Atael, Abdollah

Brown, John W.

Foot, Peter J. S.

Kingston University Research Repository

   Synthesis and properties of novel dendrimers for molecular dAbdollah Ataei 1, Peter J. S. Foot 1,*, John W. Brown1Materials Research Center, School of Life Science, PharmacyABSTRACT The primary aim of this investigation was to synthesizmodel non-polar drug (a water-insoluble dye) at a low generation number. Fairly longto help encapsulate the non-polar guest molecules efficiently at a low generation number. 3,5building block, and ethyl 6-bromohexanoate as a branching unit; two different cororder to assess the effects of the different structures on the dendrimers’ ability to encapsularelevant physico-chemical properties of these new molecules were investigated using surface tension, molar conductivity, dye encapsulation and the normal spectrometric techniques.Keywords: Dendrimer, oil red O, dye encapsulation, hyperbranched polymer, 1. INTRODUCTION  Hyperbranched polymers, and most notably dendrimers, constitute a class of macromolecules whose distinctive properties such as their degree of branching, multi-valency and defined molecular shape can make them suitable as platforms for molecular encapsulation and applications including drug delivery [1]. Dendrimers can offer two different interfacial environments [2], namely their interior and their outer surfaces. The outer surface determines molecular features such as solubility, and is where the terminal groups are located, providing an interface with the surrounding medium. When a hydrophilic substituent is introduced onto this surface (the periphery) a dendrimer becomes  2. EXPERIMENTAL SECTION Abbreviations used in this work:  DCM= dichloromethane; THF = tetrahydrofuran. The letter G is used for the generation number, and C for the number of phenyl groups in the core (2C for a biphenyl and 3C for terphenyl core). 2.1. Materials and methods.  All the synthetic chemicals used in this research were purchased from Sigma Aldrich Ltd. (UK) or Lancaster Ltd., and were used as supplied unless stated otherwise. All solvents used during the reaction, work-up and purification procedures were supplied by the Fisher Chemical Co. Electrical conductivity measurements [8] were made using a conductivity meter (Wooden Precision Apparatus Limited, CMD 830 WPA), calibrated by a standard aqueous KCl solution.   Solution surface tensions were measured using the De Nouy ring method [9,10], due to its simplicity and accuracy. In the dye encapsulation experiment, Oil Red O dye was used; it has an absorption maximum at around 518nm and has an empirical formula of C26H24N4O; the structure of this compound is shown in Figure 1. Absorbance measurements to determine the solubility profiles were made on a Kontron Uvikon 940 UVVolume 6, Issue 2, 2016, 1172-1175   Original Research Article Biointerface Research in Applied Chemistrywww.BiointerfaceResearch.com 1  & Chemistry, Kingston University London, Kingston upon Thames *corresponding author ee novel water-soluble dendrimer materials which would be able to -chain alkyl carboxylate branching units were used -dihydroxybenze centers were used, namely biphenyl and terphenyl, in te nonpolar molecules. The structures and  CMC. more hydrophilic, and potentially waterthe dendrimer can provide a nonincorporate hydrophobic molecules. It is known that having a hydrophobic interior and a hydrophilic surface can make such dendrimers good delivery vehicles for drugs with poor aqueous solubility, and some of the important ongoing work in this area is7]. The present paper reports the synthesis and properties of some novel, low-cost dendritic materials with the potential for use as drug-delivery vectors.  