The application advances of dendrimers in biomedical field

Dendrimers are a family of nano‐sized three‐dimensional polymers with unique dendritic branching structures and compact spherical geometries. In recent years, dendrimers have made a series of breakthroughs in the biomedical field. In this review, we introduce the synthesis principles, modification methods, and new materials designed based on dendrimers; discuss the importance of cytotoxicity of dendrimers for applications; and elaborate on their applications in the field of molecular assembly and cancer diagnosis and treatment. We speculate that in the near future, more new materials based on dendrimers will be applied in the biomedical field.

F I G U R E 1 (A) Two principle synthetic methods for constructing dendritic macromolecules (dendrons) Reprinted with permission. [9]opyright 2001 Elsevier.(B) Synthesis of polyamidoamine (PAMAM) dendrimers Reprinted with permission. [10]Copyright 2022 Indian Chemical Society.(C) Dimensionally scaled comparison of a series of PAMAM dendrimers with a variety of proteins, a typical lipid-bilayer membrane, and DNA.[11] Copyright 2001 Elsevier and 2005 Elsevier.nucleus of ammonia, known as "starburst dendrimers." The dendrimers consist of a single "wedge" or dendron that radiates from the centre, with each layer of concentric branching units forming a complete generation (G) in the dendrimer family, identified by a specific generation number. [5]This branching structure provides controlled increases in the molecular weight, size and number of surface groups of the dendrimer.
The unique structure and nano-size of dendrimers render their properties different from traditional polymers, facilitating their application in nano-medical research.Researchers have found that dendrimers can be used as drug and gene transport or carrier systems.Meanwhile, some dendrimers have antibacterial, fungal, [6] and cytotoxic properties and have certain medicinal value.However, further research found that dendrimers often have high cytotoxicity, which is not conducive to their application in the biomedical field.In this research, we first introduce the synthesis principles of dendrimers and discuss cytotoxicity principles and methods of reducing cytotoxicity.Second, we introduce a type of dendrimer that is harmless to cells and its current application.Next, we highlight the applications of dendrimers in molecular assembly and cancer therapy.Finally, new materials based on dendrimer design are introduced (Scheme 1).

Synthesis methods and size of dendrimers
Over the past decades, several synthetic strategies have been developed to generate multiple dendrimer families with a variety of chemical compositions, which have been widely used in various fields, including chemistry, biology, and medicine.Dendrimers are usually created using two methods.The first method is the divergence method [3] in which the core location is where the dendritic unit formation begins (root).This method assembles monomer modules into radial and branched motifs according to some dendritic rules and principles. [7]The first rule is divergent synthesis, which consists mainly of building branched polymers by increasing the branching units.The advantage of divergent synthesis is that the number and length of the branches can be controlled according to the need to obtain materials with special properties.In particular, the general steps of divergent synthesis include the synthesis of the core unit, synthesis of the S C H E M E 1 Schematic illustration of this review.The synthesis of dendrimers and their applications are included in this review.first layer of branches, increase in the number of branches and modification of dendrimers.Dendrimers synthesized by the divergent method have highly branched and highly crosslinked properties, which can introduce a large number of reactive functional groups into the polymer and have good controllability. [7]The second method is convergence, [8] which begins with the molecules that will eventually make up the surface of the branch unit (that is, from the leaves of the molecular tree) and reaches the root reaction core inward.Achieving the dendrimer structure calls for the formation of a single reaction branch unit first, followed by the reactions of numerous branch units with the multifunctional core (Figure 1B). [9,10]The second method is convergent synthesis, which involves the functionalization and polymerization of molecular centres.The advantage of convergent synthesis is that the highly branched and crosslinked polymers can be synthesized, thus obtaining materials with special properties.The general steps of synthesis by the convergent method include functionalization of the central molecule, reaction of an additional monomer and modification of the dendrimer.According to the polymer structure, reaction conditions and application requirements, the synthesis of dendrimers involves a variety of synthetic chemical methods.In addition to the divergence and convergence methods, the step polymerization method to construct polymers by step polymerization and the surface modification method to change the surface properties by introducing chemical functional groups on the surface of dendrimers have been widely used for dendrimer synthesis. [9]endrimers have attracted more attention than linear or branched polymers because the stepwise growth of dendrimers makes the products almost monodisperse, while the polymerization process of linear or branched polymers involves chain growth, and the products are statistical mixtures.Therefore, by controlling the details of the core, interior and periphery, it is possible to design dendrimers with near-perfect structures and compositions.Such macromolecules can be used as synthetic analogues of peptides or polynucleotides, and their ability to accurately regulate structural details can change their physical, chemical, biological, or rheological properties.They are ideal molecules for simulating natural proteins. [11]Dendrimers have many families whose basic characteristics are determined by their generation.Tomalia et al. analyzed the size distribution of PAMAM dendrimers in detail using computer simulation and compared it with that of other biological macromolecules.They believed that PAMAM was suitable to be used as a mimic of protein. [9,12,13]Many of the member molecules in the PAMAM polymer family are very similar in morphology and size dimensions to some common protein structures or biological assemblies (Figure 1C).For example, G3, G4 and G5 amino-terminated PAMAM are highly similar in morphology and size to insulin (approximately 3 nm), cytochrome C (approximately 4 nm), and haemoglobin (approximately 5.5 nm).The sizes of the G5 generation and G6 generation PAMAM are the same as the thickness of the biofilm phospholipid bilayer.A histone (approximately 11 × 8 nm) and DNA double helix (approximately 2.4 nm wide) could form a very stable complex to store genetic information in the nucleus of organisms, while stable complexes with similar morphology and size to the former could be formed between PAMAM of the G7-G10 generation and DNA. [14,15]The synthesized higher-order dendrimers (G13 generation, approximately 30.2 nm) can be used to mimic the capsid protein of cowpea chlorotic mottle virus (CCMV), thus providing a reference for studying the behaviour of the virus capsid protein. [16]

