Dendrimers: Therapeutic Activity and Application

P. Dinesh Kumar¹*, P. Vijayaraj Kumar²

¹Department of Biotechnology, Acharya Nagarjuna University, Guntur-522510, Andhra Pradesh, India

²Faculty of Pharmaceutical Sciences, UCSI (University College Sadaya International) University,

Jalan Menara Gading, 56000-Cheras, Kuala Lumpur, Malaysia.

*Corresponding Author E-mail: dineshclbaid@yahoo.co.in, dineshclbaid@gmail.com

ABSTRACT:

Dendrimers represent a novel class of structurally controlled macromolecules derived from a braches upon branches structural motif. These consist of highly branched moieties that radiate from a central core and synthesizes through a stepwise repetitive reaction sequence. The structural advantage of dendrimers such as size, shape, surface and interior chemistry flexibility and topology by virtue are leads to play an important role in the field of nanotechnology, pharmaceutical and medicinal chemistry. Dendrimers have emerged as highly promising drug delivery molecule because of their unique structure and properties. Solubility enhancement is an important aspect of dendrimers and this is a synergy with site specific drug delivery. The hydrophobic guests when entrapped in the hydrophobic channels of dendrimers are solubilised in aqueous solution. Extracellular matrix of tissue particularly vascular tissue it contains a high concentration of negatively charged glycosaminoglycans which are involved in regulation of cell motility cell proliferation in the regulation of enzyme activity. Extra cellular matrix can be used as a substrate for binding and retention of drug delivered intra vascularly. Recently dendrimers have caused an explosion in biomedical science and created interest in the discovery of the drugs by virtue of their therapeutic value. The dendrimer polymer suggest that they are promising drugs wound healing ,bone mineralization cartilage formation, tissue repairing topical treatment for AIDS to prevent HIV transmission .It also acts as an anti prion, anti Alzheimer, anti coagulants, anti dots, anti inflammatory and anti cancer agents.

KEYWORDS: Dendrimer, Nano-composites, Dendrimer conjugation, Dendrimer applications.

INTRODUCTION:

Dendrimer the name comes from the Greek “ɗevɗpov”/dendron meaning “tree” synonymous terms are arborols and cascade molecule^[1]. Dendrimers are repeatedly branched molecules that are characterized by structural perfection. This is based on the evolution of both symmetry and polydispersity the field of dendritic molecule can roughly be divided into

1. low molecular weight and

2. molecular weight species

The first category includes dendrimers and dendrons and the second includes dendronized polymers hyper branched polymers and brush polymers (called as bottle brushes) tailored forms and function ever realized outside of nature. Structurally dendrimers posses 3 distinct parts

1. A core

2. Branching units

3. Branches

It is usually produced in an interactive sequence of reaction steps, in each added lerative lead to a higher generation material. The size of dendrimer can be described as a function of generation (Gn where n is 0, 0.5, 1.0, and 1.5) G is number of repetition cycles. The molecular weight of the dendrimer nearly doubles with each additional generation^[2]. Furthermore, terminal groups can be modified to obtain both a charged, and hydrophilic or lipophilic function for the desired biological and drug delivery application^[3]. Following properties of dendrimer made them ideal molecule for drug delivery applications^[4]:

a. Nanoscale sizes that have similar dimensions to significant bio-building blocks,

b. Numbers of terminal surface groups (Z) appropriate for bioconjugation of drugs, signalling groups, targeting moieties or biocompatibility groups.

c. Functional groups on the surfaces were designed to augment or resist trans-cellular, epithelial or vascular biopermeability.

d. An interior void space was used to encapsulate drug molecule, metals, or imaging moieties and also reduces the drug toxicity and facilitates controlled release.

e. Positive biocompatibility patterns that are coupled with lower generation anionic or neutral polar terminal surface groups.

f. Low-immunogenicity with modified dendrimer surfaces by small functional groups or polyethylene glycol (PEG).

Dendrimers a nano particle based drug delivery system have numerous applications in many fields such as supramolecular chemistry or host–guest chemistry^[5,6], electrochemistry^[7], and photochemistry^[8], nanoparticle synthesis^[9-11] pollution management^[12-14], dye decolorization^[15,16], preparation of monomolecular membranes^[17-19], curing of epoxy resins^[20,15], catalysis^[21-24], drug delivery^[25-29], and gene transfection^[30-32]. In recent, dendrimers usage in drug delivery had attain great development and different types of dendritic macromolecules have been synthesized and investigated as a carrier for drug delivery^[28], gene delivery^[33], targeting^[34], solubilization^[35], diagnosis^[36], chemical catalysis^[37] and as multivalent ligand for interesting biological applications^[25,38].This review article intends to provide the reader with a glimpse into the synthesis, types and important applications of dendrimers.

