Dendrimers in drug delivery and targeting: Drug-dendrimer interactions and toxicity issues

ISSN : 0976-4879

Journal ofPharmacy &

BioAllied SciencesAn Official Publication of

Organization of Pharmaceutical Unity with BioAllied Sciences (OPUBS)

Vol 6/ Issue 3/ July-September 2014

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Journal of Pharmacy and Bioallied Sciences July-September 2014 Vol 6 Issue 3 139

Departments of Pharmaceutics, and 1Pharmaceutical Chemistry, J. C. D. M. College of Pharmacy, Sirsa, Haryana, India

Address for correspondence: Dr. Deepti Pandita, E-mail: [email protected]

S everal therapeutic agents suffer from various limitations like low aqueous solubility and short half‑life. Therefore it

is necessary to design a delivery system, which can deliver a drug efficiently. Conventional drug delivery systems are useful to some extent in overcoming these issues, but they have failed to prove their effectiveness in delivering many drugs. Nanoparticle assisted drug delivery provides a platform to modify the basic properties of drug molecules viz. solubility, half‑life, biocompatibility and its release characteristics. Several nanoparticle based therapeutic products have been launched in the market while some are under clinical and preclinical trials.[1] Liposomes and polymeric‑drug conjugates hold the maximum share of it. For instance, Doxil® (doxorubicin HCl liposome injection)[2] and Abraxane® (paclitaxel protein‑bound

particles for injectable suspension)[3] are administered as first‑line treatments in various cancer types. The stability and toxicity issues associated with these technologies has remained a matter of concern untilnow.[1] The traditional linear polymers viz. polyethylene glycol (PEG), polyglutamic acid, polysaccharide, poly (allylamine hydrochloride) and N‑(2‑hydroxypropyl) methylacrylamide have been reviewed as drug delivery vehicles and accepted for clinical use,[4] but these linear polymers have poorly defined chemical structures. In this scenario, many attempts have been made to alleviate the problems associated with them. Moreover, the considerable interest of scientists in delivering therapeutic, targeting and diagnostic agents together in a single system has led to the usage of the novel class of nanoparticles as multifunctional platforms. The introduction of highly branched, well‑defined molecular architectural polymers, i.e. Dendrimers, firstly in 1978 by Vogtle has provided a novel and one of the efficient nanotechnology platforms for drug delivery.[5]

Dendrimers are three‑dimensional, immensely branched, well‑organized nanoscopic macromolecules (typically 5000‑500,000 g/mol), possess low polydispersity index and have displayed an essential role in the emerging field of nanomedicine.

Dendrimers in drug delivery and targeting: Drug-dendrimer interactions and toxicity issuesKanika Madaan, Sandeep Kumar, Neelam Poonia, Viney Lather1, Deepti Pandita

ABSTRACTDendrimers are the emerging polymeric architectures that are known for their defined structures, versatility in drug delivery and high functionality whose properties resemble with biomolecules. These nanostructured macromolecules have shown their potential abilities in entrapping and/or conjugating the high molecular weight hydrophilic/hydrophobic entities by host‑guest interactions and covalent bonding (prodrug approach) respectively. Moreover, high ratio of surface groups to molecular volume has made them a promising synthetic vector for gene delivery. Owing to these properties dendrimers have fascinated the researchers in the development of new drug carriers and they have been implicated in many therapeutic and biomedical applications. Despite of their extensive applications, their use in biological systems is limited due to toxicity issues associated with them. Considering this, the present review has focused on the different strategies of their synthesis, drug delivery and targeting, gene delivery and other biomedical applications, interactions involved in formation of drug‑dendrimer complex along with characterization techniques employed for their evaluation, toxicity problems and associated approaches to alleviate their inherent toxicity.

KEY WORDS: Dendrimer, drug targeting, drug‑dendrimer interactions, gene delivery, polyamidoamine

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DOI:

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How to cite this article: Madaan K, Kumar S, Poonia N, Lather V, Pandita D. Dendrimers in drug delivery and targeting: Drug-dendrimer interactions and toxicity issues. J Pharm Bioall Sci 2014;6:139-50.

Received : 05-09-13Review completed : 29-09-13Accepted : 14-11-13

Review Article

Madaan, et al.: Dendrimers in drug delivery and targeting

140 Journal of Pharmacy and Bioallied Sciences July-September 2014 Vol 6 Issue 3

The name has actually derived from the Greek word “dendron” meaning “tree,” which indicates their unique tree‑like branching architecture. They are characterized by layers between each cascade point popularly known as “Generations.” The complete architecture can be distinguished into the inner core moiety followed by radially attached generations that possess chemical functional groups at the exterior terminal surface [Figure 1].[6,7] With the increase in generations, the molecular size and terminal surface groups amplifies that offer wide potential for multiple interactions and hence termed as highly functional [Figure 2]. This multivalency has subjected to the formation of various host‑guest complexes that offer wide applications. [Table 1] highlights the different commercialized dendrimers and dendrimer‑based products. The present review highlights the different strategies for their synthesis, recent updates in drug delivery applications, the specific interactions playing a significant role in entrapment/conjugation of drugs, the characterization techniques of dendrimers and their drug conjugates, several aspects of dendrimers like toxicity issues and other applications.

