DENDRIMERS: A NOVEL TOOL FOR DRUG DELIVERY AND TARGETING

Dendrimers are hyper-branched macromolecules having tree like structure, consisting of a core molecule and alternating layers of monomers. Due to their unique architecture these have improved physical and chemical properties like high solubility, miscibility and reactivity. Dendrimers consist of well defined size, shape, molecular weight and monodispersity. These properties formulate the dendrimers a suitable carrier in drug delivery application. These are built from number of molecular entities of colloidal particles that exists in equilibrium with the molecules or ions in nature and due to these increases the solubility of poorly soluble drugs. Dendrimers have the ability to encapsulate and bind the guest molecule can be used for solubility enhancement, sustained release and various drug delivery applications. The terminal groups are modified to attach biologically active substances for targeting purpose. Dendrimers are suitable for a wide range of targeted drug delivery, controlled drug delivery, gene delivery and industrial applications. This review gives concise information about the dendrimers, its synthesis, characterization and application in drug delivery.


INTRODUCTION
At present scenario many researchers are working to deliver the drug with improved bioavailability, enhancement in solubility, to hit the target site to produce therapeutic effects, and to avoid the toxic effects 1 . Dend rimers is one out of many approaches which focuses on the above criteria. These are hyperbranched, globular, monodisperse, three dimensional nanoscale synthetic polymers, having very well defined size, shape and definite molecular weight having high degree of surface functionality and versatility 2 . The term "Dendrimer" derived from two Greek words "Dendron" meaning tree "Meros" meaning part. The chemistry of dendrimers was introduced in 1978 by Fritz Vogtle and co-workers. Dendrimers used as a polymeric material. Dendrimers differs from traditional polymers in that they have a multi-branched, three dimensional architecture with very low polydispersity and high functionality 3 . In dendritic structures number of terminal group increases exponentially with a linear increase in the generation of dendrimer. This relationship limits the ultimate size of the dendrimer due to steric crowding of the terminal groups. Dendrons is the term used about a dendritic wedge without a core, the Dendrimer can be prepared from assembling two or more dendrons. As we shall see later, dendrons are very useful tools in the synthesis of dendrimers by the segment coupling strategy. These dendrons have been used in the creation of numerous of dendrimers having different structures and functions. The dendrimer shell is the homo-structural spatial segment between the focal points, the "generation space". The "outer shell" is the space between the last outer branching point and the surface. The "inner shells" are generally referred to as the dendrimer interior 4 . In dendrimers, the outer shell consists of a varying number of pincers created by the last focal point before reaching the dendrimer surface.

Advantages 1. Low polydispersity index
Due to stringent control during synthesis, they have lower polydispersity index. As the density of branches increases the outer most branches arrange themselves surrounding a lower density core in the form of spheres and outer surface density is more and most of the space remains hollow towards core 5 .

Improved permeability
Dendrimers can cross bio barriers like blood brain barrier, cell membrane. Dendrimers might show an enhanced permeability and retention effect which allows them to target tumour cells more effectively than small molecules 6 .

Improved loading capacity
Drug can get entrapped inside the internal cavities as well as electro statically in the surface of dendrimers. Dendrimers structures can be used to load and store a wide range of organic or inorganic molecules by encapsulation and absorption on surface 7 .

Higher Solublization Potential
Dendrimers improves the solubility, biodistribution, and efficacy of a number of therapeutics as well as being used as imaging and diagnostic molecules in animal models bearing brain tumors. Probable mechanism includes ionic interaction, hydrogen bonding and hydrophobic interactions 8 .

Increased permeability and retention effect
Dendrimers might show an enhanced permeability and retention effect (depending on their M.W) that allows them to target tumor cells more effectively than small molecules 9 . 6. High stability Dendrimers have nanoscopic particle size range from 1 to 100 nm, which makes them less susceptible for reticulum endothelium uptake. Dendrimers drug complex or conjugate shows better colloidal, biological and shelf-stability 10 .

High uniformity and purity
Dendrimers have uniform sizes range, well defined surface functionality, and negligible impurity.

Low immunogenicity
Dendrimers shows low or negligible immunogenic response when injected or used topically. The problems as found in vesicular system like chemical instability, drug leakage, aggregation and fusion during storage, solubility in physiological environment, lysis of phospholipids, purity of natural phospholipids are not present in dendritic system 9. Sustained/extended effect Dendrimers releases drug in a sustained manner.

Multifunctional platform
Multiple functional groups are present on outer surface of dendrimers, which can be used to attach vector devices for targeting to particular site in the body. Terminal groups may also be modified to reorganize specific receptors. The surface modification may allow designing dendrimers mimicking biological exoreceptors, substrates, inhibitors or cofactors 11 .

Low toxicity
Most dendrimers systems display very low cytotoxicity levels but have good biodegradability.