SYNTHESIS OF DENDRIMERS

The first synthetic procedure towards well defined branched structures was reported by vogle’ in 1978, who named this procedure a “cascade synthesis” .In the early 1980’s, Denkewalter patented the synthesis of L-lysrine-based dendrimers^[39]. The first dendritic structures that were exhaustively investigated and that received widespread attention were Tomalia’s PAMAM (polyamidoamine) dendrimers^[40] and newkome’s “arborol” systems^[41]. In the synthesis of dendrimers, monomers lead to a mono disperse polymer, tree like generational structure. There are 2 methods of dendrimer synthesis they are

1. Divergent synthesis

2. Convergent synthesis

Divergent Synthesis

The dendrimer is prepar ed from the core as the starting point and built up generation by generation. In the divergent way, problems occur from an incomplete reaction of the end groups, since these structure defects accumulate with the buildup of further generation. Divergent name is derived from the manner in which dendrimer grows outward from the core. The higher generations of divergently constructed dendrimers always contain certain structural defects. To prevent side reaction and to force reactions to completion a large excess of reagent is required

Convergent Synthesis

The convergent synthesis starts from the surface and ends up at the core, where the dendrimer segments (dendron) are coupled together. Two of these end tips are attached to a branched monomer to form a dendron and the process is repeated until a desired size is reached. These interconnected branches are then attached to a core molecule. To synthesize dendrimers is difficult because a repeated reaction which consists of many steps is needed to protect the active site even in both methods. That why these are obstacles to the synthesis of large quantities of dendrimers^[42].

Fig 1 PAMAM Dendrimers

Other alternative preparation methods have been developed that aim to reduce the number of synthetic and purification steps and increase yields, such as the double-stage convergent growth approach^[43,44], double-exponential dendrimer growth approach^[45], and orthogonal coupling^[46].

Types of dendrimers

Now days, dendrimers with different designed functionalities have become objects of particular academic and practical interest because of their unique superbranched architectures, high densities of peripheral functionalities, symmetrical shapes, and monodispersity. Here, some of the dendrimers having different functionalities are briefly described.

PAMAM dendrimer

The PAMAM (polyamidoamine) dendrimers are synthesized up to generation 10 (G10) by the divergent method starting from ammonia or ethylene diamine initiation cone reagents (fig. 1). They are constructed using a reiterative sequence consisting of a double Michael addition of methyl acrylate to a primary amino group followed by amidation of the resulting carbomethoxy intermediate with a large excess of ethylene diamine. Many surface modified PAMAM dendrimers are non-immunogenic, water-soluble and possess terminal-modifiable amine functional groups for binding various targeting or guest molecules. PAMAM dendrimers generally display concentration-dependent toxicity and haemolysis. PAMAM dendrimers are hydrolytically degradable only under harsh conditions because of their amide backbones, and hydrolysis proceeds slowly at physiological temperatures^[47]. The internal cavities of PAMAM dendrimers can host metals or guest molecules because of the unique functional architecture, which contains tertiary amines and amide linkages. PAMAM dendrimers are the most extensively reported moiety for almost all existing applications of dendrimers.

PPI dendrimers

Poly (propyleneimines) dendrimers (fig. 2) were created by Meijer at DSM of the Netherlands^[48]. PPI dendrimers up to generation 5 are synthesized by the divergent method starting from 1, 4. diamino butane. They grow by a reiterative sequence consisting of (A) a double Michael addition of acryloritrile to the primary amino groups followed by (B) Hydrogenation under pressure in the presence of Raney cobalt. Today, these PPI dendrimers are synthesized in large quantities by DSM and are commercially available. DSM uses its own designation to describe its dendrimers, where the core is diaminobutane, dendrindictes the interior dendritic branch cell; and n is the number of end groups.

Liquid crystalline dendrimers

These are mesogenic (liq. crystalline) monomers e.g. mesogen functionalized carbosilane dendrimers. Functionalization of end group of carbosilane dendrimers with 36 mesogenic units, attached through a C-5 spacer, leads to liquid crystalline dendrimers that form broad smetic A phase in the temperature range of 17–130C^[49]. Boiko et al. had synthesized first photosensitive liquid crystalline dendrimer with terminal cinnamoyl groups^[50].

Fig 2 PPI Dendrimers

They have confirmed the structure and purity of this LC dendrimer by 1H NMR and GPC methods. It was shown that such a dendrimer, under UV irradiation, can undergo E-Z isomerisation of the cinnamoyl groups and [2 + 2 ] photocycloaddition leading to the formation of a three-dimensional network.

Tecto dendrimers

Tecto-dendrimers are composed of a core dendrimer, which may or may not contain the therapeutic agent, surrounded by dendrimers of several types, each type designed to perform a function necessary to a smart therapeutic nanodevice^[51]. The Michigan Nanotechnology Institute for Medicine and Biological Sciences (M-NIMBS) are developing a tecto dendrimers which are used to perform the functions like diseased cell recognition, diagnosis of disease state, drug delivery, reporting location and reporting outcome o f therapy. The future planning was to produce a smart therapeutic nanodevice for the diseased cell like a cancer cell or a cell infected with a virus.

