A Glimpse into Dendrimers Integration in Cancer Imaging and Theranostics

Cancer is a result of abnormal cell proliferation. This pathology is a serious health problem since it is a leading cause of death worldwide. Current anti-cancer therapies rely on surgery, radiation, and chemotherapy. However, these treatments still present major associated problems, namely the absence of specificity. Thus, it is urgent to develop novel therapeutic strategies. Nanoparticles, particularly dendrimers, have been paving their way to the front line of cancer treatment, mostly for drug and gene delivery, diagnosis, and disease monitoring. This is mainly derived from their high versatility, which results from their ability to undergo distinct surface functionalization, leading to improved performance. In recent years, the anticancer and antimetastatic capacities of dendrimers have been discovered, opening new frontiers to dendrimer-based chemotherapeutics. In the present review, we summarize the intrinsic anticancer activity of different dendrimers as well as their use as nanocarriers in cancer diagnostics and treatment.


Introduction
All types of human cells may suffer an abnormal proliferation that can lead to cancer cells. Cancer classification/identification is performed according to the tissue and cell type from which the cancer cells arise. Therefore, there are multiple distinct types of cancer, which can vary significantly in their behavior and response to treatments [1]. This disease is a major public health problem and a leading cause of death worldwide in countries of all income levels.
According to the World Health Organization (WHO), the social and economic impact caused by cancer is increasing. In a 2014 report, an annual cost of EUR 1.04 trillion was estimated for global cancer expenses. The report also declared that it is important to continue investing in care and control, which will prevent a considerable number of deaths for years to come. [2]. Extended lifespan associated with environmental factors (e.g., exposure to pollution, carcinogenic agents, radiation, viruses and bacteria) and the low efficacy of available treatments, closely associated with an increased drug resistance, also contributes to cancer development [3].
Currently, the standard cancer treatments used in clinical settings are radiotherapy, surgery, and conventional chemotherapy [4]. However, these therapies present several drawbacks, such as high toxicity, due to insufficient selectivity and unspecific targeting of cancer cells, which leads to increased resistance to anticancer drugs.
It is therefore relevant to find new anticancer agents able to control tumor growth with minimal side effects. In recent years, the use of nanotechnology in cancer treatment has offered some exciting possibilities, including improvement of detection and elimination of cancer cells before tumor development. This includes the use of dendrimerbased nanotherapeutics as a novel strategy for diagnosis and therapy (theranostics) [5][6][7]. has offered some exciting possibilities, including improvement of detection and elimination of cancer cells before tumor development. This includes the use of dendrimerbased nanotherapeutics as a novel strategy for diagnosis and therapy (theranostics) [5][6][7]. Dendrimers are synthetic 3D polymers with well-defined layered architecture [8]. Owing to their high functionality and loading capacity, as well as their precisely controlled chemical composition and molecular weight, dendrimer-based anticancer therapies offer great advantages over conventional formulations. These include the use of dendrimers as advanced contrast agents (diagnosis) [6], nanocarriers (treatment) [5,9], and theranostic agents (diagnosis, treatment and disease monitoring) [7]. In the quest for new anticancer drugs using dendrimer-based therapies, Shao et al. demonstrated that dendrimers may display innate anticancer activity and anti-metastatic properties without the loading of any therapeutic agent [10]. In this study, poly(acylthiourea) G4 dendrimer (PATU-PEG) demonstrated a higher anticancer activity than Doxil ® (doxorubicin liposomal), a wellestablished chemotherapy drug, and was able to inhibit cell seeding, thus exhibiting a strong decrease in cell metastasis. This study opened new doors for anticancer dendrimer design, targeting effective and safer (with reduced toxicity) anticancer therapies that may be combined with diagnostic features, such as novel molecular imaging technologies. In summary, due to the increase relevance of dendrimers in therapy, this review highlights recent studies regarding the use of these outstanding nanoparticles in cancer theranostics. We also discuss the intracellular pathways associated with their cellular uptake and possible organelle targeting.

