Polyester Dendrimers: Smart Carriers for Drug Delivery

Polyester dendrimers have been shown to be outstanding candidates for biomedical applications. Compared to traditional polymeric drug vehicles, these biodegradable dendrimers show excellent advantages especially as drug delivery systems because they are non-toxic. Here, advances on polyester dendrimers as smart carriers for drug delivery applications have been surveyed. Both covalent and non-covalent incorporation of drugs are discussed. Abbreviations: BACPT 7-butyl-10-aminocamptothecin bis-HMPA 2,2-bis(hydroxymethyl)propanoic acid BNCT boron neutron capture therapy COMU


Introduction
The concept of using high molecular weight polymeric systems as potential drug delivery systems was initially introduced by Ringsdorf [1,2] and Kopeček [3,4].In chemotherapy, the use of high molecular weight systems results in enhanced targeting of tumor tissue and improved efficiency of the treatment [5][6][7], an effect explained in part by the enhanced permeability and retention (EPR) phenomenon observed in tumor tissues [8][9][10].The EPR effect is the property by which certain sizes of molecules such as liposomes and macromolecular drugs tend to accumulate in tumor tissues much more than they do in normal tissues [8][9][10][11].A good drug molecule must selectively target and bind the receptor microenvironment to ultimately elicit an appropriate biological response.A large number of newly developed drug molecules however, are rejected by the pharmaceutical industry and will never benefit a patient because of poor bioavailability caused by low water solubility and/or poor cell membrane permeability.In addition, a good number of launched drugs exhibit suboptimal performance for the same reasons.Consequently, efficient ways of enhancing bioavailability and biocompatibility of new drugs are of particular interest.Dendrimers offer a particularly attractive alternative as drug delivery systems as they offer a high drug-loading capacity either by encapsulation or conjugation [12][13][14][15].Encapsulation and conjugation of drugs with dendrimers have shown immense utility for delivery of hydrophobic drugs (enhancing solubility) [16,17], labile drugs (sheltering from harsh surroundings), and small molecule drugs (avoiding fast elimination and exposure to healthy tissues).Interest in the use of dendrimers for drug delivery and medicinal applications has mushroomed in the last few years  and numerous reviews have appeared on dendrimers in drug delivery and biomedical applications [50][51][52][53][54][55].
Certain unique dendritic features clearly set these compounds apart as special and optimum nanoscale carriers in medical applications.These unique features include polyvalency, high degree of branching, high solubility, globular architecture and most importantly, their well-defined architectures (monodispersity) which translate into a more consistent well-defined polymer that brings better reproducibility of results.The success of dendritic nanoparticles for drug delivery applications largely depends on the ability of scientists to design smart carriers with the ability to overcome drug leakage, immunogenicity, cytotoxicity, reticuloendothelial system uptake, and hemolytic toxicity among other shortcomings.One strategy to overcome these shortcomings is to use polyester dendrimers which have been shown to be non-toxic and biocompatible [19,[56][57][58][59][60].By attaching a drug to a suitable carrier, it is possible to enhance its aqueous solubility, increase its circulation half-life, target certain tissues, improve drug transit across biological barriers, and slow drug metabolism [61][62][63][64][65][66].Optimization of these features to maximize drug bioavailability to diseased tissues while minimizing drug exposure to healthy tissues, results in improved therapeutic efficacy.
Polyester dendrimers have been shown to be associated with various novel applications and are particularly promising as drug carriers.The use of biodegradable dendrimers emerged as a strategy to produce desirable large molecular weight carriers that achieve high accumulation and retention in diseased tissues, but allow rapid and safe elimination of non-toxic dendrimer fragments into the urine.

Oral Drug Delivery
Amongst the many routes for drug delivery, the oral route is preferred [18,[67][68][69][70], probably because of patience preference.For many existing and new drugs such as therapeutic peptides, peptidomimetics, oligonucleotides and others, oral bioavailability is in many cases below acceptable levels.To overcome this problem and to guarantee a sufficient high oral uptake, the use of efficient oral drug delivery systems is important.Transport of a dendrimer through the epithelial layer of the gastrointestinal tract depends upon the dendrimer's characteristics.Housing a drug inside a soluble dendrimer host not only solubilizes it but also allows it to bypass using a transporter protein for movement from the intestinal tract into the blood.Often drugs are not compatible with use of the protein transporter system that is designed to pass nutrients.The oral route using dendrimers looks very promising especially with anticancer and antihypertensive drugs [23,[71][72][73][74][75].

Transdermal Drug Delivery
The human skin is a readily accessible surface for drug delivery.Transdermal drug delivery can be used as an alternative route of administration to accommodate patients who cannot tolerate oral dosage forms.It is also of great advantage in patients who are nauseated or unconscious.Drugs that cause gastrointestinal upset can be good candidates for transdermal delivery because this method avoids direct effects on internal organs such as the stomach and intestine.In addition, drugs that are degraded by the enzymes and acids in the gastrointestinal system may also be good targets.However, many new drugs are hydrophobic causing low water-solubility that results in insufficient levels of drug delivered into cells.Water soluble and biocompatible dendritic species are known to improve drug solubility and plasma circulation time via transdermal formulations and to deliver drugs quickly and effectively [72,[76][77][78][79][80][81].In this regard, dendrimers have been shown to be useful as transdermal drug delivery systems for various types of medications [77,80,81], including anticancer, antiviral, nonsteroidal anti-inflammatory and antihypertensive drugs.

Ocular Drug Delivery
Ocular drug delivery has been a major challenge to pharmacologists and drug delivery researchers due to the eye's unique anatomy and physiology [82].The most common route of administration for the treatment of various ocular diseases is the topical application of drugs to the eye.Because of drainage of the excess fluid via the nasolacrimal duct and elimination by tear turnover, the intraocular bioavailability of topically administered drugs is poor.Research advances have shown that the use of drug delivery systems such dendrimers can help to overcome the many disadvantages and complications associated with ocular drug delivery [83,84].