Types of Dendrimers 1. Poly amidoamine dendrimers (PAMAM)
Dendrimers: PAMAM dendrimers represent an exciting new class of macromolecular architecture called "dense star" polymers. They have twodimensions, star like pattern. These are synthesized by the divergent method starting from ammonia or ethylenediamine initiator core reagents. These dendrimers are commercially available, usually as methanol solutions 12 .

Hybrid dendrimers
Hybrid dendrimers are hybrids of dendritic and linear polymers. These are obtained by complete mono functionalization of the peripheral amines of a "zerogeneration" polyethyleneimine dendrimer, provide structurally diverse lamellar, columnar, and cubic self organized lattices that are less readily available from other modified dendritic structures 13 .

Fréchet dendrimers
It is a more recent type of dendrimer based on polybenzyl ether hyper branched skeleton. These dendrimers usually have carboxylic acid groups as surface groups, serving as a good anchoring point for further surface functionalization, and as polar surface groups to increase the solubility of this hydrophobic dendrimer type in polar solvents or aqueous media 14 .

Tecto dendrimers
These are composed of a core dendrimer, surrounded by dendrimers of several steps (each type design) to perform a function necessary for a smart therapeutic nanodevice. Different compounds perform varied functions ranging from diseased cell recognition, diagnosis of disease state drug delivery, reporting location to reporting outcomes of therapy 15 .

Chiral dendrimers
The chirality in these dendrimers is based upon the construction of constitutionally different but chemically similar branches to chiral core. Their potential use includes as chiral hosts for enantiomeric resolutions and as chiral catalysts for asymmetric synthesis 16 .

Multiple antigen peptide dendrimers
It is a dendron-like molecular construct based upon a polylysine skeleton. Lysine with its alkyl amino sidechain serves as a good monomer for the introduction of numerous of branching points. This type of dendrimer used in biological applications, e.g. vaccine and diagnostic research 17 .

Amphiphilic dendrimers
They are built with two segregated sites of chain end, one half is electron donating and the other half is electron withdrawing 18 .

Propylene Imine (PPI) dendrimers
These dendrimers are generally poly-alkyl amines having poly-alkyl amines as end groups, and numerous tertiary trispropylene amines present in interior portion. PPI dendrimers are commercially available up to G5, and has found widespread applications in material science as well as in biology 19 .

Poly amidoamineorganosilicon (PAMAMOS) dendrimers
These are radially layered poly(amidoamineorganosilicon) dendrimers (PAMAMOS) having inverted unimolecular micelles that consist of hydrophilic, nucleophilic polyamidoamine (PAMAM) interiors and hydrophobic organosilicon (OS) exteriors. These are excellent in networks regularity and have ability to complex and encapsulate various guest species offer unprecedented potentials for new applications in nanolithography, lectronics, photonics, chemical catalysis etc. These dendrimers are exceptionally useful precursors for the preparation of honeycomb-like networks with nanoscopic PAMAM and OS domains 20 .

PROPERTIES OF DENDRIMERS 1. Size and shape
Dendrimers shows improved physical and chemical properties due to their molecular architecture. The dendrimers shape depend on the generation i.e. lower generation shows open planar elliptical shape while higher generation shows compact-spherical shape. Inherent structural defects exist when synthesizing high-generation dendrimers. The reasons include incomplete reactions and steric resistance, which cause the missing of repeating units, intramolecular cyclization, dimmer formation and retro-Michael reaction in dendrimers. Due to their nanometric scales and other properties that are similar to proteins, dendrimers are also known as artificial proteins. The dendrimer can be controlled by molecular engineering so that its size resembling to antibodies, enzymes and globular proteins 21 .

Monodispersivity
The monodispersion means that the dendrimers has a well defined molecular structure and without large individual variations, in other words, they are homogeneous unlike other polymers due to their controlled synthesis and purification processes. Dendrimers are monodisperse in nature i.e. they have isomolecular species, whose molecular size, shape and disposition of organic moieties are adjusted and controlled. Monodisperse property of dendrimers has already been extensively characterized by high performance liquid chromatography, size exclusion chromatography, mass spectrometry, gel electrophoresis, and transmission electron microscopy 22 .

Polyvalency
It shows the outer arrangement of reactive groups on the exterior of dendrimer nanostructure. Polyvalency it provides for versatile functionalization; it produce multiple interactions with biological receptor sites, for example, in the design of antiviral therapeutic agents. The polivalency is related to the quantity of reactive sites on outside of the dendrimer potential to form connections with various materials of interest. The multivalencyallows better interaction with biological targets since most of the molecular interactions occur through biological multivalent bonds 23 .

Solubility and biocompability
The solubility of dendrimers is determined by the surface functional groups, dendrimer generation, repeated units, and even the core.
Generally dendrimers have greater solubility in common solvents as compared to linear polymers. If the surface end groups are hydrophobic in nature, then dendrimers are soluble in nonpolar solvent. If the surface end groups are hydrophilic in nature and dendrimers are soluble in polar solvent 24 .