Dendrimer Nanoparticles
Multifunctional nanoparticles display great potential for drug and gene delivery, especially for cancer therapy [11,12]. Dendrimers are a class of hyperbranched synthetic polymers with a very low polydispersity, also known as "cascade polymers" [13]. Biologically, dendrimers are highly biocompatible, with a predictable biodistribution and cell-membrane-interacting features mostly determined by their size and surface charge [14]. They were first synthesized in the late 1970s by Tomalia et al., with the desire to mimic a common pattern in nature with vast potential applications [15]. Due to their hyperbranched structure, dendrimers are extremely versatile macromolecules. Their structure can be defined by three main elements: the inner core, repetitive branching units (dendrons), and terminal groups that provide surface tuning ( Figure 1) [16]. Dendrimers are classically obtained by two main approaches: a divergent or a convergent synthesis. In the first methodology, the dendrimer structure is constructed starting from a core molecule. The core reacts with monomers containing one reactive Dendrimers are classically obtained by two main approaches: a divergent or a convergent synthesis. In the first methodology, the dendrimer structure is constructed starting from a core molecule. The core reacts with monomers containing one reactive group and two dormant groups originating the first generation (G1) dendrimer. Then, the periphery of the G1 dendrimer may be activated for reaction with more monomers forming the second-generation dendrimer (G2), and so on. The divergent approach typically originates lower dendrimer generations of open structures and asymmetric shape [16]. In the convergent approach, the individual branches (dendrons) are first synthetized and then attached to a functional core molecule [17]. This methodology minimizes the occurrence of structural problems and facilitates the purification of the product [16]. Later, with the development of "click chemistry" [18], the preparation of dendronized systems became more efficient, requiring minimal purification [19]. She et al. [20], for instance, synthetized G2-poly-L-lysine dendrons that were connected at the core to a heparin sulphate moiety via "click chemistry", and doxorubicin (DOX) was conjugated to the terminal ends using acyl hydrazine. As a consequence of the dendrimer architecture, the number of peripheral groups increases exponentially with generations, which results in nanosized particles suitable for drug loading and release [21]. However, when a critical branched state is reached, dendrimers cannot grow further because of the steric restriction imposed by the increasing branch density. This phenomenon is known as the "starburst effect" and is usually observed in high generations [16].
Since the discovery of these polymers [15], a variety of dendrimers have been developed, polyamidoamine (PAMAM) being the most studied [22]. PAMAM dendrimers are synthetized by the divergent method, mostly using an ethylenediamine core, and are hydrophilic, biocompatible, and non-immunogenic. Polypropylene imine (PPI) dendrimers, along with PAMAM, have been also widely investigated. PPIs are based on a 1,4-diaminobutane core or similar molecules and grow via double Michael addition reactions [13]. Poly-L-lysine (PLL) dendrimers are amino-acid-based polymers [23] that differ from PAMAM and PPI dendrimers in shape, since they are mostly asymmetrical. They have lysines as branching units and amines as terminal groups [22]. Phosphorous-based dendrimers are another interesting and well-studied class of dendrimers [24]. The potential of phosphorous dendrimers has been largely demonstrated, especially as cancer therapeutics. Similarly, carbosilane dendrimers have been explored as antimetastatic agents when complexed with ruthenium derivatives [25].
The different classes of dendrimers and their chemical structures are summarized in Table 1. Table 1. Summary of different classes of dendrimers depicted in above section.

Classes of Dendrimers
Chemical Structure

PAMAM
Ethylenediamine-based core and terminal groups with primary amines PPL Amino acid lysine-base core and branching units PPI 1,4-Diaminobutane-based core and terminal groups with primary amines Phosphorous dendrimers P-Cl-based core, azabisphosphonates are possible terminal groups Carbosilane dendrimers Si-based dendrimer

Dendrimers General Role in Cancer
Dendrimers can be used in a vast number of theranostic applications [26][27][28][29]. It is, therefore, important to be aware of the properties they have to display in order to be employed as biomedical devices. Biocompatibility is crucial to preventing undesirable responses from the host, a property that can only be defined depending on specific applications [29]. In order to prevent bioaccumulation and consequent toxicity, biodegradability is a must. Another important aspect for the development of biomedical devices is their pharmacokinetics, namely their fate in the body after administration [29]. Additionally, the water solubility of dendrimer-drug conjugates enhance the bioavailability of poorly soluble drugs [30]. Lastly, polyvalence, i.e., the ability to support versatile surface functionalization and multiple interactions with biological receptors, is a key property for highly versatile platforms [27].
Dendrimers, as shown in several studies, have a high potential to be used as nanocarriers for both diagnostic and therapeutic approaches [31,32]. Dendrimer-drug interactions might occur in many ways and are dependent on multiple factors such as size, charge, or the chemical nature of the dendrimer/drug. The chemistry behind nanocarriers is the same used in diagnostic or therapeutic schemes, with the agent selected for conjugation being the key player. In general, dendrimers could be used as nanocarriers via two major approaches: loading or conjugation at the surface of the drug and/or target molecule. Encapsulation solves solubility problems indicated by many chemotherapeutics and drugs in general. When a drug is entrapped into the dendrimer's cavity, the polymer works as a dendritic box [33,34]; in this case, the dendrimers can cargo the drug of interest by forming structures that are stabilized via non-covalent interactions. In a different context, dendrimers could also be used as gene vectors, especially cationic dendrimers. When the strategy is dendrimer-drug conjugation, systemic effects can be reduced, increasing the efficacy of cellular targeting. This strategy also improves the half-life of the drug. The conjugate linker is also key to understanding release mechanisms. In many cases, ester and amide conjugate linkers are used that allow enzymatic or hydrolytic cleavage [34,35], easier for esters than amides [36]. Dendrimer-drug conjugation may influence the efficacy of drug itself. Importantly, these nano-polymers can cross cellular barriers by transcellular or paracellular pathways.
To use dendrimers as an alternative diagnostic tool or improve the properties of a contrast agent, it is important to guarantee some criteria. Dendrimers offer many advantages to improve the free delivery of contrast agents or drugs, including high solubility and low polydispersity, which are properties of all dendrimer classes ( Table 2).  [48][49][50] EPR: enhanced permeability and retention, BBB: blood-brain barrier.