Chiral dendrimers

In chiral dendrimers the construction of core was based on different constitution but with similar chemical branches. Asymmetric catalysis and chiral molecular recognition are the main applications of chiral, nonracemic dendrimers^[52].

PAMAMOS dendrimers

PAMAMOS (poly amidoamine-organosilicon) are radially layered, inverted unimolecular micelles that consist of hydrophilic, nucleophilic polyamidoamine (PAMAM) interiors and hydrophobic organosilicon (OS) exteriors. These are exclusively useful for the preparation of honeycomb like networks with nanoscopic PAMAM and OS domains^[53].

Hybrid dendrimers

Hybrid dendrimers are combination of dendritic and linear polymers in hybrid block or graft copolymer forms. The small dendrimer segment coupled to multiple reactive chain ends provides an opportunity to use them as surface active agents, compatibilizers or adhesives, e.g. hybrid dendritic linear polymers^[54].

Peptide dendrimers

Peptide dendrimers are defined as dendrimer containing peptides on the surface of the dendrimer frame work with amino acids as a branching (or) core unit. Biological and therapeutical relevance of the peptide dendrimers with the peptide molecule make them a potential candidate for various drug delivery systems. The main applications of the peptide dendrimers includes cancer, antimicrobials, antiviral, central nervous system, analgesia, asthma, allergy, Ca⁺² metabolism, magnetic resonance imaging (MRI), magnetic resonance angiography (MRA), fluorogenic imaging and serodiagnosis^[55,56].

Glycodendrimers

Dendrimers that incorporate carbohydrates into their structures are termed as glycodendrimers. Glycodendrimers are three types (i) carbohydrate-coated; (ii) carbohydrate centered; and (iii) fully carbohydrate-based. Glycodendrimers have been used to study the protein–carbohydrate interactions that are in many intercellular recognition events. The main applications of glycodendrimers are study of protein–carbohydrate interactions, incorporation into analytical devices, formulation of gels, targeting of MRI contrast agents, drugs and gene delivery systems^[57,58].

Applications of dendrimers

Dendrimers have attracted the most attention as potential drug delivery scaffolds due to their unique characteristics. Dendrimers have narrow polydispersity; nanometer size range of dendrimers can allow easier passage across biological barriers. Dendrimers can be used to deliver drugs either by encapsulating the drug in the dendrimer interior void spaces or by conjugation to surface functionalities. All these properties make dendrimers as suitable carrier for drug delivery.

Dendrimers in transdermal drug delivery

Now day’s dendrimers had key role for the improvement transdermal drug delivery system. Delivery of the drug via transdermal formulation is difficult because of the hydrophobic nature and inefficient cell entry. Highly water soluble dendrimer are designed which improve the drug solubility, plasma circulation, and entry to cells make efficiently delivery drug from transdermal formulation.

Nonsteroidal anti inflammatory drug (NSAIDs) used for acute and chronic rheumatoid and osteoarthritis are limited there clinical usage by adverse events such as dyspepsia, gastrointestinal bleeding and renal side effects when give orally. Transdermal formulation will overcome adverse events and also provide good therapeutic blood level maintains for longer time. But poor rate of transcutaneous delivery pulls down transdermal delivery system. Drug permeation through the skin was enhanced by PAMAM dendrimer complex with NSAIDs (Ketoprofen, Diflunisal) as skin penetration enhancers. Permeation studies on rat skin were carried out for ketoprofen and diflunisal drug. High permeation was achieved by drug dendrimer complex (ketoprofen 3.4times and diflunisal 3.2times) when compared to drug. Antinociception effect of ketoprofen shows that dendrimer complex reduced writhing for period 1-8hr but drug reduced writhing up to 4-6hr.

In another study indomethacin and PAMAM dendrimer investigated^[59]. In-vitro and in-vivo studies were carried out for PAMAM dendrimer complex. In-vivo pharmacokinetic and pharmacodynamic studies in Wistar rats showed that significant higher concentration and effective concentration could be maintain for 24h in blood by G4 dendrimer indomethacin transdermal formulation.

Various transdermal penetration enhancers based on chemical and physical approach were carried out chemical penetration enhancers such as sulfoxide, oxazolidionesis, fatty acids essential oil, pyrrllidoions, terpenes and terpenoirds were used. Inotophoresis, electrophoresis, ultrasound, gel and patch are physical penetrates which used to exchange absorption of drug ^[60-62].

Recently Zhao et al conjugated PEGylated PAMAM dendrimers for transdermal delivery of bioactive molecules delivery of bioactive by pre-treatment or co treatment technique using different vehicle lime water, chloroform isopropyal myristal chloroform water mixture and octanal water mixture emulsion. Further he reviewed the three different mechanisms which use to deliver the bioactives^[63].

In another study Welowie et al used that PAMAM dendrimers to conjugate 8-methoy psiralae (a photo sentizier for puva therapy)^[64]. Here solubility of 8-methoxypsiralane PAMAM conjugate increased. Moreover in another study solubility of riboflavin was enhanced with increase in generation of PAMAM dendrimers. Moreover diffusion of riboflavin in pig ear skin was enhancing with increase in generation.