Application of dendrimers in biomedicine
At present, various dendrimer families have been synthesized, which have potential applications in the fields of biomedicine and pharmacy.Some typical structures are shown in Figure 2 13) silanesiloxane dendrimers.9]

Application of dendrimers in the field of molecular assembly
[22] Many advances have also been made in recent years in the co-assembly of dendrimers and macromolecules, with dendrimers proven to assist in the controlled assembly and release of biological macromolecules.Liu et al. [23,24] utilized the electrostatic induction of protein assembly by the G5 generation PAMAM and found that each spherical PAMAM could simultaneously interact with two stable protein-1 (SP1) proteins to form SP1 composite nanowires of 1 μm length through endto-end connection (Figure 3A), which had good stability.They further covalently linked the porphyrin molecule to the surface of the PAMAM and introduced a selenocysteine residue onto the SP1 protein to construct a selenocysteine SP1.Utilizing the two new molecules and the new protein for assembly, a nano enzyme with multiple catalytic centres was successfully constructed, which can simul-taneously exhibit the catalytic activities of glutathione peroxidase and superoxide dismutase.
Kostiainen et al. achieved a series of results in the coassembly research of dendrimers with viruses or proteins.In 2010, they reported a study on the control of virus assembly and release by dendrimer units. [26]In this study, they did not use the complete dendrimer but used one of the branched units, a dendrimer, whose terminal was connected to the spermine chain with an ultraviolet-sensitive o-nitrobenzyl ester (positively charged under physiological conditions).The increase of dendron algebra could significantly improve its interaction and binding strength with the capsid protein of CCMV so that the capsid proteins formed tightly arranged assemblies.However, after ultraviolet light irradiation, the o-nitrobenzyl ester bond broke, and the surface of the dendritic unit dendron formed a negatively charged carboxyl group that was the same as and mutually exclusive of the electric property of the CCMV capsid protein such that the tightly arranged assemblies would disintegrate along with it, and the controlled release of CCMV would be achieved.In 2013, they synthesized an amphiphilic dendrimer [27] containing both hydrophilic and hydrophobic units.The amphiphilic dendrimer was bonded to the surface of CCMV particles by electrostatic interaction and co-assembled into a crystalline complex with a lattice constant (α = 42 nm).The results of smallangle X-ray scattering and frozen electron microscopy showed that the complex had a face-centred cubic structure and a highly ordered structure, indicating that the amphiphilic dendrimer could be used to generate inclusion bodies that mimic the nanostructures.In 2015, they used a series of systematic PAMAM dendrimers (from G2 to G7 generations) and transferrin (Apoferritin, aFT) to assemble a binary eutectic structure. [28]The crystal structure and lattice constant depend on the algebra of the dendrimers; that is, the lattice size of the eutectic can be adjusted by adding different generations of PAMAM.The same PAMAM in the assembly environment with different ionic strengths can control the assembly of aFT into two different crystal symmetries: a face-centred cubic lattice and a closely packed hexagonal lattice.The structure and stability of soft-particle co-crystals are important for applications that require highly controllable structures, such as protein-based mesoporous materials, nanoporous materials, and metamaterials.Their experimental results and proposed concepts open up new dimensions for the regulation of the structural properties of biologically relevant soft substances (such as crystal symmetry, lattice parameters, and selective functionalization) and provide a systematic insight into the crystal co-assembly of protein-polymer nanoparticles.
Setaro et al. [25] designed and synthesized negatively charged phthalocyanine (Pc) dendrimers that can be used Structure of typical dendrimers.Reprinted with permission. [17]Copyright 2015 American Chemical Society.

F I G U R E 3
Assembly of viruses or proteins by dendrimers.(A) Strategy for the assembly and optically-triggered disassembly of a hierarchical cowpea chlorotic mottle virus (CCMV)-dendron complex.Reprinted with permission. [23]Copyright 2015 American Chemical Society.(B) Cricoid SP1-based self-assembly induced by a G5 polyamidoamine (PAMAM) dendrimer.Reprinted with permission. [25]opyright 2015 American Chemical Society.
as photosensitizers to activate molecular oxygen into singlet oxygen, which is one of the main reactive species in photodynamic therapy.The amount of negative charge on the Pc surface depends on the generation of the dendrimer, while Pc polymerization generation can be regulated with the appropriate selection of the Pc metal centre and its axial substitution.These two parameters can determine the result and efficiency of the templated self-assembly process through which the viral protein can form 18 nm viral-like particles around these dendritic chromophores (Figure 3B).In this way, protein-dendrimer biohybrid nanoparticles with potential therapeutic potential are obtained, and this biohybrid assembly may play a crucial role in future nanomedical and nanotechnology applications.