Drug Delivery by Injection
Drug administration by injection encompasses intramuscular (IM), intravenous (IV), and subcutaneous (SC) drug administrations.Medication delivered via injection often acts rapidly and has essentially high bioavailability.Injections are useful for drugs that are poorly absorbed or ineffective when given orally.Injection is also an excellent way to administer drugs to patients who are nauseated or unconscious.However, because the drug is delivered to the site of action extremely rapidly with IV injection, there is a risk of overdose if the dose has been calculated incorrectly, and there is an increased risk of side effects if the drug is administered too rapidly.Numerous reports of IV administered dendrimer-drug complexes have appeared [85][86][87][88][89].For example, 2'-(benzo[1,2-c] 1,2,5oxadiazol-5(6)-yl(N 1 -oxide) methylidene)-1-methoxy methane hydrazide presents antichagasic activity but has low water solubility.Guest-host interactions with a dendrimer result in good drug solubilization.These interactions can be controlled by varying the solution pH, allowing drug deliverance [85].
To achieve positive results in the encapsulation and release of a guest drug, suitable dendrimer-guest partners must be carefully selected.For example, the complexation of opposite charged PEG block copolymers with cationic amino methacrylates or anionic styrene sulfonates has been explored [109].Polymer-drug partners with specific acid-base interactions between hydrophobic drug molecules (R 1 -COOH) and polymer segments (NH 2 -R 2 ) improved the drug loading capacity [110].Hydrogen bonding formation between the guest drug and the host polymer has also been explored [111][112][113][114][115][116].
Another innovative way to deliver a drug conjugated to or adhering to a dendrimer is to further conjugate the dendrimer to aptamers [117][118][119][120].The aptamers can be selected to bind to specific cell types, such as cancer or other disease cells with different cell-surface biomarkers.For example, carboxy-coated PAMAM dendrimers were conjugated to amino groups of the aptamers by forming activated esters from the carboxy groups [117,118,121].Such an approach could easily be adapted to carboxy-coated polyester dendrimers that would have the advantage of having low toxicity and biocompatibility associated with polyester dendrimers.

Attractive Features of Polyester Dendrimers for Drug Delivery Applications
Improving the therapeutic index of drugs is a major incentive for innovation in many therapeutic areas such as cancer, inflammatory and other infectious diseases like HIV.Polyester dendrimers constitute an attractive class of compounds because they are biodegradable and biocompatible [61].In addition, whenever they have been tested, they have been found to have low toxicity [56,122,123], unlike other dendrimers [124]; this is extremely important if these molecules are to be used as frameworks for drug delivery and other biological applications in biological systems.Like other dendrimers in general, polyester dendrimers have interior void spaces which may be used to encapsulate small molecule drugs, metals, or imaging moieties.Not only does encapsulation increase the half-life of the drug due to controlled release, it also reduces the drug toxicity due to lessened drug exposure to healthy tissues en route to the receptor microenvironment or diseased tissues.Because a prolonged therapeutic level can be achieved due to a sustained drug release [125], the frequency of dosing can be reduced which would in turn contribute to improved compliance by the patient.The surface hydroxyl groups of polyester dendrimers are responsible for their high solubility and miscibility and for their high reactivity.These highly reactive surfaces can be modified to optimize bio-distribution, receptor mediated targeting, and/ or controlled release of encapsulated active moieties or attached drugs [57].In addition, polyester dendrimers have labile ester functionalities which are steadily hydrolyzed in vivo to release entrapped or covalently attached drugs.Higher generation polyester dendrimers can easily be prepared with facile synthesis of other dendrimers and a variety of methods have been reported for ester bond formation [126][127][128][129][130][131][132][133][134][135].The synthesis of polyester dendrimers in general [135] and those derived from bis-2,2-hydroxymethylpropanoic acid (bis-HMPA) in particular [58] have been reviewed.A recent attractive strategy is the use of uronium-based coupling agents, TBTU [136], TATU [137], and COMU [138] to promote ester bond formation between carboxylic acid dendrons and polyalcoholic cores [139].This regioselective esterification of primary hydroxyls in the presence of non-protected secondary or tertiary hydroxyls (Grindley-Twibanire esterification) is efficient for the O-6 acylation on carbohydrates and it has recently been applied in the divergent construction of a second-generation mixed polyester dendrimer [139], the preparation of Lyme disease glycolipid antigens [140], and the direct synthesis of maradolipids and other trehalose 6-monoesters and 6,6'-diesters [141].The reduction in synthetic steps means less cost for the final product and in addition, the elimination of protection/deprotection steps leads to minimized chemical wastes, which is important as we move towards a greener chemistry and a greener world.