Moghmin et al show that furful permeation enhances through rat skin model using pamam dendrimers (G5) in water vehicle by pretreatment^[65].

Yang et al reported that smaller G2 pamam dendrimers penetrate the skin layers more efficiently than the larger ones (G4) ^[66]. Moreover conjugation of oleic acid to G2 dendrimers increases their 1-octanol/PBS partition coefficient, resulting in increased skin absorption and retention. Here permeation across skin layers is directly based on the size, surface charge and hydrophobicity of PAMAM dendrimers (fig. 3).

In transdermal applications nanoparticles (polysacchird and dendrimers) are used to increase the potential of transdermal drug delivery system. Permal and co had extensive research work on dendrimer application in transdermal system they reveal that physico chemical properties of dendrimers play a vital role in delivery of drug by increase the penetration^[67].

Therefore data suggested that dendrimer drug complex make transdermal delivery system was effective and might be a safe and efficacy method for treating different diseases.

Dendrimers in oral drug delivery

Traditional Oral drug-delivery system has been the dominant route for many years because of its significant advantages. A major challenge for drugs is the possibility of oral delivery, but main drawback was the limited drug transport across the intestitinal epithelium due to their large size relative to the tight epithelial barrier of the gastrointestinal tract. Duncan’s and his research group showed that macromolecules of 3nm diameters could penetrate through the rat’s intestinal membranes, which allows G2.5-G3.5-PAMAM dendrimers to transport across the intestine^[69]. Moreover the acidic nature of the GI-tract enzymes and stomach can affect the drug and the nanocarrier.

Fig 3 Schematic representation of the internalization mode of PAMAM dendrimers with different surface attachments^[66].

D’Emanuele group investigated effect of dendrimer generation and conjugation on the cytotoxicity, permeation and transport mechanism of surface-modified cationic G3-PAMAM propranolol dendrimer conjugation across Caco-2 cell monolayers^[70]. They suggested that the route of propranolol transport was initially transcellular, while the conjugate was able to bypass the P-gp efflux transporter, and they arrived as the same inference as above concerning the penetration pathway of the intestinal membrane. Najlah investigated transepithelial permeability of naproxen, a low solubility drug^[71]. Stability studies of G0 PAMAM conjugates in 50% liver homogenate was compared to that in 80% human plasma showed the lactate ester linker gave prodrug of elevated stability in plasma with sluggish hydrolysis in liver homogenate. So, these conjugations exhibit potential nanocarriers for the enrichment of oral bioavailability. The Cheng and Xu group, reviewed that a PAMAM dendrimer complex of the anti-inflammatory drug ketoprofen sustained antinoninceptive activity (inhibit rate > 50%) until 8 h of oral administration to Kunming mice, whereas this activity was absent with the free drug after 3 h^[72]. Increase in permeability and cellular uptake was produced by G4- PAMAM 7-ethyl-10-hydroxycamphtothecin complexation with respect to free 7-ethyl-10-hydroxycamphtothecin. They reported that complex has the potential to improve the oral bioavailability of drug.

Lin et al carried out study on effects of PAMAM dendrimer in intestinal absorption of poorly absorble drug such as 5(6)- carboxyfluorsin isothicynate dextran, calctitonin and insulin in rat^[73]. Drug carboxylorescin and calcitonin showed increase in absorption in rats small intestine for 0.5%w/v G2 PAMAM dendrimer complex. But fluorescine isothiocynate dextran and insulin had not produced any desirable effects. Moreover absorption in small intestine is mainly base on molecular weight of drug ie the molecular weight of drug increase absorption of drug decreases.

Recently Kolhatkar et al explored oral delivery of SN – 38 (a potent topisomers –I inhibtor) and active metabolize of irinotecan hydrochloride (cpt-11) was improved by conjugation with G4 PAMAM dendrimer.10 fold increase in caco₃ cell monolayer and 100 fold increase in cellular uptake by SN-38 and G4 PAMAM dendrimer than plain drug^[74].

Dendrimers in targeted drug delivery

There is great interest in developing new targeted delivery systems for drugs that are already on the market, especially cancer and tumor therapeutics. Most of the current chemotherapeutic agents on the market are low molecular weight agents with high pharmacokinetic volume of distribution both of which contribute to their cytotoxicity. Moreover, the low molecular weight of these chemicals makes them easily excreted, hence a higher concentration is ultimately required, and consequently a higher toxicity is unavoidable. Their low therapeutic index does not contribute favourably to this dilemma, as the needed concentration for the effective treatment must always be reached, but unfortunately the therapeutic levels are often exceeded. Additionally, these drugs when administrated alone, lack specificity and cause significant damage to noncancerous tissues. This results in serious, unwanted side effects such as bone marrow suppression, hair loss (alopecia), and the sloughing of the gut epithelial cells. Moreover most chemotherapeutic agents have poor solubility and low bioavailability, and are formulated with toxic solvents. Thus, the use of dendrimers allow for the preparation of low water soluble cancer medications in liquid formulations. Ideally, dendrimers will allow for more specific targeting of the drug, thereby improving efficacy and minimizing side effects. By using dendrimers in drug design and delivery, researchers are trying to push dendrimers to be able to deliver the drug to the targeted tissue, release the drug at a controlled rate, be a biodegradable drug delivery system, and to be able to escape from degradation processes of the body.