Application of dendrimers in cancer diagnosis and treatment
Key features that support dendrimers as potential drug carriers include their excellent cell uptake, high density of multiple functional groups (e.g.amines, hydroxyl groups and carboxylic acids), versatility, and ability to contain or conjugate a high proportion of higher relative molecular weight drugs. [29,30]First, the dendrimers can act as single-molecule micelles, capable of encapsulating and solubilizing the drug into the voids within the dendrimers.Second, if the drug is carried by dendrimers larger than 5 nm, the size of the carrier will exceed the threshold for renal filtration; hence, the kidneys cannot filter it out of the blood, and the drug will survive in the blood for a longer time.Third, tumour tissue has an enhanced permeability and retention effect.Therefore, the combination of chemotherapeutic drugs with dendrimer carriers will lead to the passive targeting of tumour tissues. [12]Additionally, the surface-modifiable functional groups of dendrimers can improve their biocompatibility and change the surface electrical properties through surface modifications, such as acetylation, polyethylene glycol (PEG) and glycosylation. [9,31]][34][35][36] As shown in Table 1, several common dendrimers can be used as drug carriers to carry a variety of anticancer drugs to achieve cancer treatment. [34]lycerol and succinic acid-based dendrimers can be used to deliver 10-hydroxycamptothecin to various cell lines. [37]Cellular uptake of the dendrimer/camptothecin conjugate was increased 16-fold compared to the free drug, and cell retention increased from 35% to 50% after 32 h. [38]imilarly, the triazine dendrimer-methotrexate complex showed good effects in vivo.Liver damage was quantified by measuring the level of alanine aminotransferase 48 h after an intraperitoneal injection of the dendrimermethotrexate complex or methotrexate solution (2 mg methotrexate/kg).The results showed that the alanine transaminase levels were decreased by 27% in mice receiving the dendrimer-methotrexate complex, indicating low toxic side effects caused by the encapsulated drug. [39]oxorubicin (DOX) was covalently coupled to the G3 polyester-PEG dendrimer via an acyl bond to improve TA B L E 1 Anticancer drugs delivered using dendrimers.Reprinted with permission. [34]Copyright 2016 Elsevier.Adriamycin, Dimeethoxycurcumin and Tamoxifen PAMAM its bioavailability. [40]The construct contained eight PEG chains and 16 pH-sensitive acyl linker sites.In vitro experiments showed that the dendrimer-DOX complex was 10-fold less toxic to colon cancer cells (CT-26) than the free drug due to slow cellular absorption and a gradual drug release.After a single intravenous injection of the dendrimer-DOX complex (20 mg DOX/kg) in subcutaneous tumour-bearing mice, a 60-day observation showed complete tumour regression with a survival rate of 100%.In addition, the dendrimer-DOX complex showed similar antitumor activity to Doxil, a clinically approved liposome doxorubicin complex, and the dendrimer-DOX complex could be stored under desiccate conditions, making it a more stable drug.