Advances in Polyester Dendrimers for Drug Delivery Applications
Fréchet and coworkers have contributed tremendously to the area of polyester dendrimers [56,57,96,97,126,[142][143][144][145][146][147].In early 2000, this group prepared and evaluated various dendritic architectures composed of a polyester dendritic scaffold based on the monomer unit bis-HMPA for their suitability as drug carriers both in vitro and in vivo [63].The systems were found to be water soluble and nontoxic.In addition, the potent anticancer drug, doxorubicin (DOX), was covalently bound via a hydrazine linkage to a high molecular weight 3-arm poly(ethylene oxide)-dendrimer hybrid as shown in Figure 1.The highly potent anticancer agent doxorubicin is a fluorescent compound, and this provides a convenient analytical tool for monitoring the biodistribution of the polymer-DOX conjugate.The polymer-DOX conjugate showed no significant accumulation in any vital organ including the liver, heart, and lungs.This is a significantly different distribution pattern than is observed for the free drug, which partitions into a variety of organs such as the liver and heart [148].The polymer-DOX conjugate had a circulation half-life of 72 min, which is significantly longer than the half-life of the free doxorubicin (~8 min).This is an indication that the dendritic form of the carrier favourably enhanced the pharmacokinetics and the biodistribution of the drug.The results of the cytotoxicity of drug-polymer conjugate in vitro suggested that polyester dendrimer-based systems do not exhibit a significant toxic effect and the drug release rates from the hydrazine linker encouraged further evaluation of the model compound as a polymeric drug carrier.In vitro evaluation of the conjugate was performed in various cancer cell lines to compare the cytotoxic activity of the bound drug in relation to the free drug.The three cell lines examined exhibited a range of sensitivity to the free doxorubicin, from 0.025 μg/mL for B16F10 to 0.62 μg/mL for the MDA-MB-435 cell lines.In all three cell lines examined, the free drug was considerably more potent than the drug-polymer conjugate; 6-fold in the B16F10 cells, 50-fold in the MDA-MB-231, and 9-fold in the MDA-MB-435 cells.In summary, the dendrimer-DOX system shows no accumulation in any vital organ examined, including the liver, heart, and lungs.The results suggest that this polyester dendritic backbone is a highly water soluble, nontoxic, and biocompatible polymer [63].
After contradictory reports [63,149], the hydrolysis kinetics of low molecular weight and polymeric doxorubicin hydrazone carboxylates were reinvestigated [150].It is now believed that DOX toxicity was likely not observed due to the specific linkage to DOX, which allowed intramolecular cyclization to produce an inactive version of DOX [150].
The system evaluated here had a molecular weight of 22,550 g/mol prior to functionalization.To further increase the circulation half-life to effectively exploit the EPR phenomenon [8-10], higher molecular weight systems were prepared and evaluated [13,56].The biological evaluation of a library of eight polyester dendrimer-poly(ethylene oxide) (PEO) bow-tie hybrids of the form shown in Figure 2 was described [56].Evaluated polymers were designed to include a range of MWs (from 20,000 to 160,000) and architectures with the number of PEO arms ranging from two to eight.For these bow-tie dendrimers, the number of PEO arms can be adjusted to provide polymers of different architectures by using dendrons of different generations.By tuning the combination of the number of PEO arms and their length, a variety of MWs can be prepared.Accordingly, a small library of eight polymers was prepared where the PEO functionalized dendron was varied from the first to the third generation (two to eight arms).PEO arms with MWs of 5000-20,000 were used to prepare polymers with MWs ranging from 20,000 to 160,000 since one goal here was to prepare long-circulating drug carriers.In order to track the polymers in vivo, some dendrimer hydroxyl groups of each bow-tie polymer were statistically converted to tyramine carbamates as shown in Scheme 1, by activation of the polymers with a limited amount of 4-nitrophenyl chloroformate, followed by excess tyramine [56].After investigating the in vitro cytotoxicity of the polymers, the in vitro biodegradability, the biodistribution, and the biodistribution in tumored mice, the following observations and conclusions were made.In vitro experiments revealed that the polymers were nontoxic to cells and were degraded to lower MW species at pH 7.4 and pH 5.0.Biodistribution studies with 125 I-radiolabeled polymers showed that all carriers with MWs of 40,000 and greater had plasma circulation times in excess of 24 h, while those with lower MWs were cleared more rapidly with significant quantities excreted in the urine.Comparison of the renal clearances for the four-arm versus eight-arm polymers indicated that the more branched polymers were excreted more slowly into the urine, a result attributed to their decreased flexibility.Polymers with "two arms" which have essentially linear architectures were rapidly taken up by the liver.The biodistribution results of two long-circulating high MW polymers in mice bearing subcutaneous B16F10 tumors indicated high levels of tumor accumulation.

Scheme 1. Functionalzation of bow-tie dendrimers for biodistribution studies.
Overall, the attractive features of this type of branched carriers including degradability, lack of toxicity, long circulation half-lives, and high levels of tumor accumulation make them very promising for therapeutic applications [56].In addition, because a long blood circulation time is a prerequisite for tumor targeting using the EPR effect [9,151], it is evident that bow ties with molecular masses >40 kDa are acceptable candidates for passive tumor targeting.Accordingly, this type of systems was further evaluated (in mice with cancerous tumors) as drug delivery systems with the highly potent doxorubicin as the attached drug [57].
For this therapeutic study in mice with cancerous tumors, a 16 bow tie with a molecular mass of 45 kDa was used because it is more branched and contains less PEO per dendron than the other prepared bow ties [57].Highly branched bow ties exhibit good steric protection of their payloads, whereas bow ties containing just enough PEO to prevent renal clearance have higher theoretical drug-loading capacities [56,57].In order to achieve a drug loading comparable to polymers or liposomes that have been previously used to deliver DOX (≈10 wt%) [152,153], the bow tie with 16 hydroxyl groups provided a sufficient drug payload given that the yield of the hydrazone formation with polyester dendrimers is roughly 50% [13,63].The attachment of doxorubicin via pH-sensitive hydrazone linkages is shown in Scheme 2 [57,63].
Several detailed experiments were conducted on the dendrimer-DOX conjugate including cytotoxicity in cell culture, biodistribution in tumor-implanted mice, and chemotherapy experiments on C-26 colon carcinoma [57].This tumor model was chosen because it represents a challenging cancer cell line that is relatively sensitive to free DOX in cell culture but not in vivo, a finding that has been attributed to the inability of the drug to attain sufficient intra-tumor concentrations [154].Additionally, a positive control experiment (using Doxil) was also conducted in order to evaluate the effectiveness of dendrimer-DOX in comparison to the FDA-approved doxorubicin carrier Doxil.Scheme 2. Functionalization of bow-tie dendrimers for therapeutic studies.