Technology Platforms

The advances and innovations of polymer science and architectural chemistry have resulted in the development of dendrimers as multifunctional highly branched polymeric systems. Various

dendritic platforms such as polyamidoamine (PAMAM), poly (propylene imine) (PPI), poly‑L‑lysine, melamine, poly (etherhydroxylamine) (PEHAM), poly (esteramine) (PEA) and polyglycerol have been synthesized and explored as drug delivery vehicles.[7,18] The synthesis of dendrimers can be traced back to 1980’s when D. Tomalia used “divergent methodology” to synthesize them. The strategy involves the coupling of monomeric molecules that possesses reactive and protective groups with the multifunctional core moiety which leads to stepwise addition of generations around the core followed by removal of protecting groups. PAMAM‑NH2 dendrimers were firstly synthesized by coupling N‑(2‑aminoethyl) acryl amide monomers to an ammonia core and today they are the most systematically synthesized and commercialized family of dendrimers using this methodology. This method offers an advantage of producing modified dendrimers by changing the end groups and in turn their physicochemical properties can be altered as per the required application. Although, this approach suffers from limitation of structural defects due to incomplete reaction of groups which can be trounced by the excessive addition of monomeric units.[6,19]

Figure 1: Schematic representation of dendrimer components. Reproduced with permission from ref.,[7] Copyright 2005, Elsevier Figure 2: Dendrimers as multifunctional nanoplatforms

Table 1: List of commercially available dendrimers and dendrimer‑based productsCommercialized dendrimers

Brand name Type of dendrimer Company Status References

Priostar® PEHAM/PEA Starpharma Marketed [8,9]

Starburst® PAMAM Starpharma Marketed [10]

Astramol® PPI Starpharma Marketed [11]

Polylysine Poly‑L‑lysine Starpharma Marketed [12]

Commercialized/pipeline dendrimer‑based products

Dendrimer Type of dendrimer Company Application Status References

Vivagel® Poly‑L‑lysine Starpharma Prevent the transmission of HIV and STDs

Clinical trials (phase 3)

[12]

Stratus CS® PAMAM Dade behring Cardiac assay diagnostic Marketed [13]

Superfect® PAMAM Qiagen Transfection agent Marketed [14,15]

Priofect™ PAMAM Starpharma Transfection agent Marketed [15]

Alert ticket™ PAMAM U.S. army lab Anthrax‑detecting agent Marketed [16]

Dendrimer‑docetaxel ND Starpharma Breast cancer treatment Preclinical [17]

Dendrimer‑oxaliplatin ND Starpharma Colon cancer treatment Preclinical [17]

ND: not define, PEHAM: Poly (etherhydroxylamine), PEA: Poly (esteramine), PAMAM: Polyamidoamine, PPI: Poly (propylene imine), HIV: Human immunodeficiency virus, STDs: Sexually transmitted diseases



Another approach “convergent methodology” of synthesizing dendrimers was pioneered by Hawker and Frechet in 1990’s. This strategy refers to the inward growth by gradually assembling surface units with reactive monomers. This approach produces more homogenous and précised molecular weight products. The deactive side products produced during reactions gets easily separated by purification, but this becomes harder in higher generations due to similarity in products and reactants. The convergent methodology suffers from one major limitation that it gives low yield in the synthesis of large structures and hence is suitable for production of only lower generation dendrimers.[20] These synthesis methodologies are presented in Figure 3.

Other approaches that have been employed for dendrimer synthesis include “Hypercore and branched monomers”; “Double exponential”; “Lego chemistry” and “Click chemistry.” The first approach involves the pre‑assembly of oligomeric species to accelerate the production of dendrimers in few steps with high yield,[21] whereas the double exponential approach follows the production of monomer unit for both divergent and convergent growth from a single functionalized group. The produced monomer units are allowed to react, which produces an orthogonally protected trimer that is grown further. Advantages of this method include accelerated synthesis of dendrimers and this method can be applied to both convergent and divergent method.[21] Lego chemistry approach employed by Tomalia and

Svenson involves the production of phosphorus dendrimers by utilizing highly functionalized core and branched monomers. Several changes have been made to the basic scheme of synthesis and more refined schemes are being developed, which involves the multiplication of terminal surface groups from 48 to 250 in one step.[22] The same authors used click chemistry method in successful production of triazole dendrimers using Cu (I)‑catalyzed click chemistry reactions. Again, this methodology is involved in hasten synthesis of dendrimers by divergent approach with high purity and excellent yield. This technique involves the reaction of two different monomeric units of complimentary functionalities that render spontaneous synthesis by avoiding the use of activating agents/protecting groups and reducing the number of steps.[19] More future work is needed to find out the cost‑effective synthesis strategies for successful commercialization of this technology.