a -visible spectrophotometer. Particle sizes of the nanoparticulate products in aqueous media were determined by means of a Malvern Instruments Zetasizer, model 3000HSN NFigure 1. Structure of Oil Red O dye2.2. Synthesis of compound 7, (HOOC) The starting material chosen to produce the chain ends with potentially hydrophilic functional groups was ethyl 6bromohexanoate (1). To help provide a hydrophobic interior containing electron-rich aromatic rings, it was decided to employ 3,5-dihydroxybenzyl alcohol as a building block. The generation growth step was started by reacting two equivalents of dihydroxybenzyl alcohol (2). This reaction produced the firstgeneration alcohol (EtO2C)2-G1end. Activation of the hydroxymethyl group at the focal point of was achieved by reaction with thionyl chloride and a catalytic amount of DMF in the presence of dry DCM, which gave the Received: 19.01.2016 / Revised: 27.01.2016 / Accepted: Page | 1172 elivery KT1 2EE (UK) -mail address:p.j.foot@kingston.ac.uk encapsulate a yl alcohol was used as a -soluble. The interior of -polar environment suitable to   reviewed in references [3-. N NOH . 6-G1-3C (Scheme 1). -1 with 3,5---OH (3) with an ethyl ester chain-3 ISSN 2069-5837 Open Access Journal  16.02.2016 / Published on-line: 15.04.2016  Synthesis and properties of novel dendrimers for molecular delivery Page | 1173 corresponding chloride (EtO2C)2-G1-Cl (4). The next stage was to react three equivalents of 4 with 1,1,1-tris(4-hydroxyphenyl)ethane (5). The resultant dendritic polyether macromolecule (EtO2C)6-G1-3C (6) was isolated, and transformation of the terminal ethyl ester groups of 6 into the desired carboxylate groups was then accomplished by alkaline hydrolysis [11]. Reaction of 6 with a large excess of potassium hydroxide was found to proceed satisfactorily in the mixed solvent system of tetrahydrofuran/water/ethanol. Acidification of the reaction mixture by a well-known literature method [12] gave the carboxylate-terminated molecule 7, (HOOC)6-G1-3C. OOBr+OHOHOHO OOHO OOOO OClO OOOButanone, refluxK2CO3Thionyl chloride, DMFDry DCM+O OO OOOOOOO OOOO OOOOOOOOHOHOH(4)KOH, EtOHWaterButanone, 18-crown-6K2CO3O OO OOHOHOOOO OHOHOO OOOOOOHOH(1) (2) (3) (4)(5) (6) (7) Scheme 1.Synthesis of compound 7, (HOOC)6-G1-3C. OOOHOOOOOOOOOOOOOOClOOOOOOOOOOOO+OOOOOOOOOOOOO OOOO OOOOOO OOOOOOOOH OH(2) (4)(8) (9) (10) (11) (12)KOH, EtOHWaterOOOOOOOOHOOHOOO OHOOHO OOOOOHO OHOOOOHOOHButanone, 18-crown-6K2CO3Thionyl chloride, DMFDry DCMButanone, 18-crown-6K2CO3Scheme 2. Synthesis of compound 12, (HOOC)8-G2-2C.  2.2.1.Spectroscopic characterisation of compound 71H (CDCl3), ppm).  2.2 (t, 12H, CH2COOH)6, J=7.4Hz); 1.41 (overlapped peaks), 12H; (CH2CH2)6, 1.6 (overlapped peaks, 24H (CH2CH2)12); 4.05 (t, 12H, (CH2OAr)6, J=6.4 Hz); 6.3 (d, CH, 4-ArH); 6.26 (t, 6H, CH, 2,6-ArH); 5.20 (s, ArCH2OAr); 6.70 (d, 6H, CH, 3,5-Ar in core); 7.1 (d, 6H, CH, 2,6-ArH in core); 12.35 (s, 6H, OH), 2.28 (q, 3H, CH3 core). 13C NMR (ppm):177.21 (COOH, carbonyl); 36 (CH2COOH); 25 (CH2CH2COOH); 25.5 (CH2CH2CH2COH); 29.3 (CH2CH2OAr); 68.82 (CH2OAr); 99.5 (CH, 4 Ar); 159 (CO, 3,5-Ar); 102 (CH, 2,6-ArC); 142 (C, 4-Ar); 71.4 (CCH2OAr); 158.0 (C, 4-Ar in core); 129.2 (CH, 2,6-Ar in core); 114.8 (CH, 3,5-ArC in core); 135 (C, 1-Ar in core); 12.8 (CH3, attached to core); 48 (C-CH3 in core).IR  max (KBr, cm-1):2950, 2972 (R-CH3); 2916, 2936 (R’-CH2R’’); 3010, 3097 (p-di-subst.); 3010, 3079 (1,3,5-tri-subst.); 2900, 3100 (C-C-COOH); 1210, 1310 (Ph-O-C, ester); 1710 (C=O)  High-resolution mass spectrometry:Found mono-isotopic mass = 1356.6478; Calculated mono-isotopic mass = 1356.6444. 2.3. Synthesis of compound 12, (HOOC)8-G2-2C (Scheme 2). Synthesis of the second-generation dendron was achieved by reacting two equivalents of (4) with one equivalent of 3,5-dihydroxybenzyl alcohol (2) in the presence of potassium carbonate, 18-crown 6 and butanone, heated under reflux to give (EtO2C)4-G2-OH (8) with four ethyl ester chain-ends. Activation of the hydroxymethyl group, the focal point of 8, by reaction with thionyl chloride and several drops of DMF as a proton scavenger, proceeded to give the corresponding chloride (EtO2C)4-G2Cl (9). The next stage was to use the biphenyl moiety of (4,4’-dihydroxybiphenyl) as a core unit (10). Two equivalents of 9 were reacted with one equivalent of biphenol core in the presence of potassium carbonate, 18-crown-6 and butanone to give (EtO2)8-G2-2C (11) with eight ester chain-ends. The transformation of the terminal ethyl ester groups of 11 into the desired carboxylate Abdogroups was accomplished by alkaline hydrolysis.with a large excess of potassium hydroxide proceeded satisfactorily in a mixed solvent system of tetrahydrofuran/water/ethanol. Acidification gave the carboxylterminated macromolecule (HOOC)8-G2-2C 12. 