In summary the antitumor effect of doxorubicin (DOX) conjugated to a biodegradable dendrimer was evaluated in mice bearing C-26 colon carcinomas.A bow tie biodegradable polyester dendrimer containing 8-10 wt% DOX was prepared.The design of the dendrimer carrier optimized blood circulation time through size and molecular architecture, drug loading through multiple attachment sites, solubility through PEGylation, and drug release through the use of pH-sensitive hydrazone linkages.In culture, dendrimer-DOX was >10 times less toxic than free DOX toward C-26 colon carcinoma cells after exposure for 72 h.Upon in vivo administration to mice bearing cancerous cells, dendrimer-DOX was eliminated from the serum with a half-life of 16 h, and its tumor uptake was nine fold higher than in vivo administered free DOX at 48 h.In addition, a single in vivo injection of dendrimer-DOX at 20 mg/kg DOX equivalents 8 days after tumor implantation caused complete tumor regression and 100% survival of the mice over the 60 days experiment.No cures were achieved in tumor implanted mice treated with free DOX at its maximum tolerated dose (6 mg/kg), drug-free dendrimer, or dendrimer-DOX in which the DOX was attached by means of a stable carbamate bond.The antitumor effect of dendrimer-DOX is similar to that of an equimolar dose of liposomal DOX (Doxil).There is no doubt that the remarkable antitumor activity of dendrimer-DOX results from the ability of the dendrimer to favorably enhance the pharmacokinetics profiles of attached doxorubicin.
In another study, Namazi and Adeli reported the synthesis and controlled release of biocompatible prodrugs of β-cyclodextrin linked with PEG-containing Ibuprofen or Indomethacin [155].The same group also prepared citric acid-polyethylene glycol-citric acid (CPEGC) triblock dendrimers as biocompatible compounds up to the third generation, and investigated them as potential drug-delivery systems [156,157].The preparation of a second-generation dendrimer of this type is shown in Scheme 3. Here, the encapsulation and the controlled release of anti-inflammatory drugs 5-aminosalisylic acid, mefenamic acid, and diclofenac were investigated [157].Citric acid and poly(ethyleneglycol) (PEG) were selected because of their good water solubility, low toxicity and biocompatibility and these moieties are widely accepted for use in drug formulations.A series of complexes/drugs from the synthesized dendrimers were prepared.It is worthy pointing out that the isolated water-soluble dendrimers were capable of binding and solubilizing non-polar hydrophobic molecules.Scheme 3. Synthesis of second-generation dendrimer for drug delivery studies [155][156][157].
During controlled release investigation, it was noticed that in all cases after approximately 350 min, the release of drugs from the complexes was approximately completed and was changed to a very slow rate [157].The ability of this type of non-toxic dendrimer to enhance pharmacokinetic profiles of encapsulated drugs is evidence that these dendrimer-drug complexes could be considered as potential drug-delivery systems.This system is a promising polymeric backbone for use as scaffolds in the development of well-defined polymeric drug carriers.
To date clinical experience with neutron capture therapy (NCT) is with the non-radioactive isotope boron-10 (BNCT).The use of other non-radioactive isotopes such as gadolinium has been limited, and to date, it has not been used clinically.BNCT has been evaluated clinically as an alternative to conventional radiation therapy for the treatment of malignant brain tumors and recurrent head and neck cancers [158][159][160][161]. Consequently, efficient methods for the delivery of boron-10 to biological tissues have been the subject of longstanding research.However, an obstacle to mainstream application of BNCT for cancer treatment has been the selective delivery of adequate boron concentrations to target tissues [162].To address this problem, high boron content species such as polyhedral borane clusters, closo-[B 10 H 10 ] 2− , closo-[B 12 H 12 ] 2− , and the isoelectronic icosahedral family of carboranes, closo-C 2 B 10 H 12 , have attracted significant attention [158].With this in mind, Adronov and coworkers prepared aliphatic polyester dendrimers based on bis-HMPA that incorporate an easily controllable number of carboranes within the interior of the dendritic structure [163].Newkome and coworkers had previously reported the production of water-soluble carborane-functionalized dendrimers, involving the reaction of alkyne moieties with decaborane to form ortho-carborane cages within the interior of cascade macromolecules [164].Here [163], It was critical to develop a bifunctional carborane synthon that matches the dual functionality of the bis-HMPA monomer, allowing it to be inserted within the dendrimer synthesis at any generation using traditional esterification reactions.This flexibility in the position of carborane insertion provided control over the boron concentration within a specific dendrimer target compound.Scheme 4 illustrates this synthetic strategy.Scheme 4. The strategy for incorporation of a carborane synthon into the polyester dendrimer [163].
Upon peripheral deprotection to liberate a polyhydroxylated dendrimer exterior, the resulting structures exhibited aqueous solubility as long as a minimum of eight hydroxyl groups per carborane were present.More importantly, irradiation of these materials with thermal neutrons resulted in emission of gamma radiation that is indicative of boron neutron capture events occurring within the carborane-containing dendrimers, indicating that these structures should serve as potential BNCT agents.