Dendrimers Role in Bioimaging and Theranostics
Since late-stage diseases are highly lethal, early detection is vital for successful treatment. Hence, the role of dendrimers in diagnosis is of great value due to their versatility and polyvalence. The most common techniques in molecular imaging used for diagnostic purposes are computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET) using contrast agents (CAs). In addition, contrast agents can also be used in theranostic approaches via molecular imaging such as photodynamic therapy (PDT), photothermal therapy (PTT) or neutron capture therapy (NCT) [51][52][53]. MRI is non-invasive and provides high-quality three-dimensional images without causing radiation damage, thus, it is used to detect solid tumors. In some body tissues, the use of CAs is needed to enhance the intensity of the signal transmitted, improving the quality of the image obtained and the sensitivity of the method. The most used and studied CAs for cancer diagnosis are gadolinium (Gd) chelates. However, CAs have some limitations, such as poor solubility, a low penetration rate, and in some cases, high toxicity. In this sense, nanoparticles and dendrimers have been emerging as good alternatives to overcome these drawbacks. For instance, conjugation of Gd chelates to a dendrimer improved circulation time and specificity in comparison to free Gd chelates [28]. Wiener et al. developed a new class of MRI-CAs with enhanced magnetic resonance properties through the conjugation of a PAMAM G6 dendrimer with a Gd (III)-DTPA by means of thiourea linkage, in which DTPA was a chelator. The authors obtained excellent MRI images of blood vessels and long blood circulation times [6]. In addition, PLL dendrimers were also developed for MRI via conjugation with Gd-DTPA [54].
With the progression of nanomedicine, one major goal has been the development of nanodevices capable of simultaneously combining therapy and diagnosis, allowing real-time monitoring of treatment progress and efficacy. This theranostic approach enables personalized medicine directing the treatment for each patient. This theranostic paradigm implies the acknowledgement that not everyone responds in the same way to a certain drug or treatment protocol [55,56]. Nanoparticles have been proposed and demonstrated as potential nanovehicles for cancer theranostics. Several nanoparticles were developed and approved for this aim, but in some cases they are not enough to overcome pharmacokinetic limitations [57]. Dendrimers, in particular, are one of the most suitable and promising platforms under this strategy, since they can be designed to overcome many of these drawbacks. For diagnostic purposes, the major contribution of dendrimers is in molecular imaging, where they are used as MRI, PET, CT and luminescent imaging agents. PAMAM dendrimers are the most studied since they were the first to be synthetized and are commercially available. They are also used in PDT or PTT techniques as well as agents of NCT.
For bioimaging and theranostics approaches, CAs are, in general, conjugated with dendrimers by the addition of a chelator agent. Dendrimers and dendrons can be conjugated with radionuclides such 18 F for PET [32]. For breast cancer MRI, a Gd-DO3A agent was conjugated with a PAMAM G6 -Cystamine dendrimer, enabling an increased concentrations of Gd-DO3A in the blood circulation [58]. For PTT applications targeting breast cancer, PAMAM G3 and PAMAM G5 were conjugated with Au nanorods@SiO 2 and MoS 2 , respectively [59,60]. For human colorectal carcinoma, PPI dendrimers were reported as MRI contrast agents by conjugation with a DTPA derivative complexed with Gd(III) [60].
Dendrimers functionalized with chelating agents can be readily labeled with radioisotopes. For example, 68 Ga was conjugated with a PAMAM G4 -DOTA with a higher retention of the conjugate in Ehrlich's ascites tumor models in pre-clinical trials [61]. Conjugation with fluorescent probes is also a reliable approach. Folic-acid-functionalized PAMAM G5 conjugated with fluorescein isothiocyanate (FITC) (FI-FA-PAMAM G5 ) were developed for targeting folate-receptor-overexpressing cancer cells. To make this nanoplatform suitable for PET, FI-FA-PAMAM G5 was conjugated with the chelator agent DOTA and 64 Cu ( 64 Cu-DOTA-FI-FA-PAMAM G5 ). The final multifunctional nanoplatform shows promising results regarding diagnosis of FR-overexpressing tumor xenografts [62]. Encapsulation with fluorescent probes is also useful for diagnostic purposes. Increasing specificity with molecular targets at the surface, probes are released from the dendritic box and delivered into the target tissue. For instance, FITC or rhodamine dyes were encapsulated in functionalized (lauroyl and propranolol) PAMAM dendrimers and used for detection of colon and breast cancers [63,64]. Other examples and techniques have been reviewed [39,65].
The conjugation strategy has also been widely explored. For breast cancer MRI, a Gd-DO3A agent was conjugated with a PAMAM G6 -Cystamine dendrimer, enabling an increased concentration of Gd-DO3A in blood circulation [58]. For PDT or PTT applications targeting breast cancer, PAMAM G3 and PAMAM G5 were conjugated with MoS 2 and Au nanorods@SiO 2 , respectively [59,60]. Dendrimers functionalized with chelating groups readily conjugate radioisotopes. For example, 68 Ga was conjugated with a PAMAM G4 -DOTA for breast cancer diagnosis, and this demonstrated a higher retention of the conjugate in the tumor tissue [61]. Table 3 summarizes the use of dendrimers for bioimaging and theranostic applications. Although dendrimers have a key role in diagnostics, there are still some concerns regarding their safety. The main issues are biodistribution and elimination, but toxicity may be relevant in the case of intrinsically cationic dendrimers, as they are dependent on size/generation/M w /charge as shown by us [49] and others [50].
The biological fate of polymers is the sum of many factors and since there is still a gap of knowledge regarding how their physicochemical properties (size, charge, degree of branching, shape, plasma coating) influence their biological fate, radiolabeling is normally required at the different steps of synthesis, as well as extensive studies on different animal models. Nevertheless, for PAMAM dendrimers, a rapid decrease in blood plasma concentration with binding to vascular surfaces and accumulation in the liver, kidneys, and spleen was observed, as well as rapid opsonization and phagocytic clearance by the reticuloendothelial system [66,67]. There is evidence that some dendrimers can still be metabolized to their native constituents, but small alterations by capping or degree of branching can render them inert to biodegradation [68].
Also relevant is the fact that some surface modifications such as preparation of conjugates made with PEG allow for longer blood circulation and renal clearance and lower toxicity [39,69].

