendron displayed tumor accu-

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mulation likely caused by passive targeting via the enhanced permeation and retention (EPR) effect (Figure 8) . Ex vivo fluorescence imaging further confirmed the obvious fluorescent signal in SKOV3 tumors of mice injected with QD-dendron-RGD2 or QD-dendron at 4 h (Supplementary Figure 13A) . At 24 h, the fluorescent signal in SKOV3 tumors of mice injected with QD-dendron-RGD2 remained high with excellent contrast, whereas there was virtually no fluorescent signal in the tumor of mice injected with QD-dendron (Supplementary Figure 13A) . The results were consistent with in vivo fluorescence imaging. The fluorescent signal in kidneys was extremely high at 4 h, which was consistent with the biodistribution of QD-dendron and consistent with a scenario of renal excretion. The region-of-interest (ROI) signal integration analysis on the ex vivo fluorescence images was then performed to semiquantitatively study the uptake ratio of QDs in each organ. At 4-h postinjection, the ROI analysis showed that the tumor uptakes of QD-dendron-RGD2 and QD-dendron under the same conditions were high, with 19.5 ± 2.2% of injected dose (ID)/g and 20.8 ± 3.5% ID/g, respectively (Supplementary Figure 13B) . By comparison, at 24 h post injection (p.i) the tumor uptakes of QDdendron-RGD2 and QD-dendron were significantly different (p < 0.05) and 7.2 ± 1.5% ID/g and 1.1 ± 0.2% ID/g, respectively (Supplementary Figure 13C) . In summary, QD-dendron and RGD-modified nanoparticles demonstrated small size, high stability, biocompatibility, favorable in vivo pharmacokinetics and successful tumor imaging properties.

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Figure 8. In vivo near-infrared fluorescence imaging. The dorsal images of SKOV3 tumor-bearing (arrows) mice (L: left side; R: right side) injected with (A) QD-dendron-RGD2 (200 pmol) and (B) QD-Dendron (200 pmol) at 0.5, 1, 4, 5, 5.5, 6, 8, 24 and 28 h, respectively [86] .



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Review Bordeianu, Dib, Garofalo et al. Miscellaneous Imaging

Upconversion NP (UCNP) as a new class of imaging agent is gaining prominence because of its unique optical properties. Multihydroxy dendritic UCNPs tailored by a representative fluorescent Rhodamine B dye and displaying enhanced water dispersibility have been recently reported to simultaneously present upconversion and downconversion luminescence in vivo [87] . Tailored biological retention and efficient clearance of PEGylated ultrasmall MnO nanoparticles as positive MRI contrast agents for molecular imaging was also made possible via a dendritic approach [88] . The detection of molecular events in the nanomolar range using T1-weighted MRI sequences requires the design of ultrasmall particles containing hundreds of paramagnetic ions per contrast agent unit. For ultrasmall MnO particles to be applied in the clinics, it is necessary to develop coatings that also enable their efficient excretion within hours. In 2014, Fortin and coworkers [88] demonstrated for the first time the possibility to use MnO particles as T1 vascular contrast agents, while enabling the excretion of >70% of all the Mn injected doses after 48 h. For this, small, biocompatible and highly hydrophilic PEGylated bisphosphonate dendrons (PDns) were grafted on MnO particles to confer colloidal stability, relaxometric performance, and fast excretion capacity. The relaxometric performance of MnO@PDns as ‘positive’ MRI contrast agents was assessed (r1 = 4.4 mM-1.s-1, r2/r1 = 8.6; 1.41 T and 37°C). Mice were injected with 1.21 μg of Mn per kg (22 μmol of Mn per kg), and scanned in MRI up to 48 h. The concentration of Mn in key organs was precisely measured by neutron activation analysis and confirmed, with MRI, the possibility to avoid RES nanoparticle sequestration through the use of phosphonate dendrons. Due to the fast kidney and hepatobiliairy clearance of MnO particles conferred by PDns, MnO nanoparticles can now be considered for promising applications in T1-weighted MRI applications requiring less toxic although highly sensitive ‘positive’ molecular contrast agents. Dual mode MRI and CT imaging was also possible with core-shell Fe3O4 @Au nanoparticles via the combination of LbL self-assembly process and dendrimer chemistry [89] . T2 relaxometry and x-ray attenuation measurements show that the formed Fe3O4 @Au NPs have an r2 relaxivity of 71.55 mM-1 s-1 and enhanced x-ray attenuation property. These properties afford their uses as contrast agent for dual mode MR/CT imaging, which has been demonstrated not only in imaging of cancer cells in vitro, but also in liver imaging via MR and subcutaneous tissue imaging via CT in vivo.