A higher percentage of anticancer pharmaceuticals currently in use are natural products and natural product analogues [165,166].Among others, two water soluble analogues of the alkaloid camptothecin are increasingly in clinical use [167][168][169][170]. Like many other pharmaceutical drug candidates, camptothecins have suboptimal properties mostly caused by their low water solubility and this resulted in their failure in early clinical trials [167].Researchers have continued to modify camptothecin analogues in an attempt to circumvent poor water solubility while retaining anticancer potency.However, dose-limiting side effects caused by the water-solubilizing functionalities include severe to life-threatening diarrhea and myelosuppression [171,172].Consequently, smart delivery vehicles for camptothecins that can reduce side effects while enhancing potency are of particular interest [23,74,[173][174][175].The work of Grinstaff and coworkers using polyester dendrimers represents an interesting example [101].Here, a biocompatible polyester dendrimer made up of the natural metabolites, glycerol and succinic acid, was prepared for the encapsulation of the antitumor camptothecins (10-hydroxycamptothecin (10HCPT) and 7-butyl-10-aminocamptothecin (BACPT)).Figure 3 illustrates an example of this type of polyester dendrimer and the encapsulation of 10-hydroxycamptothecin in the dendritic interior [101].Cytotoxicity studies of the dendrimer-drug complex toward four different human cancer cell lines, human breast adenocarcinoma (MCF-7), colorectal adenocarcinoma (HT-29), non-small cell lung carcinoma (NCI-H460), and glioblastoma (SF-268), were performed.The results with 10HCPT in HT-29 cells indicated that the dendrimer-10HCPT assembly had a 3.5-fold increase in potency relative to DMSO-dissolved 10HCPT and a 4.1-fold increase in comparison to DMSO-dissolved 10HCPT stock with subsequent dilutions made in water.

Figure 3. Encapsulation of antitumor camptothecin [101].
To determine whether the increase in anticancer activity conferred by dendrimer-encapsulated 10HCPT was observed with other cell lines, 10HCPT dissolved in DMSO and the dendrimer-encapsulated 10HCPT formulations were compared in a diverse human cancer cell panel consisting of the MCF-7 human breast adenocarcinoma, the NCIH460 human large cell lung carcinoma, and the SF-268 human astrocytoma.Interestingly, the dendrimer-encapsulated 10HCPT exhibited an improved degree of potency relative to the DMSO-dissolved drug in each cell line.Specifically, the IC 50 values for the DMSO-dissolved 10HCPT compared with the dendrimer-encapsulated 10HCPT was reduced from 72.0 to 10.1 nmol/L for the MCF-7-treated cells, from 32.4 to 16.7 nmol/L for the NCI-460-treated cells, and from 13.1 to 4.6 nmol/L for the SF-268-treated cells.
The more hydrophobic camptothecin analogue, BACPT, which possesses a 10-fold greater cytotoxic activity in most tumor cell lines when compared to 10HCPT, was also examined.The dendrimer encapsulation process allowed an enhanced aqueous solubility to the BACPT of 440 μmol/L.HCPT).Even though the dendrimer encapsulation afforded no improvement on the cytotoxicity toward HT-29 cells relative to DMSO-dissolved drug, the dendrimer-encapsulated BACPT exhibited an improved degree of potency relative to the DMSO-dissolved agent in each of the other cell lines.Comparing the DMSO-dissolved BACPT to the dendrimer-encapsulated BACPT, the IC 50 values were reduced from 26.7 to 8.3 nmol/L for the MCF-7-treated cells, from 1.2 to 0.6 nmol/L for the NCI-H460-treated cells, and from 6.6 to 1.2 nmol/L for the SF-268-treated cells.The results in this work [101] strongly suggest that these types of polyester dendrimers may be of significant utility in improving the aqueous solubility of other camptothecin analogues as well as other hydrophobic drugs with suboptimal pharmacokinetics.
The discovery and development of new and potent drugs is a time-consuming and costly process.It may take up to 15 years to develop a new drug, mostly because of lengthy clinical trials [176].A more economical and viable strategy is to devise effective delivery systems for drugs that have failed to provide optimum therapeutic benefit since controlled release of a drug at a specific target can significantly improve the effectiveness of a drug and thereby increase the therapeutic benefit [177].With this in mind, Hildgen and coworkers synthesized novel polyester-co-polyether (PEPE) dendrimers having a hydrophilic core [102].The core was synthesized using the biocompatible moieties, butanetetracarboxylic acid and aspartic acid, and the dendrons were derived from PEG, dihydroxybenzoic acid or gallic acid, and PEG monomethacrylate.Dendrimers were then obtained by coupling the dendrons to the core [102].
A second generation dendrimer of this type is shown in Figure 4.This type of dendrimer demonstrated good ability to encapsulate the guest molecule with loadings of 15.80 and 6.47% w/w for rhodamine and β-carotene respectively.The release of the encapsulated compounds was found to be slow and sustained, suggesting that these dendrimers can serve as potential drug delivery systems [102].Around the same time, the same authors investigated the influence of molecular architecture of PEPE dendrimers on the encapsulation and release of methotrexate [178].In this study, effects of alterations in the chemical structure of PEPE dendrimers on the encapsulation and release of methotrexate was investigated.A series of PEPE dendrimers of different architecture were synthesized [178].The biocompatibility of this type of dendrimers was evaluated in vitro by assessing their cytotoxicity on RAW 264.7 cells using the lactate dehydrogenase assay.Dendrimers caused no cell death even at a concentration of 250 mg/mL, suggesting that they are acceptable for pharmaceutical applications.They also showed good capacity to encapsulate methotrexate, with loading as high as 24.5% w/w.Increasing in the number of branches and the size of internal voids were shown to enhance the encapsulation.On the other hand, the absence of aromatic rings as branching units drastically reduced the loading capacity.Using spectroscopic studies, it was illustrated that physical entrapment, weak hydrogen bonding and hydrophobic interactions were the mechanisms of encapsulation.The release of methotrexate included a burst release in the first 6 h followed by a slower release over a period of 50 or 168 h.Increasing the number of branches decreased the initial burst release and in contrast, the absence of aromatic rings in the dendritic structure resulted in a very rapid release [178].Thus, this new macromolecular system exhibits promising characteristics for the development of new polymeric drug carriers.