Anticancer drug
Similarly, dendrimers can be used as delivery vehicles for nucleic acid molecules, such as siRNA and plasmid.Among them, PAMAM dendrimers are the most widely used.They usually end with a primary amine group; hence, they have a positive charge at physiological pH (7.4) and easily bind to negatively charged nucleic acids.Dong et al. [38] established a novel targeted siRNA delivery system based on the amphiphilic dendrimer AD equipped with the targeting peptide E16G6RGDK.The constructed nanoparticles were small and stable and could protect the small-interfering RNA (siRNA) from degradation (Figure 4A).In addition, the targeting moiety RGDK can bind to integrin and neuroporin-1 receptors on the surface of PC-3 prostate cancer cells, resulting in specific and enhanced siRNA cell uptake, followed by efficient siRNA endosome escape that further promotes gene silencing and produces more potent anticancer activity in castration-resistant prostate cancer models than nontargeting systems.Furthermore, the targeted delivery system caused no in vitro and in vivo toxicities and inflammation, indicating that the system can safely deliver the functional siRNA and cause gene silencing and anticancer activity.Chen et al. [41] used amphiphilic dendrimers to deliver siRNA to immune cells, such as Natural Killer cells, CD4+ T cells, B cells and macrophages, and this delivery pathway was superior to the electroporation technology, thus paving a new way for research on the function and treatment of the immune system.Immunotherapy for tumours is also a very popular research frontier.
Cheng et al. [42] used the dendrimer modified using the allylamine/zinc (II) dimer complex to bind protein through ion and coordination interactions.The best polymer carrier can effectively deliver 30 proteins and peptides into the cytoplasm while still maintaining the biological activity of the protein after intracellular release.For example, after HeLa cells were treated with the toxic protein saporin/dendrimer complex, the cells showed significant apoptosis and cell death, while the free saporin at the same concentration was non-toxic to HeLa cells.They also reported an amphiphilic dendrimer with a fluoroalkyl tail. [39]Fluorolipids are bonded to the core site of the PAMAM in the cysteamine nucleus through disulfide bonds, while phenylboronic acid is modified on the surface of the dendrimer to bind with an effective protein.The polymer has high protein binding capacity and stability and can efficiently release the carrier protein in response to glutathione.The polymer can effectively deliver the toxic saporin protein to 4T1 breast cancer cells and inhibit the growth of tumours in vivo after intravenous injection (Figure 4B).Moscariello et al. [43] constructed a protein drug carrier that could cross the blood-brain barrier by transcytosis using the hybrid material of the PAMAM dendrimer and streptavidin protein.The carrier can conveniently load biotin-modified protein drugs using a self-assembly method, can effectively enter brain glial cells and nerve cells after crossing a blood-brain barrier, does not affect the integrity of the blood-brain barrier, and shows good brain targeting effects in in vivo and in vitro experiments.
Beyond therapy, the unique properties of dendrimers, such as their monodispersity, modifiable surface functional groups and internal cavities, make them ideal vehicles for targeted delivery of therapeutic and diagnostic agents.The dendrimers can be used as diagnostic aids in a variety of molecular imaging applications, such as magnetic resonance imaging (MRI) and computed tomography (CT) angiography.In cancer treatment, imaging Applications of dendrimers and dendrons in cancer diagnosis and therapy.(A) Targeted delivery of siRNA by an E16G6RGDK-amphiphilic dendrimer.Reprinted with permission. [38]Copyright 2022 American Chemical Society.(B) Tumor growth inhibition by fluoroalkyl polyamidoamine (PAMAM) loaded with saporins.Reprinted with permission. [39]Copyright 2020 American Chemical Society.(C) Dendrimers loaded with computed tomography (CT) contrast agent for imaging of pathological tissues.Reprinted with permission. [40]Copyright 2009 National Academy of Sciences of the USA.
technology is used to diagnose and identify the location of the lesions, and the lesions are treated after identification.CT and MRI are mature imaging methods in cancer diagnosis [44] ; MRI is a non-invasive technology for the diagnosis of soft tissue tumours.Dendrimers can be used to deliver various MRI agents.For example, Langereis et al. [45] reported PPI dendrimers ending in Gd-DTPA having a size of 5-6 nm as MRI agents.Talanov et al. [46] reported a PAMAM-based nanoprobe with dual MRI and fluorescence modes.The PAMAM is covalently bonded to the Gd(III)-DTPA chelate and the near-infrared (NIR) fluorescent dye Cy5.5 unit to form a bifunctional MRI-FI reagent.Zhu et al. [47] prepared hexamer Mn(II) dendrimers containing six tyrosine-derived [Mn(EDTA)(H2O)]2 moieties coupled to a cyclic triphosphazene nucleus as MRI contrast agents.The contrast agents currently in use are cleared primarily by the kidneys; hence, prolonged exposure to contrast agents in humans with impaired renal function can lead to serious side effects.Mn(II)-containing dendrimers are potential substitutes for Gd-based contrast agents.
The dendrimers are also used to load different types of CT contrast agents, and the formed CT contrast agents have nano-sizes, which can overcome the disadvantages of small molecule iodinated contrast agents and can be used for CT imaging of different biological systems. [48]ositron emission tomography and single-photon emission CT are the most commonly used imaging techniques in nuclear medicine imaging, and effective imaging usually requires radionuclides or radiopharmaceutical-based contrast agents. [49]Small molecule radionuclide complexes or reagents can be rapidly eliminated or metabolized from organs or cannot be sufficiently accumulated in target tissues (such as tumours), which can be improved with the addition of nanoparticles or macromolecular carriers.Some dendrimer scaffolds have been developed to label various radionuclides to produce contrastenhanced and specific imaging of specific diseased tissues (Figure 4C). [40]In addition, Liu et al. combined the dendrimer and gold nano-ions to form nano-probes, which have good biocompatibility for targeted CT/MR imaging of tumours. [50]