Dendrimers as Drug Nanocarriers
The main objective of drug conjugation to a nanocarrier is to improve its efficiency by enhancing aqueous solubility; increase circulation time; stabilize the drug; confer drug release mechanisms; and enable targeted delivery to specific tissues, thus reducing side effects, possibly decreasing drug dosage, and aiding in the passage through biological barriers [26]. In addition, in cancer therapeutics it is very challenging to find adequate treatment that is highly specific for neoplastic tissues. Due to lack of specificity, anticancer drugs are administered systemically, with several side effects [34]. To overcome such problems, several nanoparticles have been used in chemotherapy, such as liposomes, nanotubes, or polymer-drug conjugates. Dendrimers such as PAMAM, PPI, PLL, and polyether have then been also investigated for the delivery of anticancer agents [71,72].
In this context, DOX-dendrimer conjugates were tested for the treatment of lung cancer. DOX-PAMAM G4 dendrimer was evaluated against a lung cancer metastasis model (B16-F10 melanoma cells). The results indicate that DOX-PAMAM G4 accumulated in lungs and the tumor burden decreased [73].
Controlled release in dendrimers is still a challenge, and the design of stimuli-responsive dendrimers is foreseen as a great solution [74]. For example, bortezomib (Btz) is a chemotherapeutic for the treatment of multiple myeloma; however, it was reported that administration of this drug can cause cardiotoxicity and thrombocytopenia [74]. Wang et al. [75] conjugated Btz with a catechol-functionalized PAMAM dendrimer via a boronate ester, which is an acid-labile group. The yielding prodrug was found to be stable at physiological pH and exhibited fast drug release due to tumor extracellular acidity. This "on-off" drug release system can improve therapeutic results, minimizing the side effects of the conventional drugs, possibly by drug conjugation (macromolecular complexes).
Taking advantage of a multifunctional platform, some authors improved molecular targeting by adding extra ligands. For instance, PAMAM dendrimers were conjugated with the antibody trastuzumab (TZ) and DOX (DOX-TZ-PAMAM) to improve specificity against breast cancer cells. The half-maximal inhibitory concentration (IC 50 ) was lower for DOZ-TZ-PAMAM than DOX-PAMAM, indicating the advantages of this strategy [77].
In another interesting approach, paclitaxel (PTX)-biotinylated PAMAM complexes were tested against ovarian cancer cells OVCAR-3 and human embryonic kidney cells HEK293T. The results revealed an increased uptake by cancer cells and a decrease in cytotoxicity for the biotinylated complexes [78].
Quintana et al. used a functionalized dendrimer to target the KB cell line. The nanodevice was composed of a generation 5 PAMAM dendrimer conjugated with folic acid (FA), FITC, and methotrexate (MTX), a chemotherapy agent and immune system suppressant (FA-FTIC-MTX-PAMAM G5 ). The human epithelial carcinoma KB cell line is known to overexpress folate receptors. The results of internalization and KB cell survival indicated an enhanced efficacy for the nanodevice when compared to free MTX [5].
PPI dendrimers are also reported as good nanocarriers for anticancer drugs [79]. These dendrimers are hemolytic, but their toxicity was found to be reduced by FA conjugation. Recently, a complex system of a PPI G5 conjugated with FA and MTX (FA-MTX-PPI G5 ) was developed. The data show a enhanced internalization and higher cytotoxicity towards the MCF-7 breast cancer cell line [80]. PPI dendrimers were also complexed with the antibody mAbK1 and PTX, a system that allowed a significant reduction in cancer activity and displayed a higher therapeutic index [81].
PPL dendrimers, being amphiphilic macromolecules, are often used as gene carriers [82] but since terminal lysines are easily modified, they can also work as nano-vehicles for anticancer drugs. PEGylated PLL containing docetaxel (DTX) is in Phase I clinical trials, being effective against different types of tumors [31]. Cationic PLL dendrimers are also useful for the encapsulation of other anticancer drugs such as 5-fluorouracil [83].
Polyurea (PURE) dendrimers (Figure 2), developed by our group, are another class of dendrimers with great potential for cancer therapeutics. PURE G4 -OMeOx 48 and PURE G4 -OEtOx 48 dendrimers, surface-coated with oligo-oxazoline PEG substitutes, were used as paclitaxel (PTX) nanocarriers. Free PTX had a IC 50 of 0.094 µM after 48 h of incubation against the hepatocellular carcinoma cell line HepG2, a value that was dramatically reduced to 1.23 nM and 1.90 nM after encapsulation into PURE G4 -OEtOx 48 and PURE G4 -OMeOx 48 , respectively [84]. The encapsulation of other anticancer agents into PURE dendrimers was also successfully achieved, as in the case of the nano-in-microparticles developed for inhalation chemotherapy [85].