The transformation of linear polymers into dendronized polymers is another avenue of polymer synthesis that has received attention.When monodisperse dendrons are attached to a linear polymer backbone, the resulting dendronized polymers have new properties and new potential applications, resulting from the almost dendritic nature of the new system.In this regard, Fréchet, Szoka and coworkers [179] synthesized rigid-rod dendronized linear polymers consisting of a poly(4-hydroxystyrene) backbone and fourth generation polyester dendrons [179].Figure 5 shows a schematic representation for this type of dendronized polymers.Both in vitro and in vivo evaluations of the polymers were then carried out to determine their suitability as drug delivery vehicles.Cytotoxicity assays indicated that these polymers are well tolerated by cells in vitro.Detailed biodistribution studies of the polymers in both non-tumored and tumored mice revealed that as for random coil linear polymers, renal clearance was a function of polymer size [179].High accumulation in organs of the reticuloendothelial system was exhibited by a dendronized polymer with a very high molecular weight (Mn = 1740 kDa), but was not as significant for smaller polymers with Mn = 67 kDa and Mn = 251 kDa.Even though polymers with degradable backbones are more suited to prevent long-term accumulation [180], these highly functionalizable, nontoxic, dendronized polymers represent a promising new scaffold for polymeric systems with pharmacokinetic properties appropriate for use as drug carriers.
Paclitaxel is a mitotic inhibitor used in cancer chemotherapy which has shown substantial clinical efficacy for various cancer types including ovarian, breast, colon, head and neck, and non-small cell lung cancers [181,182].Due to its poor solubility however, paclitaxel is usually formulated as a 1:1 mixture of Cremophor EL and ethanol, which is diluted in normal saline or dextrose solution to a final paclitaxel concentration of 5% for administration [182].This formulation has severe side effects caused by its Cremophor EL and the organic solvent [183,184].Consequently, several attempts have been made to increase the poor paclitaxel solubility (0.3 mg/mL) using various formulations or prodrug conjugates [185][186][187][188][189][190][191][192][193].Kontoyianni, Sideratou and coworkers [194] successfully functionalized commercially available hyperbranched aliphatic polyester Boltorn H40 [63,195,196] with PEG chains to afford a novel water-soluble BH40-PEG polymer exhibiting unimolecular micellar properties appropriate for application as a drug delivery system [194].After paclitaxel was encapsulated, the solubility of the anticancer drug was enhanced by a factor of 35, 110, 230, and 355 in aqueous solutions of BH40-PEG with concentrations of 10, 30, 60, and 90 mg/mL, respectively.After an initial slow release during the first 100 minutes, more than 50% of the drug was released at a steady rate that is desirable in controlled release systems and release was almost complete within 10 h.The toxicity of BH40-PEG was assessed in vitro with A549 human lung carcinoma cells and found to be nontoxic for 3 h incubation up to a 1.75 mg/mL concentration.The anticancer drug was also found to efficiently internalize in cells, primarily in the absence of foetal bovine serum, while confocal microscopy revealed the preferential localization of the drug compound in cell nuclei [194].This is another example of how polyester based macromolecules can enhance the pharmacokinetic profiles of attached or encapsulated drugs.Other recent attempts to improve paclitaxel pharmacokinetic profiles include the work of Kissel and coworkers [197].The authors aimed to formulate nanoparticles from three different hyperbranched polymers, namely unmodified dendritic polyester (Boltorn H40), a lipophilic, fatty acid modified dendritic polymer (Boltorn U3000), and an amphiphilic dendritic polymer (Boltorn W3000) (see Figure 6) for drug delivery of paclitaxel and to investigate their properties.Boltorn series hyperbranched aliphatic polyesters have great potential for applications in the biomedical field [198] due to their low toxicity, immunogenicity [56], and biodegradability [63].Here [197], a solvent displacement method allowed preparation of nanoparticles from all three hyperbranched polymers.The lipophilic Boltorn U3000 formed the biggest nanoparticles while the amphiphilic Boltorn W3000 formed the smallest ones.Degradation profiles were investigated by short time pH-stat titration.Boltorn H40 showed a faster degradation rate than the fatty acid containing polymers.For Boltorn H40, degradation rate was also investigated in longer term mass loss studies resulting in 30% degradation during 3 weeks.Cytotoxicity studies for the nanoparticles revealed low cytotoxicity for all three polymers.All three types of nanoparticles were then loaded with paclitaxel and their release profiles were studied.Sizes and zeta potentials remained stable after loading and did not change significantly.Boltorn U3000 and W3000 represent interesting candidates for drug delivery application due to their high loading efficiency.16) Boltorn U3000; and (17) Boltorn W3000 [197].
The work of Wang and Xu [199] also illustrates the potential of Boltorn series hyperbranched aliphatic polyesters as drug delivery systems.Here, a commercially available dendrimer-like hyperbranched polymer was used as a starting material in the development of a facile synthetic route for the construction of a folic acid-based multivalent targeted drug delivery system [199].In this system, fluorescein was incorporated to act as the imaging reagent, folic acid as the targeting reagent, and methotrexate as the chemotherapeutic reagent.This method used the unpaired hydroxyls on the Boltorn dendrimer to conjugate with fluorescein, thereby making good use of the defect in the polyester.Folic acid, methotrexate, and fluorescein were all successfully attached to the polyester as illustrated in Figure 7.Even though the application of this multivalent targeted drug delivery system in clinical drugs was not reported, this system exemplifies a potentially inexpensive and well-defined multivalent targeted drug delivery carrier [199].

Figure 7.
The synthesis of the folic acid-based targeted drug delivery system [199].
Recently, Nyström and coworkers modified Boltorn H30 and H40 using an average of 5 PEG chains prior to the encapsulation of DOX for delivery to breast cancer cells [200].DOX-loaded H30-PEG10k nanoparticles exhibited controlled release over longer periods of time and flow cytometry and confocal scanning laser microscopy studies indicated that the cancer cells could internalize the DOX-loaded H30-PEG10k nanoparticles.This contributed to the sustained drug release, and induced more apoptosis than free DOX.These findings are further indications that Boltorn based nanoparticles may offer an alternative strategy for delivering drugs to cancer cells.