Application of dendrimers in gene therapy
Dendrimers can serve as multifunctional carriers to deliver therapeutic genes into the patient's body.In addition to small drug particles, dendrimers are widely used for DNA delivery.For example, PAMAM dendrimers with terminal amino groups can interact with the phosphate groups of nucleic acids.Therefore, PAMAM dendrimers have been used as genetic material carriers. [51]PAMAM dendrimers and polycations can form complexes with DNA through sequence-independent electrostatic interactions, where the anionic phosphate groups of DNA interact with the amino groups on the cationic surface.These opposite charges neutralize each other, resulting in changes in physical, chemical and biological properties.54] Tai et al. developed a novel gene delivery carrier based on PAMAM dendrimers, called Pamam-peg-EpDT3 NP, for the targeted delivery of long noncoding RNA MEG3 plasmid to castration-resistant prostate cancer (CRPC) cells.The carrier exhibited strong targeting and anti-CRPC activity both in vitro and in vivo, showing potential as a gene therapy drug. [55]owever, the ability to deliver specific parts of DNA molecules to the desired locations within cells still faces many challenges.To maintain the activity of DNA during the dehydration process, researchers have encapsulated dendrimer/DNA complexes within water-soluble polymers and deposited them onto functional polymer membranes with rapid degradation rates to mediate gene transfection.Based on this approach, PAMAM/DNA complexes have been used to encapsulate functional biodegradable polymer membranes for matrix-mediated gene delivery.Studies have shown that rapidly degradable functional polymers have great potential in local transfection. [56,57]n recent years, siRNA has shown great potential as a novel therapeutic agent in the field of gene therapy, but its application requires convenient delivery systems.Dendrimers, as a special class of synthetic macromolecules, have emerged as an exciting delivery platform due to their unique dendritic structure and controllability at the nanoscale.The use of dendrimer nanocarriers for delivering emerging nucleic acid drugs based on RNA interference (RNAi) has become a hot topic.The delivery of RNAi therapeutic drugs should not only be efficient but also targeted to the correct sites to achieve high efficacy and low toxicity.Dendrimers can also be used for gene editing.For example, dendrimers can serve as carriers for the CRISPR/Cas9 gene editing system, delivering it to target cells for precise gene editing. [58]astly, dendrimers can be employed in genetic diagnostics.Dendrimers can be used to prepare probes with multiple fluorescent labels for the simultaneous detection of multiple genes.Additionally, dendrimers can be utilized to fabricate gene chips for large-scale gene screening and diagnostics.Kim et al. designed fluorescent dendrimer nanoprobes with various organic dyes and fluorophores for targeted biomolecule labelling, significantly enhancing the fluorescence stability, probe brightness and localization precision of high-resolution fluorescence imaging. [59]

Cytotoxicity of polyphenylene dendrimers and their applications
Polyphenylene dendrimers (PPDs) are a class of dendrimers with special structures.[62] In recent years, polarity-based studies have generated a series of PPDs with different structures and functions, which broke the inherent impression of their strong hydrophobicity, making them applicable to fields such as transition metal catalysis, nanodrug carriers, and volatile organic compound sensing. [60] large number of different functional groups can be introduced to modify the core, framework, and molecular surface of PPD.Due to the rigidity of the PPD structure, the functional groups can maintain a relatively accurate position, [63] which provides the possibility to accurately control and study its function (Figure 5A).
Hammer, Brenton et al. [64] published an interesting article on the interaction with biological media by performing different chemical modifications on the surfaces of polyphenylene dendrimers to better understand which surface groups determine the properties of these macromolecules.The authors selected 1,3,6,8-tetraethylpyrene as the core, which could emit blue fluorescence to serve as a fluorescent marker to monitor cell uptake, while the ethyl group provided a four-armed dendrimer with a spherical shape.They provided a general method for synthesizing dendrimers by modifying various polar and non-polar groups in different proportions on the surface of the dendrimers.Changing the non-polar group on the surface of the PPD from propyl to other groups (hexyl, phenyl, isobutyl and isopropyl) does not influence the performance of the PPD.In another study of the importance of the balance between polar and non-polar groups on the surface of dendrimers for cell viability, the authors conducted a 72 h cytotoxicity study of PPD on HLC cells at concentrations of 5×10 −6 M. All PPDs with 1:1 and 2:1 ratios of polar to non-polar groups were found to be almost non-cytotoxic, and each dendrimer sample had approximately 100% cell viability.This result suggests that PPD is harmless to cells (Figure 5B).
Because PPD has several benzene rings, it is less relevant to biological applications (cell uptake, nano-drug carriers, gene delivery, etc.).][67][68] PPD has been introduced into a series of biological application scenarios by modifying these materials through various methods.For example, the surface functionalization of G1 and G2 PPD with amine groups and their subsequent coupling to poly(L-lysine) Cterminal activated carboxylic acid groups [69,70] enhances the water solubility of the materials by binding polypeptides to the surface of the dendrimer and promotes their biocompatibility. [71]he incorporation of polar groups on the PPD surface provides an effective way to adjust the solubility and F I G U R E 5 (A) General synthesis of polyphenylene dendrimers (PPDs) with "patched" surfaces with various chemical modifications.(B) Toxicity studies on HLC cells for dendrimers with a 1:1 and 2:1 ratio of polar to non-polar groups.Reprinted with permission. [64]Copyright 2017 Wiley-VCH.