Dendrimers as Gene Nanocarriers
Therapeutic nucleic acids are designed to trigger or suppress the expre

Dendrimers as Gene Nanocarriers
Therapeutic nucleic acids are designed to trigger or suppress the expression of specific genes that are responsible for the biosynthesis of different proteins [86]. Nucleic acid therapy for cancer treatment has a lot of potential since nucleic acids are highly biocompatible and have high specificity compared to chemotherapies. However, nucleic acids are large hydrophilic molecules that cannot penetrate cell membranes and are vulnerable to enzymatic degradation; therefore, delivery systems are necessary to obtain good therapeutic outcomes [22].
Several distinctive properties of dendrimers make them a better option than other cationic polymers: a high density of positive charges provides multiple attaching sites for (negatively charged) nucleic acid molecules; the complexation of nucleic acids with dendrimers protects them from nuclease degradation [22]; and the abundance of tertiary amines facilitates endosomal disruption though a "proton sponge" effect, with consequent nucleic acids release [22,87].
Xu et al. [88] constructed a targeted delivery system by conjugating FA onto a PAMAM G4 surface to function as a DNA plasmid gene carrier for gene delivery into head and neck cancer cells. FA receptors are overexpressed in cancer cells, and because of that, FA is often used to target these cells [5]. The PAMAM G4 -FA/plasmid polyplexes were taken up by receptor-mediated endocytosis. The authors verified that the conjugate exhibited a high tumor uptake and a highly localized retention in the tumors. By associating an anticancer drug with oligonucleotides, a synergistic effect was obtained [22]. Han et al. [89] combined drug and gene delivery by using a PAMAM dendrimer with encapsulated DOX associated with small interfering RNAs (siRNAs) targeting major vault protein (MVP), which is related to multidrug resistance. siRNAs suppress expression of carcinogenic genes by targeting messenger RNA (mRNA) expression [82]. PAMAM dendrimers were functionalized by a polysaccharide hyaluronic acid (HA) to increase tumor specificity since HA receptors are overexpressed in some types of cancer. For instance, it was observed that codelivery of siRNA and DOX by PAMAM-HA caused an increase in cytotoxicity in MCF-7 cells, since the delivery of siRNA allowed DOX to access the cell nucleus more easily [89].
Our group also prepared PURE G4 -siRNA dendriplexes [90]. In this work, the goal was to develop a tool for gene silencing. The mechanism proposed for siRNA releasing was based on a "proton sponge" mechanism that allowed the dendriplex to escape from the endosome and deliver siRNA to the cell.
Applied to gene therapy, PPI dendrimers were modified with maltose (mal-PPI) and conjugated to single-chain fragment variables (scFvs). This system works as a carrier for siRNAs and improves siRNA delivery in EGFRvIII-positive tumors [91].
The use of dendrimers and polymeric nanoparticle-aptamer bioconjugates can also be very useful for the development of effective delivery systems [92,93]. In this sense, it was found that PEGylated PAMAMs, functionalized with the anti-PSMA aptamer and loaded with the tumor suppressors non-coding miR-15a and miR-16-1, induce cell death of prostate cancer LNCaP and PC3 cell models [94]. Figure 3 and Table 4 summarizes the different strategies to use dendrimers as nanovesicles for drug and gene delivery.

Dendrimers as Intrinsic Anticancer Drugs
As highlighted in the previous sections, dendrimers have a remarkable cap act as therapeutics nanocarriers. Interestingly, a few studies extended the conv role of these NPs to a new paradigm: the investigation of these macromolecules Ester and amide groups are inserted in the surface to allow conjugation. When in the cell, these linkages are both cleaved by hydrolysis or enzymes. Another way is to directly conjugate dendrimers with specific groups such disulfide or thioketal linkers, which are cleaved by glutathione or reactive oxygen species (ROS). (B) Drugs and/or diagnostic agents are encapsulated into the hydrophobic pocket of dendrimers, a more efficient process in the case of hydrophobic compounds. The dendritic box or unimolecular micelle is promoted by guest-host interactions. Multi-molecular micelles are formed when conjugated dendrimers form agglomerates. (C) This is another way to use dendrimers as delivery systems, taking advantage of electrostatic interactions. Nucleic acids interact with the cationic surface groups (e.g, protonated amines) of the dendrimers.