The various types of delivery vehicles studied to date, including linear polymers, micellar assemblies, liposomes, and polymersomes, do not possess all the desired design features [201] one would want in an efficient drug carrier.Features such as a long blood circulation time, high tumor accumulation, high drug loading capacity, low toxicity, low polydispersity index, and simple preparation are necessary for a suitable drug carrier.Considering the above criteria, PEGylated dendrimers constitute an attractive option, because their size and degree of branching can be precisely controlled and they possess multiple functional appendages for the attachment of both drugs and solubilizing groups.
To minimize the hydrolytic susceptibility of the ester bond during synthesis of drug conjugates, Fréchet, Szoka and coworkers reported an elegant synthesis that combines the biocompatibility of bis-HMPA dendrimers with the robustness of polyamide dendrimers, yielding a hybrid scaffold capable of translation into clinical studies [98].A drug loaded PEGylated ester-amide dendrimer is shown in Figure 8.The biodistribution of the ester-amide dendrimer was determined in C26 tumored female Balb/C mice.Mice were injected with 8 mg Dox eq/kg, formulated as Doxil or compound 19.After 48 h, Dox had significantly accumulated within the tumor compared to insignificant amount in vital organs.This observation is important because lowering Dox accumulation in the vital organs is important for reducing toxicity, while uptake by tumor tissue must be maintained to promote treatment efficacy.For therapeutic studies, a dose-response experiment was performed in C26 tumored Balb/C mice, and four treatment groups were investigated, Doxil (20 mg Dox/kg) and compound 19 (10,15, and 20 mg Dox/kg).Dose-dependent survival was observed and all three groups treated with loaded PEGylated ester-amide dendrimer 19 showed significant tumor growth delay and prolonged survival.Mice treated with compound 19 at 20 mg Dox/kg had 9 out of 10 mice tumor free at the end of 60 days.The results in this study and earlier studies [57] confirm that Dox-loaded PEGylated dendrimer carriers are as effective as Doxil against the C26 tumor model.
In another recent study, Malkoch, Fadeel and coworkers elegantly evaluated the biocompatibility of a library of aliphatic polyester dendrimers based on bis-HMPA [59].In addition, dendrimers with two different chemical surfaces (neutral with hydroxyl end group and anionic with carboxylic end group) and dendrons corresponding to the structural fragments of the dendrimers were also evaluated.
Commercially available PAMAM dendrimers with cationic (amine) or neutral (hydroxyl) end groups were also included for comparison.In vitro studies were conducted in human cervical cancer and acute monocytic leukemia cells differentiated into macrophage-like cells as well as in primary human monocyte-derived macrophages.The entire hydroxyl functional bis-HMPA dendrimer library demonstrated excellent biocompatibility, whereas the cationic, but not the neutral PAMAM exerted dose-dependent cytotoxicity in cell lines and primary macrophages.Studies to evaluate material stability as a function of pH, temperature, and time, demonstrated that the stability of the 4th generation hydroxyl functional bis-HMPA dendrimer increased at acidic pH.This is further indication that bis-HMPA polyester dendrimers are degradable and non-cytotoxic.
Polymeric micelles can be formed in solution only above the critical micelle concentration.However, micelles formed from amphiphilic block copolymers have attracted attention in drug delivery because of their ability to decrease unwanted side effects, prolong the circulation time, and reduce uptake by the reticuloendothelial system (RES) [202][203][204][205]. On the other hand, micelles injected into the body are usually subjected to severe dilution and this normally leads to their dissociation and a rapid release of physically encapsulated drugs.Consequently, their effectiveness in drug delivery and their in vivo application are considerably reduced [206].The use of amphiphilic polymeric unimolecular micelles can help to eliminate problems associated with the dissociation and the large sizes of polymeric micelles.Dendrimer-like star polymers composed of a hydrophobic star polymer core and a hydrophilic dendron shell, have a potential to deliver drugs more effectively via appropriate structure-tuning.Wang and coworkers synthesized well-defined folate-functionalized dendrimer-like star polymers, by combining living ring-opening polymerization (ROP) of L-lactide and dendrimer synthesis [207,208].Here [207,208], a poly(L-lactide) star polymer was selected as the hydrophobic core and non-toxic biodegradable dendrons based on bis-HMPA were used as the shell [208,209].Surface functionalization using carboxylic acid groups allowed further conjugation with PEG oligomers for water solubility enhancement.These dendrimer-like star polymer nanoparticles were then functionalized further with folic acid because it is known that folic acid is non-immunogenic and has a strong binding affinity to the folate receptors, which are overexpressed on the surface of many human tumor cells [210][211][212].Scheme 5 shows the functionalization of this type of dendrimer-like star polymers using folic acid and the fluorescent probe, Hilyte488.Hilyte488 was attached to the G1-g3-FA conjugate to form G1-g3-FA-Hiyte488. UV−vis spectroscopy was employed to determine the number of Hilyte488 units incorporated per G1-g3-FA.In vitro results showed that FA-functionalized and anticancer drug-loaded degradable dendrimer-like star polymer (DLSP) could specifically target and kill human KB cells [209].