Jesus and group had explored the possibility of a 2, 2-bis (hydroxymethyl) propanoic acid based dendritic scaffold as a delivery carrier for doxorubicin in vitro and in vivo^[75]. The dendrimer doxorubicin formulation covalently bound through a hydrazone linkage to a high molecular weight 3-arm polyethylene oxide; exhibits reduced cytotoxicity in vitro. However, in vivo biodistribution experiments showed minimal accumulation in vital organs, including the liver and heart, and increased half-life of doxorubicin compared to the free drug. Thus, it was hypothesized that proper choices of nanocarrier systems can increase the circulation half-life to effectively exploit the enhanced permeation retention (EPR) effect phenomenon and thus have tremendous potential to increase the efficacy of the drug to a greater extent. Malik et al synthesized cisplatin PAMAM dendrimer conjugate^[76]. The conjugate shows increased solubility, reduced toxicity and EPR properties. It was observed that this formulation showed superior activity over cisplatin when injected into mice bearing B16F10 tumor cells.

Zhou and colleagues synthesized (‘time-sequenced propagation technique’)poly (amide-amine) based dendrimers and conjugated with 1-bromoacetyl-5- fluorouracil to form dendrimer–5FU conjugates^[77]. In vitro studies revealed that the release of 5FU depends on the dendrimer generation, and indicated that this could be a promising carrier for the antitumor drugs. A study by Lee group showed the viability of polyester-based dendrimer–PEO–doxorubicin conjugate to substantially inhibit the progression of DOX-insensitive C-26 tumor subcutaneously implanted in BALB/c mice^[78]. This dendrimer–PEO–doxorubicin conjugate also showed the capability to eliminate the tumours as compared to drug. Bhadra et al used PEGylated PAMAM dendrimers for the incorporation of 5FU^[79]. It was observed that this is formulation is appropriate for prolonged delivery of anticancer drugs by in vitro and blood-level studies in albino rats, without producing any significant hematological disturbances. Drug leakage and hemolytic toxicity were reduced by PEGylation there by improve drug-loading capacity and stability. Asthana et al flurbiprofen PAMAM dendrimer formulation was synthesized and observed initial rapid release (more that 40% till 3rd hour) followed by slow release of loaded drug^[80]. In vivo study was performed in albino rats, using carrageenan induced paw edema model, revealed 75% inhibition at 4^th hour that was maintained above 50% till 8th hour. Dendritic formulation compared to free drugs showed 2-fold and 3-fold increased in mean residence time and terminal half-life, respectively. Choi and colleagues synthesized oligonucleotides linkage PAMAM dendrimers conjugated with the folic acid and fluorescein isothiocyanate for targeting the tumor cells and imaging respectively^[81]. DNA-assembled dendrimer conjugates were evaluated in vitro to detecting tumor cell-specific binding and internalization. These DNA-assembled dendrimer conjugates may allow the combination of different drugs with different targeting and imaging agents. Bhadra et al produced PPI dendrimers galactose conjugated load with primaquine phosphate, a liver schizonticide^[82]. In vivo evaluation of these formulations in Sprague–Dawley (SD) rats indicated that the primaquine phosphate accumulated mainly in liver were 30.7±2.6%, 25.7±2.89% and 50.7±5.9% for free primaquine, uncoated PPI dendrimer-primaquine and galactose coated PPI dendrimer-primaquine respectively. But after 2 h, drugs were found in blood for free primaquine 18.5±0.89%, uncoated PPI dendrimer-primaquine 25.7±2.89% and galactose coated PPI dendrimer-primaquine 7.8±0.76% respectively. These results showed that galactose coating could endure the dendrimers with more effectively targeting ability and reduce the hematological toxicity and hemolytic toxicity. During another study, folic acid was conjugated to dendrimers as targeting agent and then coupled with a model drug Methotrexate^[83]. These conjugates were injected to immunodeficient mice bearing Human KB tumors and evaluated. Biocompatibility of these macromolecules was found from animal weight examination and histopathology of the liver, spleen, and kidney after administration of these conjugates. Folic acid targeted dendrimers and Methotrexate conjugate was found to be much more effective than free Methotrexate as well as dendrimer- Methotrexate conjugate in this study. Confocal microscopy images obtained of tumors after 15 h of i.v. injection showed a considerable number of fluorescent cells with targeted dye-conjugates. Further conformation were analysed with isolated cell suspension of tumor cell.