Dendrimers as Intrinsic Anticancer Drugs
As highlighted in the previous sections, dendrimers have a remarkable capacity to act as therapeutics nanocarriers. Interestingly, a few studies extended the conventional role of these NPs to a new paradigm: the investigation of these macromolecules as anticancer agents (Table 5), taking advantage of intrinsic properties introduced by rational design. Xiao Zhang et al. evaluated the therapeutic potential of tryptophane-rich peptide dendrimers (TRPDs) against various tumor cell lines [95]. The results show that TRPDs display a high cytotoxicity towards various tumor cell lines and inhibited the proliferation of tumor cells using a BALB/c mice in vivo model. Another work demonstrated that two different CPP44 peptide-dendrimer conjugates were efficient against acute leukemia [96]. The co-administration of peptide dendrimers, composed of lysine and arginine residues, was shown to induce a significant enhancement of DOX and gemcitabine chemotherapeutic action in a mouse model of pancreatic ductal adenocarcinoma [97]. This work paved the way for the use of dendrimers against tumors with hypovascular and hypopermeable characteristics, including pancreatic ductal adenocarcinoma (a highly lethal and therapeutically resistant cancer).
Other dendrimer classes such as phosphorus and carbosilane dendrimers were shown to potentially act as anticancer agents [24,25]. These dendrimers were tested against several in vitro cancer cell models such as the solid tumor KB cell line, liquid tumor HL-60 cell line (phosphorus dendrimers), and the HeLa, HT-29, MCF-7, and MDA-MB-231 cell lines (carbosilane dendrimers).
Ornithine dendrimers also demonstrated good potential as anticancer drugs. His-and Pro-rich amphiphilic bola-type dendrimers were studied against U87 and T98G glioblastoma cells. Both ornithine-type dendrimers were efficient against both cell lines, showing a cytostatic effect [98].
In addition, Shao et al. demonstrated that dendrimers possess both intrinsic anticancer and antimetastatic properties [10]. In their study, a PEGylated polyacylthiourea (PATU G4 -PEG) dendrimer was investigated. Upon performing tumor growth inhibition studies, these dendrimers, without conventional therapeutic agents in their composition, exhibited potent anticancer activity. One of the major advantages of PATU G4 -PEG is the fact that it presents a low acute and subacute in vivo toxicity, since the major organs of the tested mice did not show significant damages or lesions after treatment and since, after five consecutive days post-treatment, there were no substantial changes in mouse serum chemistry. Using Doxil ® , an established first-line anticancer PEGylated liposome formulation as the positive control, the authors demonstrated that PATU G4 -PEG had a higher anticancer activity in the animal model. As a possible mechanism of action, they proposed that these NPs can sequestrate copper, allowing the inhibition of angiogenesis and cellular proliferation. The authors also reported the inhibition of cell seeding and consequently a strong decrease in cell metastasis by comparing with mice treated with PBS as a control group and mice treated with ammonium tetrathiomolybdate (TM), a strong copper-depleting agent for cancer treatment under clinical trials [10]. Although further studies are needed to vali-date the mechanisms of action, this study hypothesized a central role of dendrimers in cancer therapies, changing the way we look at dendrimers and opening new trends for future research.