This folate-functionalized degradable amphiphilic dendrimer-like star polymer (FA-DLSP) hybrid formed unimolecular micelles in the aqueous solution with a mean particle size of about 15 nm as determined by dynamic light scattering and transmission electron microscopy.To study the feasibility of the dendrimer-like micelles as potential nanocarriers for targeted drug delivery, the anticancer agent DOX was encapsulated in the hydrophobic core, and the loading content was determined by UV/VIS analysis to be 4% by weight.The DOX loaded dendrimer-like micelles demonstrated a sustained release of DOX due to the hydrophobic interaction between the polymer core and the drug molecules [207].The hydrolytic degradation in vitro was monitored by weight loss and proton nuclear magnetic resonance spectroscopy to gain insight into the degradation mechanism of the micelles.It was found that the degradation was pH-dependent and started from the hydrophilic shell and moved gradually to the hydrophobic core.Flow cytometry and confocal microscope studies revealed that the cellular binding of the FA-DLSP hybrid against human KB cells with overexpressed folate-receptors was about twice that of the neat DLSP (without FA).The in vitro cellular cytotoxicity indicated that the FA-DLSP micelles (without DOX) had good biocompatibility with human KB cells, whereas DOX loaded micelles exhibited a similar degree of cytotoxicity against human KB cells as that of free DOX.These results clearly show that this type of dendrimer-like unimolecular micelles are a promising nanosize anticancer drug delivery system with excellent targeting properties [207].Scheme 5. Functionalization of dendrimer-like poly(L-lactide) (PLLA) star polymer for drug delivery studies [209].
Polymer vesicles commonly referred to as polymersomes have recently received significant attention for biological applications [213][214][215][216][217][218][219][220][221].Polymersomes can be readily accessed from a wide range of block copolymers and they typically exhibit much lower critical aggregation concentrations and enhanced thermodynamic and kinetic stabilities [222,223].Recently, Gillies and coworkers exploited the multivalent and multifunctional capabilities of polymersomes in a dendritic sialopolymersome system designed to interact with the influenza virus at two different stages in the infection process [103].First, the sialic acid N-acetylneuraminic acid (Neu5Ac) was conjugated to the polymersome surface in order to inhibit the binding of viral hemagglutinin to sialic acids on host cells, thus preventing viral entry.Second, the neuraminidase inhibitor zanamivir was encapsulated into the polymersome core in order to prevent the release of progeny virus from the host cells, thus inhibiting viral replication.With the aim of maximizing multivalent effects at the polymersome surface, polyester dendrons functionalized with Neu5Ac were synthesized and conjugated to polymersomes.The binding of the resulting dendritic sialopolymersomes to Limax flavus agglutinin was studied and compared to the sialodendron and a monovalent Neu5Ac derivative using an enzyme-linked lectin inhibition assay.The results of this study revealed that while the sialodendron exhibited a 17-fold enhancement (per sialoside) relative to the small molecule, the dendritic sialopolymersomes resulted in an almost 2000-fold enhancement in binding affinity.It was also found that encapsulation of zanamivir into the dendritic sialopolymersomes could be performed with the same efficiency as for naked polymersomes to provide a drug loading of about 35 wt%.Drug release rates were similar for both systems with sustained release over a period of 4 days.The results described in this paper indicate that multifunctional polymersome systems can be used for the interaction with and inhibition of influenza viruses [103].
Polyester dendrimers are often synthesized by repeated esterification of bis-HMPA dendron followed by deprotection.This synthetic strategy is straightforward, but when the protection/deprotection reactions are incomplete, defects and thus polydispersity are introduced.Shen and coworkers [19] suggest that aliphatic polyester dendrimers without heterocyclics have better biocompatibility and biodegradability for translational nanocarriers.Using the highly efficient thiol/acrylate Michael addition reactions, a strategy for the synthesis of bis-HMPA-based dendrimers without any protection/deprotection steps was developed.Scheme 6 shows the preparation of a dendrimer bearing 128 terminal hydroxyl groups in five steps.To investigate the potential of this type of dendrimers as drug carriers, G5-128 OH was pegylated to obtain a water-soluble biocompatible dendrimer capable of encapsulation and controlled release of a hydrophobic anticancer drug, doxorubicin.The esterification of G5-128 OH using PEG2k-COOH and the loading of G5-PEG with DOX is shown in Scheme 7. Scheme 6. Efficient synthesis of a fifth generation aliphatic polyester dendrimer [19].
Evaluation of the G5-PEG/ DOX complex revealed that DOX could be released with a slight burst at pH 7.4 and 37 °C followed by a very slow release.About 40% of the DOX was released in 24 h and less than 60% in 100 h.This sustained release is an improvement and is in contrast to the usually severe burst release of most micellar drug carriers [224,225].DOX release was greatly enhanced at acidic pH.Upon evaluation of the cytotoxicity of free DOX, G5-PEG/ DOX (15.2 wt% DOX), and G5-PEG to ovarian cancer cells, it was found that G5-PEG was not toxic even at high doses.The IC 50 of the DOX in the G5-PEG/ DOX to SKOV-3 ovarian cancer cells was 0.085 μg/mL, not significant different from that of free DOX (0.056 μg/mL).Further evaluations of this type of dendrimers as drug carriers are currently underway [19].Scheme 7. Preparation of G5-PEG and its loading with DOX [19].

Conclusions
Various types of delivery vehicles including linear polymers, micellar assemblies, liposomes, polymersomes, and dendrimers have been studied in an effort to identify an ideal drug carrier.The use of dendrimers as drug carriers by encapsulating hydrophobic drugs is an attractive method for delivering highly active pharmaceutical compounds that may not be in clinical use due to their limited water solubility and suboptimal pharmacokinetics.There has been a substantial interest in the area of polyester dendrimers as potential drug delivery carriers because of their relatively easy preparation, biodegradability, and biocompatibility.Encapsulation and conjugation of drugs with polyester dendrimers have shown immense employment for the improvement of pharmacokinetic profiles of hydrophobic and labile drugs.vitro and in vivo studies have shown that, in contrast to other dendrimers and polymers, the polyester dendrimer scaffold is hydrolytically degradable and less toxic and does not accumulate in vital organs.Activity in the evaluation of polyester dendrimers as drug carriers has intensified over the last few years and the trend is expected to continue.

Figure 4 .
Figure 4.A novel second-generation dendrimer with a hydrophilic interior [102].The letters m and n indicate the number of ethylene glycol repeats.