Toxicity
Some dendrimer systems display very low toxicity levelswith dendrimers carrying anionic groups being less toxic than those carrying cationic groups. Dendrimers may cause toxicity mainly attributed to the interaction of the cationic dendrimers surface with negative biological load membranes damaging cellular membranes causing hemolytic toxicity and cytotoxicity. Therefore, PAMAM dendrimers are more cationic than anionic cytotoxic 25 .  . Electrochemistry gives information about the possibility of interaction of electroactive end groups. c). Electrophoresis used for the assessment of purify and homogeneity of several type of water soluble dendrimers. 5. Rheology, physical properties 28 a). Intrinsic viscosity-used as analytical probe of the morphological structure of dendrimers. b). Differential scanning calorimetry (DSC)-used to detect the glass transition temperature which depends on the moleculer weight, entangment and chain composition of polymers. c). Dielectric spectroscopy (DS)-gives information about molecular dynamic processes (α, β)

Drug and dendrimers interactions
Dendrimers interact with drug molecules physically by absorption on surface by electrostatic interactions or by conjugation with the surface groups for covalent bonding or by encapsulation of the drug into the cavities of the dendrimers. In most cases, however, the conjugated dendritic assembly functions as pro-drug "where, upon internalization into the target cell, the conjugate must be liberated to activate the drug.

Viscosity
In solution dendrimers form a tightly packed ball, which influences its rheological properties. The intrinsic viscosity dendrimers solution does not exhibit linear relationship with mass 26 .

Immunogenicity
It is one of the crucial biological properties of the dendrimers. As per research, unmodified amino terminated PAMAM dendrimers are presenting no or only weak immunogenicity of the G3-G7. PAMAM dendrimers with polyethylene glycol chains decrease immunogenicity and offers longer lifetime in the blood stream in comparison to un-modified dendrimers.

Loading capacity (molecular container property)
This property makes dendrimers very suitable as drug delivery vehicles and also appropriate for obtaining electro-optic or magnetic devices. In addition to carrying materials on their surface, the internal cavities of dendritic structures can be used to carry and/ or store a wide range of metals, organic, or inorganic molecules by encapsulation and absorption. The appropriate type (and degree) of functionalisation will results in the desired loading capacity 27 .

METHODS OF SYNTHESIS
There are two different methods of dendrimer synthesis, divergent synthesis and convergent synthesis 1. Divergent Method In this method, dendrimers grows outwards from the focal core, using a pair of basic operations that consist of coupling of building blocks, and de protection or modification of end functionalities of the periphery to create new reactive surface functionalities; this pair of basic operations is often referred to as the growth of a generation.

Figure 2: The divergent method
Each step of the reaction must be determined to full completion to prevent mistakes in the dendrimer, which can grounds trailing generations (some branches are shorter than the others). This process is repeated until the desired number of generations is obtained.To minimize these side reactions and imperfections, it"s recommended to use a large excess of reagents 28 .

Convergent Method
It is an alternative method to the divergent approach for producing precisely controlled dendritic architectures. The convergent approach overcomes some of the problems associated with the divergent method. In the convergent approach, the dendrimer is constructed stepwise. Starting from the end groups and progressing inwards. When the growing branched polymeric arms, called dendrons, are large enough, they are attached to a multifunctional core molecule.

Figure 3: The convergent method
This method makes it very much easier to eliminate impurities and shorter twigs along the way, so that the final dendrimer is more mono-disperse. The convergent approach does not allow the formation of high generations because steric problems occur in the reactions of the dendrons and the core molecule.
Reactions under a convergent approach need a relatively longer time compared to that of the divergent method 29 .

APPLICATION OF DENDRIMERS
Specific properties such as unparalled molecular uniformity, multifunctional surface and presence of internal cavities makes dendrimers suitable for a variety of high technology uses and are as follows: (a). Pharmaceutical applications 1. Dendrimers in ocular drug delivery The nanosize, ease of preparation, functionalization, and possibility to attach multiple surface groups renders dendrimers as suitable alternative vehicle of ophthalmic drug delivery. PAMAM dendrimers with carboxylic or hydroxyl surface groups, improves residence time and enhance bioavailability of pilocarpine in the eye. Also some of the phosphorus containing dendrimerswith quaternary ammonium core and terminal carboxylic groups has successfully reported for ocular drug delivery of carteolol 32 .

Dendrimers in pulmonary drug delivery
Dendrimers have also been reported for pulmonary drug delivery as well. A previous study was performed, by measuring plasma anti-factor Xa activity using PAMAM dendrimers in enhancing pulmonary absorption of Enoxaparin, and by observing prevention efficacy of deep vein thrombosis in a rodent model. it was observed that G2 and G3 generation positively charged PAMAM dendrimers increased the relative bioavailability of Enoxaparin by 40% while G2.5 PAMAM half generation dendrimers containing negatively charged carboxylic groups had no effect 33 .