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Conclusion & future perspective Nanoparticles functionalized with dendrimers or dendrons themselves multifunctional lead to suitable properties for in vivo applications in nanomedicine as evidenced by this review. Indeed, over the last decade a combination of imaging modalities and several biocompatible and/or biodegradable dendrimers or dendrimer–nanoparticle conjugates have produced bioimaging probes and nanomedicine tools which have prolonged plasma half-lives, enhanced stability, reduced toxicity and improved target specificity. However, biomedical applications of those nanoobjects are related to their proper characterization and reproducibility such that they can be translated forward to clinical evaluation. Much work remains to be done. The dendrimer–nanoparticles conjugates published so far are in an extremely competitive environment, but often provide sufficiently original synthetic approaches to stand out from all the researches performed in this field. Indeed, for example, the use of metal oxide nanoparticles of sizes below 15 nm, varying in shape (spherical, cubic and core-shell), composition and coated with a thin dendritic layer whose thickness can be finely tuned and adapted to the NP nanometer size is a very innovative approach. The systematic in vitro and in vivo bioassessment (fate, behavior and excretion pathways) of all these dendrimer–nanoparticles conjugates is also a concern, and without being fundamentally innovative in itself, constitutes a fundamental approach that is currently undeveloped, although some groups started this type of approach. Parameters to be evaluated are numerous (core/shell composition, choice of the metal, morphology, size, dendrimers or dendrons’ nature, and so on). Basically it would also be wise to focus on the behavioral aspects of these conjugates in biological fluids and in particular to study their interaction with proteins. Also, other fundamental studies are still needed, for example, the selection of coupling agents allowing strong binding and high grafting rate by preserving both molecules and NPs properties. Moreover, quantitative evaluation of the functions at the surface of NPs will be among the mandatory studies. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.



Review

Executive summary Dendrimers & dendrons • A dendritic approach to in vivo efficient targeting combines several advantages such as increasing sharply the binding ratio of the nanoobject to the target tissue by increasing the number of biological effectors within the same size-controlled nanoobject (multivalent system); allowing a multimodal imaging (MRI, PET and single photon emission computed tomography [SPECT]) through complexation of diverse metallic ions; having favorable biodistribution properties (elimination of the nontargeted complexes by a renal way). • The small size of the dendritic objects is mandatory as it allows envisaging a crossing of the capillary barrier, a urinary elimination and a favorable biodistribution. • Dendrimers with MRI capabilities have been developed; dendritic macromolecules have also been investigated as carriers for therapeutic drugs, gene therapy, and radiation sensitizers.

Dendronized magnetic iron oxides • To be used as MRI contrast agents, magnetic nanoparticles (NPs) should exhibit high-saturation magnetization and be functionalized with molecules favoring water diffusion around and close to the magnetic core and leading to biocompatible stable suspensions in physiological media with an average hydrodynamic size smaller than 50 nm, ensuring a high blood-circulation time. • Iron oxide NPs are also developed for magnetic hyperthermia (MH). When exposed to alternating magnetic fields of appropriate amplitude and frequency, these NPs release heat locally (where they are concentrated), which reduces the cancer cells viability.

Dendrimer-encapsulated gold nanoparticles • The activity and utilization efficiency of AuNPs in material and biological applications are primarily influenced by their shape, size, and size distribution, which are strongly dependent on the synthesis methods and preparation procedures. • As a kind of template for the preparation of gold nanoparticles, dendrimers could satisfy the controllability requirements due to their hyperbranched 3D structure, good monodispersity and nanosized (even smaller) internal cavities.

Dendronized quantum dots • Compared with traditional ultraviolet excitation of QDs, near-infrared (NIR) excitation has many advantages such as being less harmful, showing little blinking effects, no autofluorescence and deep penetration tissue. • Photochemical, thermal, and chemical stability of CdSe dendron-nanocrystals are exceptionally good in comparison to nanocrystals coated by single-chain thiol ligands. The thin, about 1–2 nm, but closely packed and tangled ligand shell provides sufficient stability for the ‘dendron-protected nanocrystals’ to withstand the rigors of the coupling chemistry and the standard separation/purification techniques.

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