Kostas Kostarelos et al studied the complexation of the chemotherapeutic drug doxorubicin (DOX) with the novel sixth-generation cationic poly-L-lysine dendrimer^[84]. DOX- dendrimer complex (at 1:10 molar ratio) has enhanced penetration into prostate 3D multicellular tumor spheroids (MTS) compared to the free DOX. Moreover DOX_DM complexes achieved a significantly higher cytotoxicity in DU145 MTS system compared to the free drug. Further incubation of MTS with low DOX concentration (1 μM) complexed with dendrimer led to a significant delay in MTS growth compared to untreated MTS or MTS treated with free DOX. DOX-dendrimer complex achieved good retention in a Calu-6 lung cancer xenograft model in tumor-bearing mice.

Xiangyang Shi et al report here a general approach to using multifunctional poly(amidoamine) (PAMAM) dendrimer-based platform to encapsulate a potential anticancer drug 2-methoxyestradiol (2-ME) for targeted cancer therapy^[85]. Release studies showed that 2-ME complexed with the multifunctional dendrimers released in a sustained manner. 3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay in conjunction with cell morphology observation demonstrates that the dendrimer G5-2-ME complexes can specifically target and display specific therapeutic efficacy to cancer cells overexpressing high-affinity FAR. This study suggests that multifunctional dendrimers may be used as a general drug carrier to encapsulate various cancer drugs for targeted therapy of different types of cancer.

Doxorubicin (DOX), an effective anticancer drug, was used by Umesh Gupta et al to develop and explore the anticancer potential of the dendrimer based formulations^[86]. DOX was loaded (approximately 26 and 65%) to the PPI dendrimers as well as folate conjugated PPI (PPI–FA) dendrimers, respectively. In vitro drug release of the formulation was found to be faster in the acidic media than at the higher pH. The prepared formulation displayed a higher cell uptake in MCF-7 cancer cell lines as evidenced by fluorescence studies. The results suggested that, in future, folic acid conjugated PPI dendrimers may emerge as a better choice for anticancer drug targeting.

Garcia-Vallejo and co used Leb-conjugated poly(amido amine) (PAMAM) dendrimers to characterize the optimal level of multivalency necessary to achieve the desired internalization, lysosomal delivery, Ag-specific T cell proliferation, and cytokine response^[87]. Increasing DC-SIGN ligand multivalency directly translated in an enhanced binding, which might also be interesting for blocking purposes. Internalization, routing to lysosomal compartments, antigen presentation and cytokine response could be optimally achieved with glycopeptide dendrimers carrying 16–32 glycan units. This report provides the basis for the design of efficient targeting of peptide antigens for the immunotherapy of cancer, autoimmunity and infectious diseases.

El-Sayed et al reported that the coupling of N-acetylgalactosamine (NAcGal) to generation 5 (G5) of poly(amidoamine) (PAMAM-NH2) dendrimers via peptide and thiourea linkages and produced NAcGal-targeted carriers used for targeted delivery of chemotherapeutic agents into hepatic cancer cells^[88]. Result showed that uptake of NAcGal-targeted G5 dendrimers into hepatic cancer cells occurs via ASGPR-mediated endocytosis. Further internalization of these targeted carriers increased with the increase in G5 concentration and incubation time following MichaeliseMenten kinetics characteristic of receptor-mediated endocytosis. Based on the result G5-NAcGal conjugates function as targeted carriers for selective delivery of chemotherapeutic agents into hepatic cancer cells (fig. 4).

Fig 4 A schematic drawing showing the composition of a drug-loaded G5-NAcGal conjugate binding to the ASGPR expressed on the surface of hepatic cancer cells (e.g. HepG2), which triggers receptor-mediated endocytosis of these G5-NAcGal conjugates followed by endosomal escape and release of the therapeutic cargo into the cytoplasm while the ASGPR recycles back to the cell surface ^[88].

Arun Kumar Gupta et al synthesised 4.0 G PAMAM dendrimer and conjugated with Gallic acid [GA] for cancer targeted drug delivery system^[89]. The Cytotoxicity study revealed that the conjugate is active against MCF-7 cell line and might act synergistically with anti-cancer drug and gallic acid–dendrimer conjugate might be a promising nano-platform for cancer targeting and cancer diagnosis.

Dendrimers in gene delivery

Gene therapy is an approach that aims to cure inherited and acquired diseases by correcting the overexpression or underexpression of defective genes. The success of gene therapy is largely dependent upon the development of a vector that delivers and efficiently expresses a therapeutic gene in a specific cell population. To administer a therapeutic gene (genetic medicine) into the body of the patient, a delivery system is required. These medications include gene therapy, DNA vaccination, ribozymes, and antisense oligonucleotides. In gene therapy, successful DNA transfer results in the production of therapeutic protein that is encoded by the transgene. Viruses and chitosan have been discarded due to severe toxicity problems. Then, the difficulty is that some nonviral synthetic vectors are insufficiently efficient to transfer genes into the interior of the nucleus. Also, the carrier must be released from the endosome following endocytosis. Dendrimers are much more stable than liposomes and present the advantage of precise design of the size, monodispersity, generation, and nature of termini. The most common dendrimers used today in gene delivery are polyethyleneimine and fractured PAMAM dendrimers.