F I G U R E 6 Polyphenylene dendrimers and their application. (A) Synthesis strategies of polyphenylene dendrimers (PPDs)
. Reprinted with permission. [60]Copyright 2015 Royal Society of Chemistry.(B) Photo-tuned opening and closing of PPDs.Reprinted with permission. [74]opyright 2011 American Chemical Society (C) PPD-based drug carriers.Reprinted with permission. [18]Copyright 2014 WILEY-VCH.functionality of the dendrimers.By performing a "posterior" atom transfer radical polymerization (ATRP) on the PPD surface, a water-soluble PPD similar to the surface functionalized organic nanoparticles can be created and controlled by varying the number of amine groups and the degree of polymerization during the ATRP reaction.Mullen et al. [72,73] found that PPD could be used as a staining agent for extracellular matrix in animal tissues at physiological pH, and the introduction of cationic substances on the surface of PPD enabled it to effectively cross the cell membrane.This method has been demonstrated to be very valuable for the synthesis of water-soluble core-shell dendrimers.
Alternate polar (sulfonate) and non-polar (n-propyl) groups may also be introduced into the periphery of the PPD for surface modification purposes.Mullen et al. synthesized G1-G3 generation PPD with eight (G1 generation) to 32 (G3 generation) sulfonic acid groups and an n-propyl group on the surface. [68,71]This configuration results in a non-polar dendrimer having polar plaques at the periphery, and the number, order, position, and distance of the functional groups can be determined by the synthetic modification of organic structural units.Furthermore, the shape-persistent nature of the PPD provides stable nanophase separation between the patterned polar and non-polar portions, thereby creating attractive and repulsive forces with the solvent molecules.Thus, such a periphery imparts a unique surface polarity to the PPD, resulting in unprecedented solubility in solvents ranging from toluene to water.
Adenovirus type 5 (Ad5) is a familiar nucleic acid or drug carrier, and its practical application is limited by the protein cap formed in the human body and the related toxicity.Wu, Wagner et al. innovatively adopted the "corona" composed of amphiphilic PPD to bind to the facade of Ad5, and this unique structure effectively prevented endogenous blood coagulation factor X from binding to the virus surface because PPD masked the binding sites on the virus surface.Furthermore, PPD also affected the binding and adsorption of Ad5 to other proteins in the serum.PPD regulates the distribution of Ad5 in organisms; in particular, it reduces its distribution in the liver and increases its distribution in the heart.Notably, the corona structure formed also affected the specific interaction of Ad5 with the cellular coxsackie-adenovirus receptor. [75,76]Wagner et al. used a series of PPD-coated liposomal nanocarriers with different surface characteristics to explore the effects of the amphiphilic surface, surface charge and shape durability on the adsorption of serum proteins and the formation of protein caps and change and affect the fate and orientation of liposomal nanocarriers in a biological fluid environment by controlling the façade characteristics of PPDs.They demonstrated that the surface charge and hydrophobicity of PPDs played an important function in the formation of the liposome protein cap, effectively reducing the adsorption of opsonin and the complement protein, thus inhibiting the cell uptake caused by the related immune response. [77]These results suggest that researchers can control the biological characteristics of nanodrug carriers by modifying the facade of PPD.

1.4
New composite nanomaterials based on dendrimers

Dendrimer-nucleic acid composite nanomaterial
The dendrimer (especially the PAMAM) and the DNA short chain or nanostructure are combined to form a composite material, and the composite material is applied to the fields of molecular diagnosis and cell labelling.Single nucleotide polymorphism (SNP) analysis based on DNA chips is very important for the correlation between genetic variation and the individual phenotype and localization of pathogenic genes. [78]To develop a more suitable surface modification method for oligonucleotide immobilization, Benters et al. [79] applied PAMAM as an intermediate medium for DNA surface immobilization and constructed a DNA chip with a PAMAM linkage system.PAMAM, containing 64 primary amino groups at the periphery, was covalently attached to a silylated glass carrier, followed by modification of the PAMAM with glutaric anhydride and activation with N-hydroxysuccinimide.The PAMAMlinked system not only showed very high efficiency in the immobilization of para-aminated DNA oligomers but also high stability during repeated use and regeneration.DNA chips prepared using the PAMAM surface showed significantly enhanced signal strength when compared to conventionally used triethoxysilane or polylysine slides, thus enabling higher sensitivity for SNP hybridization analysis.Li et al. [80] constructed an Au-PAMAM-DNA nanocomposite probe to detect SNP in the vangl1 gene (Figure 7A).Through the affinity of amino-Au, nano-gold can be stably and dispersedly encapsulated in PAMAM, which can load a large number of nucleic acid detection probes.In the presence of target DNA, hybridization between nucleic acid probes and target DNA would result in a conformational change in the Au-PAMAM-DNA, and then the biotin molecule at the 5′ end would be exposed, such that it could combine with the PPy/streptavidin membrane on the surface of the electrode, and the electron transfer rate on the surface of the electrode would be significantly changed to obtain the signal.
The electrochemical sensor method for detecting gene mutations has the advantages of a simple design and low cost.The ssDNA probe is assembled on the surface of a chemical electrode and can hybridize with a target mutation gene to trigger an electrochemical signal; however, only a single ssDNA is modified on the surface of the electrode, and the high sensitivity requirement cannot be met.Based on the DNA-PAMAM conjugate, Zhu et al. [81] developed a novel, sensitive DNA biosensor based on electrochemical impedance spectroscopy (Figure 7B).The ssDNA to the gold electrode could hybridize with the G4.5 generation PAMAM-targeted DNA complex in solution to anchor the PAMAM with a carboxyl group at the periphery to the surface of the gold electrode.As the G4.5 generation, PAMAM has a high negative charge and spatial resistance, the PAMAM-dsDNA gives rise to greater resistance to electron transfer from the solution to the electrode surface.Therefore, the sensitivity of the method to the detection of target DNA is increased by two orders of magnitude.
In addition, the dendrimer can also be coupled with a nucleic acid aptamer to form a composite multifunctional nanomaterial, which has high affinity and specificity for tumour cell marker molecules, and target cancer F I G U R E 7 Nanocomposites of dendrimer-DNA and their applications.(A) Au-PAMAM-DNA nanocomposite probe for single nucleotide polymorphism (SNP) detection.Reprinted with permission. [80]Copyright 2016 Elsevier.(B) DNA biosensors based on DNA-PAMAM and electrochemical impedance spectroscopy.Reprinted with permission. [81]Copyright 2009 Royal Society of Chemistry.(C) Sgc8c-PAMAM nanocomposite for cancer cell identification.Reprinted with permission. [82]Copyright 2009 WILEY-VCH.(D) Construction of PAMAM-ssDNA oligomers based on click reaction.Reprinted with permission. [83]Copyright 2010 American Chemical Society.cells can be identified by connecting with multiple fluorophore molecules (Figure 7C). [82]However, with the DNA origami structure as the template, the ring polymerization of the PAMAM-DNA conjugate on the DNA origami could construct a more complex dendrimer structure.Liu et al. [83] covalently linked the PAMAM dendrimer to ssDNA through a "click" reaction and then hybridized with the long template DNA to form an oligomer of the dendrimer (Figure 7D).
At present, the application of dendrimer/nucleic acid composite nanomaterials in cancer treatment remains to be developed; however, its initial application in the field of molecular diagnosis has laid the foundation for and provided a reference for therapeutic applications.It is hoped that more biomedical-related functions will be explored in the near future.