Dendrimers for Drug Delivery and Targeting

The precise control over the distribution of drugs is highly valuable to abolish the typical drawbacks of traditional medicine. Thus, a drug delivery system should be designed to attain the site‑specific delivery preferentially. In recent years, improved pharmacokinetics, biodistribution and controlled release of the drug to the specific targeted site has been achieved with polymer based drug delivery.[23] Unlike traditional polymers, dendrimers

Figure 3: Schematic representation of dendrimer synthesis by (a) Divergent and (b) Convergent methodologies

b

a



have received considerable attention in biological applications due to their high water solubility,[24] biocompatibility,[25] polyvalency[12] and precise molecular weight.[26] These features make them an ideal carrier for drug delivery and targeting applications. For investigating dendrimers as drug delivery vehicles, their biopermeability across the biological membranes should be considered. A study by Kitchens et al. reviewed the cationic PAMAM‑NH2 (G0‑G4) dendrimers and evaluated its permeability across Caco‑2 cell monolayers as a function of dendrimer generation, concentration and incubation time, for oral drug delivery. Various parameters viz. transepithelial electrical resistance, 14C‑mannitol permeability and leakage of lactate dehydrogenase enzyme were studied and it was suggested that these amine terminated PAMAM dendrimers could cross the biological membranes possibly by paracellular and endocytosis pathways.[27] Moreover, by optimizing the size

and surface charge, these dendritic platforms can be developed into oral delivery systems.

Different categories of drugs that have been incorporated in these versatile nanoscopic carriers as summarized in Table 2. Recently, a dendrimer based prodrug has been developed for paclitaxel (P‑gp efflux substrate) that has focused on enhancement of permeability and transportation of drug across cellular barriers. The highly functional lauryl‑modified G3 PAMAM dendrimer‑paclitaxel conjugates demonstrated good stability under physiological conditions and 12‑fold greater permeability across Caco‑2 cell and porcine brain endothelial cells monolayers than paclitaxel alone. Finally, the authors concluded that surface‑modified G3 PAMAM dendrimers could be considered as potential nanocarriers for poorly water‑soluble P‑gp efflux transporter drugs.[32] Earlier Ooya et al.

Table 2: Different therapeutic moieties studied using dendrimer platformDrug Pharmacology Dendrimer type Application Reference

10‑hydroxycamptothecin Anticancer Carboxylated poly (glycerol‑ succinic acid)

High cytotoxicity [28]

7‑butyl‑10‑aminocamptothecin Anticancer G4.5 poly (glycerol‑succinic acid)‑COONa

Increased aqueous solubility and 16 fold increased cellular uptake

[29]

Paclitaxel Anticancer Polyglycerol (G4 and G5) Increased aqueous solubility [30]

G4 PAMAM Increased aqueous solubility, enhanced cytotoxicity

[31]

G3 PAMAM Improved permeability (12 fold) [32]

G2 PAMAM Increased aqueous solubility (3700 fold) [33]

Methotrexate Anticancer G2.5 and G3 PAMAM 24‑fold increment in cytotoxicity [34]

G5 PAMAM Targeted delivery [35]

G1, G1.5 and G2.5 PAMAM Enhanced anticancer activity [24]

Doxorubicin Anticancer G4 PAMAM Improved cytotoxicity [36]

G5 polylysine Prolonged plasma exposure and diminished drug toxicity

[37]

2, 2 bis (hydroxymethyl) propionic acid (G3)

Long term antitumor activity [38]

Famotidine Antiulcer G5 PPI Improved solubility [39]

Indomethacin NSAIDs G5 PPI Improved solubility [39]

Amphotericin B Antifungal G5 PPI Improved solubility [39]

Rifampicin Antitubercular Mannosylated PPI Sustained release and targeted delivery [40]

Lamivudine Anti‑HIV Mannosylated PPI Prolonged drug release up to 144 h [41]

Efavirenz Anti‑HIV PPI Targeted delivery [42]

Furosemide Diuretic G4 PAMAM Increased solubility and sustained release [43]

Etoposide Anticancer PAMAM High loading capacity [44]

Erythromycin Bactericidal antibiotic

G4 PAMAM Sustained release and improved activity [45]

Zidovudine Anti‑HIV G4 PPI Targeted delivery [46]

Ketoprofen NSAIDs PAMAM Improvement of drug permeation through skin

[47]

Diflunisal NSAIDs PAMAM Improvement of drug permeation through skin

[47]

5‑Fluorouracil Anticancer G4 PAMAM Targeted drug delivery [48]

Propanolol Anti‑hypertensive

Lauroyl‑G3 PAMAM Enhanced basal transport of propranolol [49]

Naproxen NSAIDs G0 PAMAM High permeability across Caco‑2 monolayers

[50]

Pilocarpine Antiglaucoma G2 and G4 PAMAM Prolonged corneal residence time [51]

Niclosamide Anthelmintic G0 to G3 PAMAM Improved solubility and sustained release [52]

Sulfa‑methoxazole Bacteriostatic antibiotic

G3 PAMAM Increased anti‑bacterial activity [53]

Nifedipine Anti‑ hypertension G5 PAMAM Enhanced solubility [54]

Brimonidine Antiglaucoma G3 PAMAM Improved solubility and tissue uptake [55]

Timolol maleate Antiglaucoma G3 PAMAM Improved solubility and tissue uptake [55]