2.3.1. Spectroscopic characterisation of compound 12 1H (CDCl3, ppm):2.2 (t, 16H, (CH2COOH)1.46 (overlapped peaks, 16H, (CH2CH2)4; 1.66 (overlapped peaks, 32H, (CH2CH2CH2)8); 3.89 (t, 16H, (CH2OAr)(CH, 6H,4-Ar)6; 6.26 (t,12 H, CH, 2,6(s,12H,ArCH2OAr)4; 6.78 (d,4H,CH, in biphenyl core); 7.32 (d, 4H, CH in biphenyl core)4); 12.4 (d, 8H, COOH 13C NMR (ppm):34.32 (CH(CH2CH2COOR)8; 25.07 (CH2CH2CH2COOR); 28.99  3. RESULTS SECTION 3.1. Solubility data for compounds 7 and 12.  Figure 2 shows the pH profiles (UV absorbance data) for compounds 7 and 12. The maximum solubility of both compounds were achieved at pH 7.4, and their solubilabsorptivity at that pH are shown in Table 1.  Table 1. Absorptivity and solubility of dendrimers 7 and 12 at pH 7.4Compound name Absorptivity /dm-3.mol-1.cm-1 (HOOC)6-G1-3C  (7) 5.04 (HOOC)8-G2-2C  (12) 30.63 Figure 2. pH-dependence of the UV absorbance of compounds and 12 (red) at λ=278nm. 3.2. CMC results and micellarproperties in aqueous media The critical micelle concentrations (CMC) of the products were measured by three different methods in order to determine whether they were forming micelles. It should be recalled that the dye used in this experiment for encapsulation was the waterinsoluble Oil Red O dye, which is known to have a moderate solubility in ethanol [13]. Studies of surface tension, molar conductivity and dye encapsulation for compounds revealed that both compounds had a CMC, which indicates that many units of these molecules come together to form micellar structures rather than forming unimolecular micelles. This was supported by simple molecular modelling using Quantum CAChe software (Fujitsu Ltd.), together with Zetasizer measurements, which indicated that the micellar structures for these two substances were much larger than the average size of the llah Ataei, Peter J. S. Foot, John W. Brown  Reaction of 11 -. 8, J=7.2Hz); 4, J=6.2 Hz); 6.30 -Ar); 5.2 )8. 2COOR); 24.8 (CH2CH2OAr); 65.9 (CH2OAr); 99.5 (Ar); 158 (COR, 3,5-Ar); 102.5 (71.4 (ArCH2OAr); 161.5 (C, 3,5in inner ring); 143 (C, 1-Ar in inner ring); 159.5 (biphenyl); 129 (C, 2,6 in biphenyl); 14.8 (173.2 (C, in carbonyl).  IR  max (KBr,cm-1):Broad peak 3200, 3600 (COOH), shoulder in region about 2800(C=O), 1596.68 (C=C), 1456.54 (Caryl ether), 1162(O-C=C), 824 (C High-resolution mass monoisotopic mass: 1830.83341830.8342. ity and molar . Solubility  /mol.dm-3 0.140 0.05  7 (blue) . -7 and 12  individual dendrimer molecules. The CMC value found by dye encapsulation was greater than those obtained from measurements of surface tension and molecular conductivity; this seems reasonable, due to the likely expansion of the miceencapsulate a dye. Table 2. Average CMC values found by two different methods for compounds 7 and 12. CMC Method (HOOC)6-/mol.dmSurface tension 1.1 x10Molar conductivity 1.4 x 10Average CMC 1.25 x 10Table 3.CMC values by dye-encapsulation for compounds 7 CMC Method (HOOC)6-G1/mol.dmDye encapsulation 7.15 x 10Table 4. Sizes of compounds 7 & 12 from modeDendrimer molecule Size of molecule modelled in flat conformation/nm (HOOC)6-G1-3C, (7) 5006  (HOOC)8-G2-2C, (12) 6146    The CMC value found by dye encapsulation was greater than those obtained from measurements of surface tension and molecular conductivity; this seems reasonable, due to the likely expansion of the micelles after they encapsulate a dye. Compounds 7 and 12 both had good solubilitpH 7.4 and they were able to encapsulate the waterand improve its solubility. In order to have more information about the properties of these macromolecules it is useful to compare their CMC values to those previously reported for wellknown surfactants. (The CMCs of the compounds in Table 5 are average values obtained by several methods such as surface tension and molar conductivity).Page | 1174 CH, 1-Ar); 99.3 (CH, 4-CH, 2.6-Ar); 142.2 (CCH2OAr); -Ar inner ring); 103.5 (C, 2,6-Ar C, 4 in C, 3,5 in biphenyl core); -3000 (acid characteristic), 1707 -H), 1374.56, 1295.62 (CO, -H