Binding of DNA to polyamidoamine Starburst dendrimers through ethidium bromide binding and fluorescence were done by Chen et al^[90]. The ethidium bromide did not readily displace the dendrimer, and only intercalated with unbound regions. Increase of DNA regions from 3.2 base pairs for a G2 dendrimer to 106 base pairs for a G7 dendrimer.

Protonable natures of nitrogen present in Polypropylene dendrimers made them ideal DNA binding agents. A study of polypropyleneimine (PPI) dendrimers found that DNA binding increased as dendrimer generation increased. But increase cytotoxicity in higher generation dendrimers pull down their usage in gene therapy.

In surface treatment of PPI dendrimers with methylated quaternary amines showed improved DNA complexation and decreased cytotoxicity. A study by Kukowska-Latallo et al reported that high levels of gene expression were found by intravenous administration of G9 PAMAM dendrimer-complexes pCF1CAT plasmid into rats in the lung tissues^[91]. Kihara et al synthesized a-cyclodextrin surfaced G3 PAMAM dendrimer conjugate^[92]. Further intravenous administration of this conjugate showed high level transgene expression in spleen. Based on above study, Wada et al synthesized a new gene transfection agent by conjugation of mannose to this hybrid material^[93]. After intravenous injection of the new hybrid material bearing mannose ligand showed higher transfection activity than dendrimer alone and the hybrid material without mannose ligand in the kidney.

Mamede et al used 111In-oligo/G4100 and 111In-oligo/G4-bt-Av100 as gene transfer vectors and in vivo biodistribution evaluation showed more accumulation in kidney and lung when compare to liver^[94]. Furthers authors summarized that the positively charged DNA/dendrimer complexes condensed to form complexes of several nanometres and resulted in uptake by lung tissues. Protonable natures of nitrogen present in Polypropylene dendrimers made them ideal DNA binding agents.

A study of polypropyleneimine (PPI) dendrimers found that DNA binding increased as dendrimer generation increased. However increase cytotoxicity in higher generation dendrimers pulls down their usage in gene therapy.

A study by Schatzlein et al. showed surface treatment of PPI dendrimers with methylated quaternary amines improved the DNA complexation and decreased cytotoxicity^[95].

Different generations of PPI dendrimers as transfection agents and target gene efficiently expressed in the liver were studied by Dufes and groups^[96]. They demonstrated that intravenous administration of a gene medicine and G3 PPI dendrimer complex could result in intratumoural transgene expression and regression of the established tumours in all the experimental animals.

Various modified PPI dendrimers were used as effective transfection agents for catalytic DNA enzymes by Tack group. Intravenous administration of G4 PPI dendrimer-PEG conjugate and DNA complex into Nude mice showed high gene transfection efficacy and nuclear uptake.

Zhongwei et al synthesis and characterized arginine functionalized peptide dendrimer-based vectors ranging from 5th generation (G5A) to 6th generation (G6A) via click chemistry, and their use for gene transfection in vitro and in vivo^[97]. In vitro studies showed that the functionalized peptide dendrimers provided serum independent and high transfection efficiency on all studied cells, as over 2 fold higher than that of branched polyetherimide (PEI) in the presence of serum. Dendrimer G5A with molecular weight of 17 kDa demonstrated 6-fold transfection activity than PEI in breast tumor models, as well as good biosafety proved by in vitro and in vivo toxicity evaluation. However, G6A with molecular weight of 46 kDa showed much higher cytotoxicity.

Li Ming Zhang and co tested the star-shaped polymer consisting of β-cyclodextrin core and poly(amidoamine) (PAMAM) dendron arms [β-CD-(D3)7] as the vector to transfect the human neu-roblastoma SH-SY5Y cells^[98]. The human neuroblastoma SH-SY5Y cells, β -CD-(D3)7/pDNA complex demonstrated a lower toxicity compared to those of PAMAM (G 4)/pDNA complex. When the N/P ratio was over 20, it was observed that PAMAM had a faster increment in toxicity compared to β-CD-(D3)7. Fluorescent image, confocal microscopy image and flow cytometry showed that β -CD-(D3)7/pDNA complexes had significantly higher transgene activity than that of PAMAM/pDNA complexes. These results indicated that β-CD-(D3)7 might be a promising candidate for neurotypic cells gene delivery with the characteristics of good biocompatibility, relatively high gene transfection capability and potential in vivo gene delivery ability.