PAMAM: Polyamidoamine, PPI: Poly (propylene imine), HIV: Human immunodeficiency virus, NSAIDs: Non‑steroidal anti‑inflammatory drugs



reported 400 times increase in the solubility of paclitaxel when incorporated in polyglycerol dendrimers.[30]

The targeted delivery of chemotherapeutics is essential to reduce the side effects significantly associated with conventional therapy, where healthy tissues such as liver, spleen, kidneys and bone marrow can accumulate the toxic levels of drug. The site specific delivery of the drug could be achieved by surface modification of dendrimers employing various targeting moieties such as folic acid (FA), peptides, monoclonal antibodies and sugar groups.[56] Several successful active and passive targeting attempts were accomplished by engineering the branching units and surface groups of dendrimers. Patri et al. conjugated FA to G5 PAMAM dendrimer for the targeted delivery of methotrexate and observed receptor mediated drug delivery that demonstrated high specificity for KB cells overexpressing folate receptors and showed slower drug release.[35] The authors further conjugated the PhiPhiLux G1 D2, an apoptotic sensor to FA attached PAMAM dendrimers which showed 5 fold enhanced fluorescence, attributed to successful delivery of drug with cell‑killing efficacy.[57] FA conjugated G5 PAMAM dendrimers for the targeted delivery of 2‑methoxyestradiol to cancer cells, as confirmed by MTT assay, were recently reported.[58] Sideratou et al. prepared folate functionalized PEG coated nanocarrier based on fourth generation diaminobutane PPI dendrimers for targeted delivery of etoposide.[59] The encapsulation of the drug inside the dendrimeric scaffold resulted in enhanced solubility of etoposide, further the in vitro release of encapsulated drug from these functionalized dendrimers was found to be sustained and comparable to the non‑functionalized dendrimers. These folate PEGylated dendrimers exhibited specificity for folate receptor with low toxicity. Furthermore, successful conjugation of FA to fifth generation PPI dendrimers was explored and its potential in targeted delivery of an anticancer drug doxorubicin was investigated. The results revealed that FA conjugated dendrimers displayed higher cell uptake in MCF‑7 cancer cell lines and significantly reduced the toxicity.[60]

Further, Patri et al. group explored the usage of monoclonal antibodies for site‑specific delivery by conjugating prostate specific membrane antigen (PSMA) J591, an anti‑PSMA to G5 PAMAM dendrimers that showed targeted delivery in all prostate and non‑prostate tumors.[61] Methotrexate bound to G5 PAMAM conjugate containing cetuximab[62] or anti‑ human epidermal growth factor receptor‑2[63] monoclonal antibodies have demonstrated their potential in tumor targeted drug delivery.

Using glycosylated dendrimers, terminally modified with sugar moieties is another strategy for targeting applications. In a study, colchicine was successfully conjugated with glycodendrimer that showed selective 20‑100 times inhibition of HeLa cells than healthy cells.[64] A study done by Ciolkowski et al. examined the influence of dendrimers’ surface modification on the strength of interaction with proteins. This study was carried out using PPI G4 and G3.5 PAMAM dendrimers as a drug carrier and hen egg white lysozyme as a model protein. As shown by differential scanning calorimetry (DSC) and circular dichroism studies the increasing maltose modification on the surface of PPI dendrimers resulted in diminishing ability of dendrimer to

interact with lysozyme because the surface charge of complexes turned neutral from cationic.[65] In addition, a comparably strong influence exerted on lysozyme by positively charged PPI dendrimer and negatively charged PAMAM dendrimer.

Dendrimers for Gene Delivery

Gene transfer is an invaluable experimental tool that involves the introduction of new genetic material to host for treatment of diseases. Various vectors and physical methods are reported for in vivo delivery of therapeutic nucleic acids. Commonly two approaches viz. viral and non‑viral based are being used to facilitate the transfer of genetic material successfully to target cells. Although viral carriers can achieve rapid transfection, but immunological and oncologic adverse effects associated with these vectors has remained a topic of concern. Non‑viral gene delivery vectors offer the usage of natural/synthetic molecules or physical forces to transfer genetic material to targeted cells. Several advantages such as ease of fabrication, targeting ability, potential for repeat administration and low immune response have led to the usage of non‑viral vectors preferably for gene therapy. Santos et al. extensively reviewed various physical and chemical methods for non‑viral gene delivery, dendrimers based vectors and their applications in tissue engineering and regeneration.[66] Among various commercially available dendrimers, PAMAM dendrimers have received the most attention as potential non‑viral gene delivery agents due to their cationic nature which enables deoxyribonucleic acid (DNA) binding at physiological pH.[67] Pandita et al. prepared dendrimer based gene delivery vectors taking advantage of the cationic nature and the “proton‑sponge” effect of these dendrimers. Arginine‑glycine‑aspartic (RGD) nanoclusters were formed by conjugation of G5 and G6 PAMAM dendrimers with a varying number of peptides containing the RGD motif, in view of its targeting capabilities [Figure 4a]. Authors reported that the system wherein G6 PAMAM dendrimer was conjugated to eight peptide arms (RGD8‑G6) enhanced the gene expression in mesenchymal stem cells and presented a 2‑fold higher bone morphogenetic protein‑2 expression in comparison to the G6 native dendrimer.[68] Various published literature suggests that functionalized dendrimers are much less toxic than the native dendrimers. Same group synthesized a new family of gene delivery vectors consisting of G5 PAMAM dendrimer core randomly linked to hydrophobic chains (with varying chain length and numbers) [Figure 4b]. In vitro studies revealed a remarkable capacity of these vectors for internalizing pDNA with very low levels of cytotoxicity, being this effect positively correlated with the CH2 content present in the hydrophobic moiety. The results demonstrated that vectors containing the smallest hydrophobic chains (La1‑G5 and La2‑G5 functionalized dendrimers) showed the higher gene expression efficiency.[69] Further, functionalized PAMAM dendrimers exhibited low cytotoxicity and receptor‑mediated gene delivery into mesenchymal stem cells and transfection efficiencies superior to those presented by native dendrimers and by partially degraded dendrimers.[70] A low affinity mesenchymal stem cells binding peptide and a high affinity mesenchymal stem cells binding peptide were conjugated with PAMAM dendrimers in the studies, providing for a mechanism of cell specific recognition [Figure 4c].