Dendrimers Cellular Uptake and Mechanism of Action at Cell Organelle Level
Dendrimers and other nanosized particles naturally accumulate in tumor sites by a possible enhanced permeability and retention (EPR) effect. This happens because the tumor microenvironment is rich in blood vessels due to increased angiogenesis. Using the blood vessels surrounding tumors, the nanoparticles can be driven into the tumors, crossing cellular barriers by transcellular or paracellular pathways [34,79]. The main question at this point is: how does the intracellular transportation of these macromolecules occur? In the case of dendrimers, their entry into target cells via direct penetration or endocytosis pathways has been described [99] (Figure 4). Importantly, the biodistribution and retention of the nanoparticles are the foremost determinants, since their diffusion and adhesion properties significantly depend on size and uptake efficiencies [100]. NPs possess biomimetic features due to their size, which is in the same range of biomolecules such as antibodies, nucleic acids, proteins, and membrane receptors. Cellular uptake, toxicity, targeting, and intracellular trafficking of NPs can be optimized by tuning physicochemical properties of NP such as size, shape, and surface properties [101]. These properties determine their uptake into mammalian cells, normally achieved by endocytosis, a form of active transport that can be classified into two major categories: phagocytosis and pinocytosis [101].
J. Mol. Sci. 2023, 23, x FOR PEER REVIEW 12 o active transport that can be classified into two major categories: phagocytosis and pino tosis [101]. Briefly, phagocytosis occurs in specialized cells, designated phagocytes, and cons of the internalization of large particles such as debris, bacteria or other large-size solu In this process, the target particle is coated with specific molecules (opsonins) that trig its internalization and is then ingested by the cell and compartmentalized to a phagoso (plasma-membrane-derived vesicle). In the intracellular space, the phagosome fuses w the lysosome and the particle is digested at acidic pH. On the other hand, pinocytosis continuous process that consists in the formation of a plasma membrane invagination capture small droplets of extracellular fluid and the molecules dissolved in it, which be, for instance, biomolecules and nutrients. Pinocytosis can be subcategorized into cla rin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis, pending on the aim and specificity of the process. Kitchens et al. studied the cellular uptake of dendrimers. Their work demonstra that cationic and anionic PAMAM dendrimers enter the cells by a endocytosis mechan [102]. Using lysosomal marker protein 1 (LAMP1), the authors observed that PAMAM NH2 and PAMAMG1.5-CO2H dendrimers co-localized at the lysosome level at differ time points depending on dendrimer surface charge. The authors also observed a decre in PAMAMG4-NH2 uptake when the endocytic pathway was blocked using different hibitors (brefeldin A, colchicine, filipin) [103]. In HeLa cells (cervical cancer cells), P MAM dendrimers were shown to be internalized by two major mechanisms: clathrinpendent endocytosis and macropinocytosis [104].
In addition to endocytosis, the uptake of polycationic polymers by cancer cells follow a direct penetration pathway (with or without pore formation). Seungpyo Hong Figure 4. Schematic representation of dendrimer intracellular transportation. In general, dendrimers enter the target cells via direct penetration or endocytosis pathways. At cell level, dendrimers that follow the endocytic pathway are released from endosomes and migrate to lysosomes. Then these macromolecules can be released from lysosomes through a ''proton-sponge" effect as shown for poly(propylene imine) dendrimers. Adapted from [79].
Briefly, phagocytosis occurs in specialized cells, designated phagocytes, and consists of the internalization of large particles such as debris, bacteria or other large-size solutes. In this process, the target particle is coated with specific molecules (opsonins) that trigger its internalization and is then ingested by the cell and compartmentalized to a phagosome (plasma-membrane-derived vesicle). In the intracellular space, the phagosome fuses with the lysosome and the particle is digested at acidic pH. On the other hand, pinocytosis is a continuous process that consists in the formation of a plasma membrane invagination to capture small droplets of extracellular fluid and the molecules dissolved in it, which can be, for instance, biomolecules and nutrients. Pinocytosis can be subcategorized into clathrinmediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis, depending on the aim and specificity of the process.
Kitchens et al. studied the cellular uptake of dendrimers. Their work demonstrated that cationic and anionic PAMAM dendrimers enter the cells by a endocytosis mechanism [102]. Using lysosomal marker protein 1 (LAMP1), the authors observed that PAMAM G2 -NH 2 and PAMAM G1.5 -CO 2 H dendrimers co-localized at the lysosome level at different time points depending on dendrimer surface charge. The authors also observed a decrease in PAMAM G4 -NH 2 uptake when the endocytic pathway was blocked using different inhibitors (brefeldin A, colchicine, filipin) [103]. In HeLa cells (cervical cancer cells), PAMAM dendrimers were shown to be internalized by two major mechanisms: clathrin-dependent endocytosis and macropinocytosis [104].
In addition to endocytosis, the uptake of polycationic polymers by cancer cells can follow a direct penetration pathway (with or without pore formation). Seungpyo Hong et al. reported the interaction of cationic dendrimers with supported lipid bilayers. The main conclusion was that these NPs can enter a cell membrane model by inducing pore formation. Applying atomic force microscopy (AFM), the authors observed that cationic PAMAM dendrimers can form holes/pores in the lipidic membrane and can also remove lipidic molecules from existing membrane defects. The authors also evaluated cell membrane recovery after dendrimer removal and found that hole/pore formation can be reversible [105]. The pore-forming ability of cationic molecules such as dendrimers and peptides can be used to develop formulations for anticancer therapy, as this mechanism of action can potentially lead to cell death [106,107].
Importantly, positively charged surface groups produce a localized charge density that is known to influence the interaction (and consequent toxicity) of dendrimers with cell membranes that possess a relevant content of negatively charged groups (as observed for the plasma membrane of cancer cells) [49]. Cellular permeability is also affected by the dendrimer generation (i.e., size). For instance, PAMAM G2 shows higher permeability rates than PAMAM G4 [108].
Dendrimers can then be designed to target a specific organelle or tissue (with a distinct mechanism of action) and induce, for instance, less side effects to normal/healthy cells. Chemical modifications in the dendrimer core or surface can improve dendrimercell interactions, as illustrated bellow ( Figure 5). Aleksandra Szwed et al. studied the interaction mechanisms of hybrid carbosilane-viologen-phosphorus dendrimers (SMT1 and SMT2) with two different murine cell lines. These dendrimers present two distinct cationic groups (internal and outer) that are specific for mitochondria, inducing perturbations in mitochondrial membrane potential and the formation of reactive oxigen species (ROS) [109].
In another study, the impact and target of PAMAM dendrimer generation (G4, G5, G6) on mitochondria and other cellular organelles such as lysosomes was evaluated in HaCaT (human epidermal keratinocyte cells) and SW480 (primary adenocarcinoma) cell lines [63]. In general, SW4810 cells were more sensitive to these cationic dendrimers than HaCaT cells. After treatment, the production of ROS was higher in SW480 and reached a maximum after 4 h of exposure. These results suggest the specificity of PAMAM dendrimers towards cancer cell mitochondria in comparison to normal cells [110]. The increase in ROS and the expression of apoptotic markers such as BAX and PARP are well reported for the activity of dendrimers used in anticancer strategies [111].
The therapeutic potential of TRPDs in several in vitro models (HepG2, MCF-7 or SKOV3 cells) was evaluated as described above [95]. The results show a good cytotoxic profile against chemoresistance cell lines (MCF-7/ADR and SKOV3/ADR cells), and it was found that the mechanism of action is related to significant supramolecular interactions with DNA through the tryptophan residues [95].

Figure 5.
Schematic representation of potential intracellular cellular organelles (including plasma membrane, lipid droplets, cell nucleus, and mitochondria) targeted by dendrimers used in anti cancer strategies. Cationic surface of cationic modified dendrimers is known to strongly interac with negatively charged lipids present in cancer cell membranes. Cationic dendrimers are also shown to specifically target mitochondria.