A comparative gene transfection study between PAMAM G4 dendrimers and the surface modified dendrimers was conducted in HEK 293T, GM7373 and NCI H157G cell lines by Srinath Palakurthi and co^[99]. Effect of excess of ornithine (100µM) on transfection efficiency of the ornithine-conjugated PAMAMG4 dendrimers was investigated in separate experiment. Transfection efficiency of PAMAMG4-ORN60 dendriplexes was slightly higher in cancer cells (NCI H157G) as compared to HEK 293T cells. Transfection efficiency of the PAMAMG4-ORN60 dendrimers decreased in presence of excess of ornithine while there was no effect on the parent PAMAMG4 dendrimers. It may be concluded that the ornithine-conjugated dendrimers possess the potential to be novel gene carrier.

Helena Tomás et al synthesis G5 PAMAM dendrimers and complex with plasmid DNA for gene delivery^[100]. Gene expression in MSCs, a cell type with relevancy in the regenerative medicine clinical context, is also enhanced using the new vectors but, in this case, the higher efficiency is shown by the vectors containing the smallest hydrophobic chains.

Zhongwei et al report the synthesis and characterization of different generations of dendritic poly(L-lysine) vectors, and their use for in vitro gene transfection^[101]. The higher generations tended to produce the greater positive potentials, indicating a stronger potency of the complexes to interact with negatively charged cell membranes. In vitro and in vivo cytotoxicity evaluations showed good biocompatibility of the dendrimers and their complexes over the different N/P ratios studied. In vitro gene transfection revealed higher efficiency of G5 than other dendrimers and insensitive variation to the presence of serum. Given its similar transfection efficiency to PEI but lower toxicity to cultured cells, dendrimer G5 could be a better candidate for gene delivery.

Based on these results, we concluded that dendrimers were promising gene vectors which might be able to deliver gene into liver, spleen, lung, kidney, and even the tumor at therapeutic levels, and the intravenous administration route should be a suitable route in these applications.

Dendrimers in pulmonary drug delivery

Bai and groups investigated Enoxaparin PAMAM dendrimers complex for pulmonary drug delivery^[102]. In this research enoxparin- PAMAM dendrimer complex were formulated and evaluated for the drug enachment. The dendrimer formulation was administered into lungs of anaesthetized rats and drug absorption was observed by measuring plasma anti-factor Xa activity, and by observing prevention efficacy of deep vein thrombosis in a rodent model. Bioavailability of enoxaparin was increased to 40% in G2 and G3 PAMAM dendrimers which are positively charged. They reported that positively charged dendrimers are suitable carrier for pulmonary delivery of Enoxaparin.

C. A. Lemere and coworkers described the boosting effect with intranasal dendrimeric Aβ1-15 (16 copies of Aβ1-15 on a lysine tree) but not Aβ1-15 peptide affording immune response following a single injection of Aβ1-40/42 in heterozygous APP-tg mice^[103].

Kannan et al carried study on in-vivo efficacy of methylprednisolon conjugate G4 PAMAM dendrimers showed good lung anti inflammation potency^[104]. Further methylprednisolon-G4-PAMAM dendrimers conjugate at the dose of 5mg/kg improved the airway delivery in pulmonary inflammatory model based on a 11 fold enchament of eosinophil lung accumulation following five daily inhalation exposure of sensitized mice to allergen and albumin. Here allergen induced inflammation reduced by drug loaded dendrimer conjugate was mainly base on improved drug residence time in the lung.

Yammoto et al carried out invivo pulmonary absorption on for G0-G3 PAMAM dendrimers conjugates of insulin and calction^[105]. Here absorption of insulin and calction was increased by PAMAM dendrimers conjugates. Moreover absorption rate was increased as generation of PAMAM increases.

To target regional lung deposition dendrimers emerged has very powerful carries in nano size. Review paper by carvalho et al and choi et al has explained the important and influence of particle size, charge, and coating on lung deposition^[106,107]. Dendrimers posses characteristic to emerge as nanocarrier for delivery bioactives through inhalation route.

CONCLUSIONS:

The application of dendrimers to drug delivery system has experienced rapid growth. Dendrimers are expected to play key role in pharmaceutical field as drug carriers. Dendrimers role in the biomedical applications is widely expanded. The supramolecular properties of the dendrimers made them major agent to delivery drugs and other function. As per reviewed in this article dendrimers are widely used in encapsulation various drugs and to deliver the drug to the targeted site. More over high level of controllable features of dendrimers such as size, shape, branching length and surface modifications make them an ideal drug carrier. Further dendrimers offer generation number and terminal groups and the chance to introduce two or more functional group types at the periphery are mammoth advantages of dendrimers over polymers. Few drawbacks like toxicity, localization, bio-distribution and costly synthesis step pull them down. In spite of above drawbacks, several dendrimers have already been commercialized, and some are in clinical trials. To make dendrimers commercial successful tool for drug delivery more research work has to be done on cost effective synthesis, toxicity reduction and drug conjugation. As reviewed in this article dendrimer moiety hold great promise and potential tool for drug delivery system.

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Received on 09.11.2013 Modified on 15.01.2014

Accepted on 28.01.2014 ©A&V Publications All right reserved

Res. J. Pharm. Dosage Form. and Tech. 6(2):April- June 2014; Page 12-133