The antigen‑presenting cells specific delivery of DNA by peptide functionalized G5 PAMAM dendrimers is also described. In vivo study on mice suggested that functionalized PAMAM dendrimer complexes can be used for effective targeting to antigen‑presenting cells so as to achieve high transfection efficiency.[71] More recently, Bai et al. investigated the efficacy of arginine‑modified PAMAM dendrimers in delivering human interferon beta gene on a xenograft brain tumor mice model. The study revealed that functionalized dendrimers reduce the tumor size effectively, which confirms the tumor targeting potential of peptide functionalized dendrimers.[72]

Recently, Shan et al. reported a novel non‑viral gene delivery vector based on dendrimers‑entrapped gold nanoparticles synthesized using amine‑terminated G5. NH2 dendrimers as templates with different Au atom/dendrimer molar ratios, which showed higher transfection efficiency than that of dendrimers without Au nanoparticles entrapped.[73] Ferenc et al. investigated dendriplexes formed by short interfering RNA (siRNA) and cationic phosphorus dendrimers. The siRNA conjugated dendriplexes showed good stability as shown by laser doppler

electrophoresis and circular dichroism spectra.[74] Previously, Drzewinska et al. functionalized PPI dendrimers by maltose or maltotriose conjugation and interacted it with anti‑human immunodeficiency virus antisense oligonucleotides to form dendriplexes. These carbohydrate‑modified PPI G4 dendrimers protected oligonucleotides from nucleolytic degradation at neutral pH but in an acidic environment, the protective effect of these dendrimers was weaker.[75] A high molecular weight bioreducable polymer was synthesized by Nam et al. for gene delivery by incorporating arginine‑grafted poly (disulfide amine) into the PAMAM dendrimer. The resultant polyplexes significantly enhanced the cellular uptake and were less susceptible to reducing agents, ensuing superior transfection efficiency.[76]

Drug‑Dendrimer Interactions

Dendrimers offer several mechanisms of interactions with different classes of drugs. The different types of drug‑dendrimer interactions proposed for drug delivery are presented in Figure 5.[77] Broadly these interactions can be categorized under two categories viz. physical and chemical bonding.

Figure 4: Schematic representation of polyamidoamine dendrimer‑based gene delivery vectors. (a) Schematic illustration of the synthesis of dendrimer/RGD conjugates (pathway A + B) and of the indirect estimation of the number of pyridyldithiol (PDP) units per dendrimer by spectrophotometry (pathway A + C). Reproduced with permission from ref.,[68] Copyright 2011, American Chemical Society. (b) Strategy of synthesis followed in dendrimer surface functionalization with alkyl chains (activation of the fatty acid + conjugation reaction). Reproduced with permission from ref.,[69] Copyright 2010, Elsevier. (c) The two‑step reaction for the synthesis of peptide‑functionalized G5 PAMAM dendrimers (pathway A + B), and for indirect estimation of the number of peptide units by spectrophotometry (pathway A + C). Reproduced with permission from ref.,[70] Copyright 2010, American Chemical Society

c

b

a



Physical bonding

Incorporation of small organic molecules may be a result of non‑bonding interactions with specific groups within dendrimer, i.e. just physical entrapment. Dendrimers can be used as dendritic boxes and unimolecular micelles (dendrimer‑drug networks) for the incorporation of hydrophobic/hydrophilic molecules by host guest interactions inside their empty cavities (nanoscale containers) present around core.[78,79] Jansen et al. were the first to entrap the rose bengal dye molecules in PPI dendrimers by using tert‑butyloxycarbonyl (t‑Boc) groups and led to the production of stable dendritic box that possess the bulky amino groups on the dendrimer surface.[80] The dendritic unimolecular micelles contain the hydrophobic cores surrounded by hydrophilic shells and they offer an advantage over conventional polymeric micelles such that the micellar structure is maintained at all the concentrations because the hydrophobic segments are covalently connected.