Dendrimers in transdermal drug delivery
Dendrimers may improve drug properties such as solubility and plasma circulation time via transdermal formulations and to deliver drugs efficiently due to it"s highly water soluble and biocompatible nature. Drug permeation can be improved through the skin when PAMAM dendrimer complex with NSAIDs like ketoprofen, diflunisal and enhanced bioavailability of PAMAM dendrimers by using indomethacin as the model drug in transdermal drug application 29 .

Dendrimers as Nano-Drugs
Dendrimers as nano-Drugs, useful as antiviral drugs against the herpes simplex virus can potentially prevent/reduce transmission of HIV and other sexually transmitted diseases (STDs) when poly (lysine) dendrimers modified with sulfonated naphthyl groups.

Dendrimers for controlled release drug delivery
Encapsulation of 5-fluorouracil into PAMAM dendrimers(G=4) modified with carboxy methyl PEG5000 surface chains revealed reasonable drug loading, a reduced release rate and reduced haemolytic toxicity. Controlled release of the Flurbiprofen achieved by formation of complex with amine terminated generation 4 (G4) PAMAM dendrimers 26 .

Dendrimers in oral drug delivery
Oral drug delivery studies using the human colon adenocarcinoma cell line, which have indicated that low generation PAMAM dendrimers cross cell membrane through a combination of two processes, i.e. paracellular transport and adsorptive endocytosis. Increase in the cytotoxicity and permeation of dendrimers when increase in the concentration and generation 28 .

Dendrimers as bio mimetic artificial proteins
Dendrimers are often referred to as "artificial proteins" due to their dimensional length scaling, narrow size distribution, and other bio mimetic properties. For examples PAMAM family, they closely match the sizes and contours of many important proteins and bio assemblies like insulin (3 nm), cytochrome C (4 nm), and haemoglobin (5.5 nm) are approximately the same size and shape as ammonia-core PAMAM dendrimers generations 3, 4 and 5, respectively.

Dendrimers as solubility enhancer
Dendrimers are unimolecular micellar nature, due to have hydrophilic exteriors and hydrophilic interiors and form covalent as well as non-covalent complexes with drug molecules and hydrophobes, and enhance its solubilisation behaviour.

Dendrimers for boron neutron capture therapy
The radiation energy generated from the capture reaction of low-energy thermal neutrons by 10B atoms has been used successfully for the selective destruction of tissue. Dendrimers are a very fascinating compound for use as boron carriers due to their well defined structure and multivalency 33 .

(c). Diagnostic applications 1. Dendrimers as molecular probes
Dendrimers are fascinating molecules to use as molecular probes because of their distinct morphology and unique characteristics. For Example, the immobilization of sensor units on the surface of dendrimers is a very efficient way to generate an integrated molecular probe, because of their large surface area and high density of surface functionalities 34 .

Dendrimers as X-ray contrast agents
Dendrimers are currently under investigation as potential polymeric X-ray contrast agents. Potential dendritic X-ray contrast agents using various organo metallic complexes such as bismuth and tin are used to obtain a high resolution X-ray image, several diseases or organs, such as arteriosclerotic vasculature, tumors, infarcts, kidneys or efferent urinary etc. In a study Krause and co-workers synthesized a number of potential dendritic X-ray contrast agents using various organo metallic complexes such as bismuth and tin 35 .

Dendrimers as MRI contrast agents
A number of research groups have explored the use of dendrimers as a new class of high molecular weight MRI contrast agents. Introduction of target specific moieties to the dendritic MRI contrast agents, to improve the pharmacokinetic properties of dendrimer contrast agents, for example folate conjugated Gd (III)-DTPA PAMAM dendrimer, which increased the longitudinal relaxation rate of tumor cells expressing the high affinity folate receptor 36 .

CONCLUSION
Present review discussed very elaborately about characterization techniques and applications of dendrimers. The dendrimers have a promising future in various pharmaceutical applications and diagnostic field in the coming years as they possess unique properties, such as high degree of branching, multi valency, globular architecture and well defined molecular weight, there by offering new scaffolds for drug delivery. They provide platforms for drug attachment and have the ability to encapsulate or bind drugs via several mechanisms. At present many drugs are facing problems of poor solubility, bioavailability and permeability. Dendrimers can work as a useful tool for optimizing drug delivery of such problematic drugs. Review concludes that there is need of some more research on effects of dendrimers to remove the ambiguity about the safety of dendrimers and to make them an excellent substitute for polymers in the future as it is one of the youngest and exciting fields of polymers researches, where all branches of science can take part and hence, deserves more intensive attention.