Other composites based on dendrimers
Dendrimers have a wide range of applications in the diagnosis and treatment of cancer, particularly in the delivery of drugs, nucleic acids, and proteins as nanocarriers.However, the properties of simple dendrimers are not ideal, which are usually hindered by the problems of low delivery efficiency and severe cytotoxicity. [84,85]To break these obstacles, it is often necessary to modify dendrimers with various functional ligands to obtain composite nanomaterials.Modified ligands usually include sugars, lipids, amino acids, proteins/polypeptides, polymers, and nanoparticles. [85]Cyclodextrin (CD) is a cyclic oligosac-charide (denoted α-, β-, and γ-CD) formed by connecting six, seven, and eight glucosyl groups through R-1 and four linkages.The internal cavity of CD can encapsulate hydrophobic molecules with various sizes matching, such as cholesterol and cholic acid. [86]CD coupling to dendrimers can improve their affinity for the cell membrane and enhance the solubility, stability, and biocompatibility of cationic dendrimers.Previous studies have shown that α-, β-, and γ-CD coupled with G2 generation PAMAM can significantly improve the transfection of NIH3T3 and RAW264.7 cells. [75]Among them, G2 PAMAM-α-CD is the most effective transfection vector, which is approximately one hundred times more efficient than G2 PAMAM alone or the mixture of G2 PAMAM and α-CD.G2 PAMAMα-CD is better than the commercial transfection reagent Lipofectin in cell transfection.Shah et al. [87] reported that a β-CD-modified PAMAM could simultaneously load siRNA and hydrophobic small molecule retinoic acid and deliver maintenance formic acid and siRNA to neural stem cells and enhance their neuronal differentiation (Figure 8A).Weil et al. [43,88,90] utilized the biotin-streptavidin system or chemical method to connect the PAMAM-dendron with the functional protein to form a complex to expand the application scenarios of the functional protein.The C3bot1 protein (C3) from Clostridium botulinum is an effective and specific Rho inhibitor and a candidate drug for the treatment of many diseases (such as cardiovascular diseases, cancer and nervous system diseases).However, the entry efficiency of the C3 protein into most mammalian cells is extremely low, which significantly limits F I G U R E 8 Dendrimer nanocomposites and their applications.(A) Cyclodextrin (CD)-dendrimer composite materials.Reprinted with permission. [87]Copyright 2013 American Chemical Society.(B) Polyamidoamine (PAMAM)-protein composite materials.Reprinted with permission. [88]Copyright 2016 WILEY-VCH.(C) PAMAM-AuNP composite materials.Reprinted with permission. [89]Copyright 2013 Elsevier.its therapeutic value. [91]They performed supramolecular self-assembly of C3 with PAMAM-dendron-converted protein [88] to form a dendron-converted streptavidin (D3SA)-C3 complex with an AB structure and dendronconverted human serum albumin-C3 complex with an ABC structure, thereby promoting cell uptake of the C3 protein (Figure 8B).They used a biological orthogonal connection method based on a boric acid/salicylic acid hydroxamic acid ester complexation to self-assemble the PAMAM-dendron onto the enzyme through an acidunstable linkage, forming a dendritic supramolecular protecting group with space requirements on the periphery of the enzyme and producing a pH-responsive dendrimerenzyme hybrid similar to a zymogen. [90]This protecting group shields most of the binding sites of the protein in a highly reversible manner and is responsive to changes between pH of 7.4 and 5.0.In addition, the formation of the PAMAM shell gives the enzyme the ability to efficiently transmembrane and localize to intracellular acidic regions, making it stable at neutral pH and released in acidic lysosomes.Proteases (trypsin, papain and DNase I) that can only be activated intracellularly can significantly reduce cell viability.The method for creating the high-efficiency zymogen hybridization provides guidance for the construction of the intelligent protein therapeutic agent.
The dendrimers can also be easily grafted onto the nanoparticles to form multifunctional materials, thus reducing cytotoxicity. [92]The PAMAM is often combined with multi-walled carbon nanotubes, [93] gold nanorods, [94] gold nanoparticles (Figure 8C), [89,95] Fe3O4, [96] MnO nanorods, [97] mesoporous silicon nanoparticles [98] and others to jointly transport siRNA, short hairpin RNA and other functional nucleic acid molecules.The nanogold material has a natural photothermal effect, and the composite material formed by the nanogold material and PAMAM not only improves the gene therapy capability of the loaded molecules but can also realize photothermal therapy under the irradiation of NIR light(Scheme 1).