Chemical bonding

Alternatively, the exploitation of well‑defined multivalent aspect of dendrimers allows the attachment of drug molecules to its periphery that result in complex formation. The resultant complexes are formed either due to the electrostatic interactions between the drug and dendrimer or conjugation of the drug to dendrimer molecule. Through electrostatic interactions, various ionizable drugs form complexes with the large number of ionizable terminal surface groups of dendrimers. In PAMAM dendrimers, both primary amine and tertiary amine groups present on the surface and within the core respectively are titrable and has pKa values of 10.7 and 6.5 respectively.[81] Hence, they possess the ionizable groups terminally as well as within their core and thus offer potential sites for drug interactions. Many of the drugs such as ibuprofen,[82] piroxicam,[83] indomethacin,[84] benzoic acid[85] have shown to form stable complexes through electrostatic interactions.

Moreover, the drugs can be covalently conjugated to dendrimers through some spacers that may include PEG, p‑amino benzoic acid, p‑amino hippuric acid and lauryl chains etc., or biodegradable linkages such as amide or ester bonds. This prodrug approach has been found to increase the stability of drugs and has affected their release kinetics significantly. Several researchers have successfully conjugated

penicillin V,[86] venlafaxine,[87] 5‑aminosalicylic acid,[88] naproxen,[50] propranolol[49] with PAMAM dendrimers. The results have shown the enhanced solubility and controlled release of drugs from these complexes in comparison to the pure drug. Apart from these, many anticancer drugs viz. cisplatin,[89] doxorubicin,[37] epirubicin,[90] methotrexate[24] and paclitaxel[33] have also been conjugated and used for drug targeting. For example, epirubicin prodrug was developed by conjugating it with PEG dendrimers containing aminoadipic acid as branching molecules. These conjugates exhibited increased blood residence time and showed improved therapeutic action. The study revealed that PEG dendrimers increased the stability of bound drug toward chemical degradation and hence can be used potentially in the development of prodrugs of large molecules.[90]

Physicochemical Characterization of Dendrimers

Various analytical techniques have been reported in literature to analyze the physicochemical parameters of dendrimers. It includes spectroscopic, dynamic light scattering (DLS), microscopic, chromatographic, rheological, calorimetric and electrophoretic characterization: Nuclear magnetic resonance (NMR), infrared, ultraviolet (UV)‑Visible, fluorescence and mass spectroscopy; small angle X‑ray scattering, small angle neutron scattering, laser light scattering; atomic force microscopy (AFM), transmission electron microscopy (TEM); size exclusion chromatography, high performance liquid chromatography (HPLC); DSC, temperature modulated calorimetry and dielectric spectroscopy; Polyacrylamide gel electrophoresis (PAGE) and capillary electrophoresis [Figure 6].[91,92] All these dendrimer characterization techniques can also be used to study the dendrimer‑drug conjugates/complexes. Encapsulation of drug molecules or nanoparticles by dendrimers can be characterized by TEM, UV‑Visible and Fourier transform infrared spectroscopy. Moreover, NMR and mass spectroscopic techniques are used to study the nature of complexes. Huang et al. characterized the silybin‑PAMAM complexes by NMR and HPLC methods. Chemical shifts of methylene protons of the G2 dendrimers were determined by NMR spectroscopy which apparently was changed with the addition of silybin.[93] The downfield chemical shift in the outermost layer of the G2 dendrimer and the interior methylene protons confirmed the electrostatic interactions between the amine functional groups of the dendrimer and the phenolic hydroxyl groups of silybin; and the encapsulation of silybin molecules by hydrophobic interactions, respectively.

Figure 5: Different types of drug‑dendrimer interactions. The darkened oval represents an active substance. Reproduced with permission from ref.,[77] Copyright 2002, Elsevier



HPLC method was used to determine the solubility of silybin and plasma drug concentration of silybin.

The shape and size of paclitaxel solubilized micelles of a linear‑dendritic copolymer (BE‑PAMAM), formed by conjugating the poly (butylene oxide) (B)‑poly (ethylene oxide) (E) block copolymer B16E42(BE) with a G2 PAMAM dendrimer were examined by DLS and cryo‑TEM. The studies indicated that spherical micelles got transformed to worm‑like micelles of the BE copolymer as a result of encapsulation of paclitaxel molecules.[33] Morilla et al. explored the potential of different generations of PAMAM dendrimers (G4, G5, G6 and G7) to deliver siRNA.[94] The formed complexes were investigated for three variables: The ionic strength of the medium (with or without 150 mM NaCl), G4, G5, G6 and G7 dendrimer generations and N/P ratio, i.e. nitrogen amines in PAMAM/phosphate in siRNA. Various analytical techniques viz. PAGE, DLS, TEM and AFM were used to characterize these variables. PAGE study revealed that in order to complexate the whole available siRNA molecules, a minimum N/P ratio of 5 (for G4, G5) and 10 (for G6, G7), was required. The sizes obtained using DLS, TEM and AFM provided the inverse relationship between size of the formed complex and its binding affinity; and also between the size of the complex and ionic strength of preparation media. Finally, the measured zeta potential of small siRNA PAMAM dendrimer complex was found to reduce from + 25 mV to 0, when drops of sodium chloride were added to the medium due to the formation of larger complexes or even aggregates. The results together indicated that ionic strength of the media was the main determinant of the size of siRNA‑dendrimer complex and responsible for the inhibition of enhanced green fluorescent protein expression (silencing activity).