bend). spectrometry:Calculated ; Found monoisotopic mass: lles after they G1-3C (7) -3 (HOOC)8-G2-2C(12) /mol.dm-3 -4 1.5 x 10-4 -4 1.1 x 10-4 -4 1.3 x 10-4 and 12. -3C (7) -3 (HOOC)8-G2-2C (12) /mol.dm-3 -4 7.75 x 10-4 lling and from Zetasizer. Size of micelle found by Zetasizer/nm Polydispersity Index 136  1.52 1247  1.12  y in water at -insoluble dye - Synthesis and properties of novel dendrimers for molecular delivery Page | 1175 Table 5. Relative molar masses and CMC for dendritic macromolecules and two well-known surfactants. Surfactant or Dendrimer CMC found  / mol.dm-3 Molar mass/Da Pluronic P105 4.3 x 10-4       [14] 6500  Hexadecylpyridinium bromide 7.3 x 10-4     [15] 384  (HOOC)6-G1-3C 3.3 x 10-4 1357  (HOOC)8-G2-2C 3.6 x 10-4 1832    Pluronic co-polymers (also known as Synperonic P105 or Poloxamer P105) have been used extensively in a variety of pharmaceutical applications including the delivery of low molecular mass drugs and polypeptides [16]. The Pluronic (P105) tri-block copolymer used for comparison here was composed of polyethylene oxide (POE) and polypropylene oxide (PPO) segments. It had a relative molar mass of 6500 [17]. The other surfactant used, hexadecylpyridinium bromide, a cationic surfactant comprising a pyridinium head and a hexadecyl tail. Pluronic P105 has the highest molar mass and hexadecylpyridinium bromide the lowest. The CMCs of our dendritic macromolecules lie in between these two, which could be due to their compact structure. Pluronic P105 had the highest CMC due to its straight-chain conformation.   4. CONCLUSIONS  It has been widely documented that the solubility of hydrophobic compounds in water can be enhanced by the addition of surfactant molecules at a concentration above their CMC [18], by an encapsulation process. Investigation of compounds 7 and 12 in the present work has revealed that they encapsulate the water-insoluble ORO dye and greatly improve its solubility. Further modification of the structure of these compounds may enhance their potential to be used as molecular delivery agents.  OH OH Figure 3. Precursor for terphenyl core.  In future, synthesis of next-generation dendrimers will be attempted, although it is likely that as the generations increase, the synthesis will become more difficult. The physical and chemical properties of the new dendritic compounds (such as solubility, formation of micelles and dye uptake) will be investigated. A feature of the continuing investigations is to make bespoke cores such as linear terphenyls which could be expected to produce larger dendrimers with bigger cavities.   To increase the potential size and encapsulation capacity of the dendrimer, it is planned to increase the length of the dendrons by reacting our current dendrons with spacers; one spacer type is based on para-substituted phenyl systems to effectively lengthen the “trunk” section of the dendrimer tree. O CH2ClR Figure 4. An example of a linking spacer.  5. REFERENCES [1]Cheng Y., Xu Z., Ma M., Xu T.,Dendrimers as drug carriers: Applications in different routes of drug administration,Journal of Pharmaceutical Science, 97, 1, 123-143, 2008. [2]Tomalia D.A., Naylor A.M., Goddard W.A. III., Starburst Dendrimers: Molecular-Level Control of Size, Shape, Surface Chemistry, Topology, and Flexibility from Atoms to Macroscopic Matter,AngewandteChemie, InternationalEnglish Edition,29, 138-175,1990. 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[17] Alexandridis P., Athanassiou V., Hatton T.A., Pluronic-P105 PEO-PPO-PEO block-copolymer in aqueous urea solutions – micelle formation, structure and microenvironment,Langmuir, 11,7, 2442-2450,1995. [18] Clint J. H.,Surfactant aggregation, CRC Chapman and Hall, New York, 1992.   5. ACKNOWLEDGEMENTS  The authors are grateful to Kingston University London, for partial support of this research.  © 2016 by the authors. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/). 

Synthesis and properties of novel dendrimers for molecular delivery

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Synthesis and properties of novel dendrimers for molecular delivery

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