Conclusions
In the last decades, many cases of toxicity associated with distinct chemotherapeutics have been reported, such as the case of paclitaxel (PTX), a microtubule stabilizing agen commonly used to treat breast and ovarian cancer. High doses of PTX were found to in duce neutropenia, i.e., low concentration of neutrophils, neuropathy, and loss of hair in patients with metastatic breast cancer. It has also been demonstrated that the anticance agent doxorubicin (DOX) can promote ovarian toxicity effects in cancer patients. In addi tion to high toxicity levels, one of the major problems associated with conventional chem otherapy is drug resistance, either because the initial tumor fails to respond to the treat ment or because it acquires resistance during relapse. Therefore, chemoresistance is a crit ical limitation to the application of chemotherapy and it must be considered when design ing new therapeutics.
To surpass these shortcomings, several types of dendrimers and other nanocarriers have been already reported and are revolutionizing the field of nanomedicine. In this re gard, dendrimers were found to be extraordinary nanocarriers for drug and nucleic acids and their use allowed an increase in therapeutic efficacy. Their use as nanocarriers has many advantages including increased drug solubilization and drug bioavailability and allowing for nucleic acid transportation. In addition, the fact that dendrimers (at least PA MAM dendrimers) can cross the blood-brain barrier makes them extremely appealing fo a variety of related therapeutics, such as the treatment of glioblastomas.
In the last decade, dendrimers have started to be explored as intrinsic anticance drugs. For that, several groups used cationic dendrimers as mimics of cationic anticance peptides. This strategy takes advantage of the enhanced electrostatic interactions between cationic dendrimers and cancer cells. This is due to the cancer cells having negatively charged surfaces arising from lactate overproduction and altered membrane glycosylation patterns. In contrast, healthy mammalian cell membranes are largely zwitterionic. Im portantly, anticancer dendrimers demonstrated the capacity to strongly decrease cell me tastasis.
In a different but complementary scenario, dendrimers are also powerful tools fo the bioimaging of cancer cells, helping medical diagnosis. . Schematic representation of potential intracellular cellular organelles (including plasma membrane, lipid droplets, cell nucleus, and mitochondria) targeted by dendrimers used in anticancer strategies. Cationic surface of cationic modified dendrimers is known to strongly interact with negatively charged lipids present in cancer cell membranes. Cationic dendrimers are also shown to specifically target mitochondria.
One of the main patterns of cancer cells is the dysregulation of lipid metabolism, leading to a high content of lipid droplets (LDs). LDs are subcellular organelles with nano to micron diameter sizes with the ability to store high amounts of cholesterol or fatty acids, thus allowing cancer cells to avoid cytotoxic processes. The high fatty acid content can also provide an extra source of energy to cancer cells. Importantly, LDs are also associated with proliferation, invasion, metastasis, and chemoresistance processes. Thus, LDs can be viewed as future cancer hallmarks. Intracellular targeting of lipid droplets is also now becoming an imaging tool to monitor cancer cells and the targets of innovative therapeutic approaches [112]. Using "click" chemistry, multivalent niacin-polymeric (including dendrimer) conjugates were shown to target LDs [113]. The production of nitric oxide (NO) following treatment was significantly increased and a reduction in a LD relevant enzyme (diacylglycerol acyltransferase, DGAT2) involved in triglyceride biosynthesis was observed [112]. This strategy can be applied in the future for the treatment of cancer and other diseases that involve the accumulation of triglycerides/LDs.

Conclusions
In the last decades, many cases of toxicity associated with distinct chemotherapeutics have been reported, such as the case of paclitaxel (PTX), a microtubule stabilizing agent commonly used to treat breast and ovarian cancer. High doses of PTX were found to induce neutropenia, i.e., low concentration of neutrophils, neuropathy, and loss of hair in patients with metastatic breast cancer. It has also been demonstrated that the anticancer agent doxorubicin (DOX) can promote ovarian toxicity effects in cancer patients. In addition to high toxicity levels, one of the major problems associated with conventional chemotherapy is drug resistance, either because the initial tumor fails to respond to the treatment or because it acquires resistance during relapse. Therefore, chemoresistance is a critical limitation to the application of chemotherapy and it must be considered when designing new therapeutics.
To surpass these shortcomings, several types of dendrimers and other nanocarriers have been already reported and are revolutionizing the field of nanomedicine. In this regard, dendrimers were found to be extraordinary nanocarriers for drug and nucleic acids, and their use allowed an increase in therapeutic efficacy. Their use as nanocarriers has many advantages including increased drug solubilization and drug bioavailability and allowing for nucleic acid transportation. In addition, the fact that dendrimers (at least PAMAM dendrimers) can cross the blood-brain barrier makes them extremely appealing for a variety of related therapeutics, such as the treatment of glioblastomas.
In the last decade, dendrimers have started to be explored as intrinsic anticancer drugs. For that, several groups used cationic dendrimers as mimics of cationic anticancer peptides. This strategy takes advantage of the enhanced electrostatic interactions between cationic dendrimers and cancer cells. This is due to the cancer cells having negatively charged surfaces arising from lactate overproduction and altered membrane glycosylation patterns. In contrast, healthy mammalian cell membranes are largely zwitterionic. Importantly, anticancer dendrimers demonstrated the capacity to strongly decrease cell metastasis.
In a different but complementary scenario, dendrimers are also powerful tools for the bioimaging of cancer cells, helping medical diagnosis.
Despite all their potential in cancer treatment, dendrimers can display significant cytotoxicity and trigger hemolysis. In most cases, toxicity is related to the strong cationic characteristics associated with amine-terminated dendrimers, and this was found to be size/charge/M w dependent. High charge and strong interaction with negatively charged cell surfaces can cause destabilization and lysis. Nonetheless, PEGylation has been shown to reduce toxicity and improve pharmacokinetics. Additionally, dendrimer modifications with targeting agents, such as tumor-specific antibodies or folic acid, decrease the number of reactive groups on the surface of dendrimers, thus reducing their toxicity and increasing their specificity.
In summary, dendrimers are now foreseen as outstanding chemotherapeutics, either via nanoformulation (drug nanocarriers, drug nanoconjugates, dendriplexes) or as polymer drugs (innate anticancer and anti-metastatic agents). This versatility is certainly a key driving force for their translation to clinics in the near future.