Recently, PAMAM and PPI dendrimers were synthesized by introducing 1,3 propane sultone in tertiary amine of these dendrimers and 1H NMR studies were performed to calculate their degrees of acetylation and interior‑functionalization. The spectrum obtained using correlation spectroscopy revealed J‑coupled protons (geminal or vicinal protons) via cross‑correlation peaks off the diagonal, which was further used

to confirm the structure of the synthesized dendrimers. Further, 1H‑1H nuclear overhauser effect spectroscopy (NOESY) was used to characterize the inclusion structure of the synthesized dendrimer and methotrexate sodium complexes that mainly revealed the spatial conformation of dendrimer/drug complexes. The presence of nuclear overhauser effect signals between guest protons and dendrimer interior protons in a 1H‑1H NOESY spectrum suggested the encapsulation of the guest molecules within the pockets of dendrimers.[95]

Toxicity Issues

It is well‑known that cationic macromolecules interact with negative biological membranes that result in their destabilization and cause cell lysis.[96,97] The nanosized particles like dendrimers have the ability to interact with nanometric cellular components such as cell membrane, cell organelles and proteins.[98] Similar to macromolecules, dendrimers with cationic surface groups tend to interact with lipid bilayer, increases the permeability and decreases the integrity of biological membrane. The mechanism involved is the leakage of cyotosolic proteins such as luciferase and lactate dehydrogenase, on interaction of dendrimers with the cell membrane that finally causes its disruption and cell lysis.[99,100] Typical representatives of this class of carriers are PAMAM‑(Starburst™), PPI and polylysine‑dendrimers. Among them, PAMAM dendrimers are shown to cause concentration and generation dependent cytotoxicity and hemolysis.[99,101] In an article in 2003, Jevprasesphant et al., investigated the cytotoxicity of PAMAM dendrimers using Caco‑2 cells and concluded that anionic or half generation dendrimers show significantly low toxicity in comparison to their respective cationic family.[102] Further, the in vitro cytotoxicity of cationic melamine dendrimers having surface groups such as amine, guanidine, carboxylate, sulfonate or phosphonate were reported and it was concluded that cationic dendrimers were much more cytotoxic than anionic or PEGylated dendrimers.[99] In a study, membrane disruption and loss of Caco‑2 microvilli was reported when the cells were treated with 0.1 mM or higher concentration of G4‑NH2, whereas the cells remained unaffected when treated with G3.5‑COOH at the same concentration.[103] Extensive in vitro studies have been performed to assess the toxicity of various modified or native dendrimers on different cell lines. Few scientists also report the essential in vivo toxicity of dendrimers. According to the very first in vivo toxicological study, dose of 10 mg/kg appeared to be non‑toxic up to fifth generation of PAMAM dendrimers, when G3, G5 and G7 generations were injected into mice and all the tested generations were found to be non‑immunogenic.[104,105] Recently, Jones et al. performed nano‑toxicological studies on amine‑terminated PAMAM dendrimers that revealed that intravenous administration was lethal to mice and caused disseminated intravascular coagulation‑like condition. Using flow cytometry and microscopic analysis it was demonstrated that cationic fluorescein isothiocyanate labeled G7 PAMAM dendrimers caused platelet disruption, whereas neutral (hydroxyl terminated) and anionic (carboxyl terminated) PAMAM dendrimers did not alter platelet morphology or their function.[106] This recent study is in consistent with the previous studies and justifies that the anionic dendrimers are non‑toxic

Figure 6: Various analytical techniques employed for the physicochemical characterization of dendrimers



in comparison to cationic ones. Tathagata and co‑workers also investigated the toxicity profile of the fifth generation PPI dendrimers and some of its surface engineered derivatives. Functionalization with t‑Boc, mannose and tuftsin led to the drastic reduction in the toxicity of PPI, this was attributed to the masking of primary amino groups responsible for the positive charge and thus the associated toxicity. Both PPI and surface engineered derivatives did not show any evidence of immunogenicity. Finally, it was concluded from these observations that functionalization of dendrimers leads to the drastic reduction of toxicity and increased biocompatibility.[107]

The oral acute toxicity study on PAMAM dendrimers in immune competent CD‑1 mice was done taking size and charge of dendrimers in consideration. The results revealed that cationic dendrimers caused more toxicity than their anionic counterparts. Maximum tolerated doses of dendrimers was determined by a dose escalation study and it was concluded that the dendrimers were safe for oral administration except for G7‑NH2 and G7‑OH, which exhibited signs of toxicity at relatively low dosage.[108] In general, two approaches are being reviewed to overcome the in vitro toxicity issues of dendrimer based nanosystems. Either biocompatible/biodegradable dendrimers viz. polyether, polyester or polyether‑imine, polyether‑copolyester, phosphate, citric acid, melamine, peptide or triazine dendrimers can be synthesized or the peripheral positively charged groups can be masked by acetylation and PEGylation. Moreover, the surface groups can be functionalized with carbohydrate, amino acid, antibody and FA that abates the overall positive charge and further the toxic effect of primary amines/imines.[109] Hence, it can be concluded that neutral and anionic dendrimers do not interact with biological membranes and are mostly compatible for implied clinical relevance.