DISCUSSION
Currently, dendrimers are far from being considered for clinical or hospital applications.Dendrimers face numerous obstacles in transitioning from the laboratory to the clinic.First, biocompatibility is an important consideration for the clinical application of dendrimers.Due to their unique structure and chemical properties, dendrimers may induce toxic and immune reactions.Comprehensive in vitro and in vivo studies are needed to evaluate the interactions between dendrimers and cells, tissues and the immune system to address this issue.Additionally, the development and adoption of standardized evaluation methods and criteria would help ensure consistency and comparability in the assessments.Drug delivery efficiency is another factor limiting the clinical application of dendrimers.The branched structure of dendrimers provides multiple drug loading sites, but the loading and release processes of drugs may be restricted, affecting the delivery efficiency.To address this issue, improvements in the dendrimer design and synthesis methods are needed to enhance drug loading and controlled release efficiency.Moreover, customized dendrimer designs can be pursued for specific drug delivery targets to achieve better delivery outcomes.
Another challenge is the in vivo stability of dendrimers.Within a biological system, dendrimers may undergo degradation, metabolism, or excretion, which can impact their persistence and therapeutic effects.To enhance the in vivo stability of dendrimers, appropriate dendrimer materials and chemical modification strategies can be employed to enhance the stability and resistance to degradation.Furthermore, suitable dendrimer structures and branching densities can be selected to balance the stability and the requirements of drug delivery in different application scenarios.
In addition, bulk production and standardization are also barriers to the clinical application of dendrimers.The synthesis of dendrimers typically involves complex chemical processes, and the production scale is relatively small, lacking standardized production methods.To overcome this problem, more efficient synthesis methods and processes need to be developed to achieve large-scale production and standardization of dendrimers.This may involve establishing standardized operating procedures, implementing quality control testing methods, enforcing rigorous quality control and assurance, and conducting continuous monitoring and evaluation.By ensuring consistency and quality in production, the reliability and feasibility of dendrimers can be improved, thereby promoting their clinical application.
Another important obstacle is the safety assessment of dendrimers.Comprehensive safety assessments must be conducted before applying dendrimers in clinical settings.This involves in vitro and in vivo studies, including evaluations of cell toxicity, immune reactions, biodegradability and other aspects.Additionally, clinical observational studies and long-term monitoring are crucial means of assessing the safety of dendrimers.Establishing standardized assessment methods and criteria and encouraging collaboration and compliance may contribute to reliable and trustworthy safety assessment results for dendrimers.
Finally, market regulation is another key factor influencing the clinical application of dendrimers.Relevant legislation and regulatory requirements, clinical trial procedures, registration and approval processes and market access requirements all need to be considered and complied with.Simplifying approval procedures, providing support and training and promoting collaboration and compliance can help mitigate the impact of market regulation on the clinical application of dendrimers.

CONCLUSION
In this paper, the basic characteristics, synthesis methods, and modification methods of dendrimers are summarized, and their applications in the fields of biomedicine and material science are introduced.Dendrimers have excellent characteristics, including a precisely controllable size and structure, being able to accommodate and protect small molecules, controlled release and biocompatibility.For example, the core, framework and molecular surface of polyphenylene dendrimers can be modified by introducing a large number of different groups.The modification of surface amphiphilic groups can endow them with good water solubility, the ability to interact with biological molecules and good endocytosis ability.Hence, research on biomolecule assembly and drug carriers can be carried out.Therefore, compared with the traditional metal nanoparticles, the synthesis method and the modification method are simple and biocompatible.The in-depth study and continuous improvement of dendrimers may give them great potential in the field of biomedicine.They have previously been developed as nanocarriers for anti-cancer, anti-infectious, anti-inflammatory, and ophthalmic drugs, nucleic acids in gene therapy and MRI imaging agents.Although dendrimers show great potential in gene therapy and other fields, they still face some challenges and difficulties in clinical application, mainly including the following aspects.1) The synthesis process is complex.Dendrimers have a complex structure; the synthesis process requires a high degree of chemical technology, and the synthesis efficiency is difficult to control; hence, the preparation cost is high.
2) The diversity of the structures makes quality control difficult.Dendrimers have a variety of structures and often have multiple reaction sites; hence, it is difficult to control their structure and quality, which also limits their application in the field of medicine.3) There is a lack of standardized preparation and quality control methods.Due to the complex structure of dendrimers, preparation is difficult, and the lack of unified quality control standards results in difficulties in large-scale preparation and clinical applications.In summary, although dendrimers have a high potential in the biomedical field, the above problems need to be solved to better apply dendrimers in the biomedical field and make a greater contribution to human health.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflict of interest.