Other Applications

The versatility of dendrimers also explains their importance in photodynamic therapy (PDT), boron neutron capture therapy and bioimaging applications.

Dendrimers in PDT

PDT refers to treatment of tumor cells by photosensitizers that on irradiation of light of appropriate wavelength pass their excess energy to nearby molecular oxygen to form reactive oxygen species such as singlet oxygen and free radicals which are toxic to cells and tissues. Dendrimers have been used as promising carriers for improved delivery of 5‑aminolevulinic acid (a natural precursor of photosensitizer protoporphyrin 1X) that increases the accumulation of porphyrin in cells, which further results in toxicity.[110‑112] Recently, polymeric micelles encapsulating dendrimer phthalocyanine have been developed as a photosensitizer formulation for enhanced photodynamic effect.[113]

More recently, G3 PAMAM grafted porous hollow silica nanoparticles (PHSNPs) were successfully fabricated as photosensitive drug carriers for PDT. The attachment of gluconic acid (GA) to this system was thereafter followed, for

surface charge tuning. PAMAM‑functionalized outer layer with a large number of amino groups provided high loading amount of aluminum phthalocyanine tetrasulfonate (AlPcS4), its retarded premature release and effective release to target tissue. The study demonstrated that irradiation of the AlPcS4 entrapped GA‑G3‑PHSNPs with light resulted in efficient generation of singlet oxygen that produced significant damage to tumor cells and suggested as effective photosensitizer formulation for PDT applications.[114]

Dendrimers in boron neutron capture therapy

Boron therapy is concerned with the treatment of cancers that is based on Boron capture reaction.[115] The applicability of PAMAM dendrimers in investigating intratumoral delivery of agents for neutron capture therapy is remarkable in biomedical science. In a study by Wu et al. functionalized G5 PAMAM dendrimers and conjugated cetuximab specific to EGF receptor to starburst dendrimers that carried around 1100 boron atoms. The in vivo results revealed that accumulation of conjugates were 10 times higher in brain tumor tissues in comparison to healthy brain tissues. This study was first to demonstrate the efficacy of a boronated monoclonal antibody for boron neutron capture therapy of an intracerebral glioma.[116] Dendrimer based boron neutron capture therapy in combination with EGF receptor targeting moieties can enhance the boron uptake in tumor tissues.[117]

Dendrimers as imaging agents

Gadolinium (III) (Gd) the paramagnetic contrast agent for magnetic resonance imaging has been successfully complexed with PAMAM dendrimers over the last decades for visualizing both tumor vasculature and lymphatic involvement.[118,119] Recently, a tumor targeting biodegradable contrast agent has been developed from Gd chelates and PEG conjugated polyester dendrimer which contains FA at the distal end. This dendrimer conjugate contrasting agent has shown to produce enhanced contrast than commercially available product Magnevist®. Furthermore, it’s retention in all the organs was low resulting in reduction of Gd induced toxicity. Thus, these positive outcomes, i.e. enhanced magnetic resonance imaging contrast and low Gd retention makes this dendrimer based biodegradable carrier a promising imaging agent.[120]

Outlook

Last decade has witnessed the amplified growth of usage of dendrimers in biological systems. The exclusive properties viz. well‑defined structure, polyvalent character and monodispersity make them effective nanosystems for drug delivery applications. The current review has emphasized on the considerable opportunities that dendrimers offer in resolving fundamental issues of solubility, drug delivery and targeting of drug molecules. In spite of this, their use is constrained due to their high manufacturing cost and further they are not subjected to GRAS status due to the inherent toxicity issues associated with them. As highlighted in the present work, many attempts have been made to resolve the problems pertaining to safety,



toxicity and efficacy of dendrimers and more research is going on to develop the functionalized moieties pertaining to its high potential as a nanocarrier. Besides drug delivery, dendrimers have been found to have a great emphasis in gene delivery, boron neutron capture therapy, PDT and as magnetic resonance imaging contrast agents. The use of dendrimers in the clinic has still not reached the success of linear polymers and several applications remain to be explored for its industrial as well as biomedical applications. With improved synthesis, further understandings of their unique characteristics and recognition of new applications, dendrimers will become promising candidates for further exploitation in drug discovery and clinical applications.

Acknowledgement

The authors would like to thank the Science and Engineering Research Board, Department of Science and Technology, Government of India for funding through the project SR/FT/LS‑145/2011.

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Source of Support: The Science and Engineering Research Board, Department of Science and Technology,

Government of India is acknowledged for funding through the project SR/FT/LS-145/2011,

Conflict of Interest: None declared.

Dendrimers in drug delivery and targeting: Drug-dendrimer interactions and toxicity issues

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