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Chapter 7

Synthesisand Characterization ofDualNanodeliverySystemsContaining Vitamin forCosmeticsand

PharmaceuticalsRajesh Kumar1,2,*,Rahul Tyagi1, ArthurC Watterson1,

VirinderS. Parmar1, 2, and Jayant Kumar21Institute forNano-ScienceEngineering andTechnologyand Center

for Advanced Materials,Departmentsof Chemistry andPhysics,UniversityofMassachusetts,Lowell, MA 1854

2Bioorganic Laboratory,Departmentof Chemistry, University of Delhi,Delhi 110 007, India

*Corresponding author: rkumar26@gmail.com

Vi t amin isknownfor itsabilityto capture free radicals andtherefore to help combat their potentialskindamaging effects.Unfortunately it is difficult for it to penetrate and spreadthrough the outer layers of the skinon its own. To get aroundthis problem, dual nano-delivery systems (DND's) havingcovalently attached vitamin have been designed andsynthesized using novel chemo-enzymatic routes. Thesesynthetic nanocarriers are unique as they have the ability tofurther encapsulate vitamin and thus offerhigh loadingandtransport capability forvitaminE. Further,thesenanocarriersoffer both fast and slow release of vitamin E. Thesenanocarriers also converthighly l ipophi l ic vitamin to water-soluble form and thus can be used in aqueous preparations forcosmeticsand pharmaceuticalapplications.

2007American ChemicalSociety 139

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140There has been a major change in the development of cosmetic products in

the recent years. Vitamins and other bioactivesubstancesare increasingly beingused alongside conventional ingredients. The immediate cause is increasingknowledge of the 'stress environment' to which the skin, as the human body'slargest organ, is exposed. Stress factors include environmental influences suchas ultraviolet radiation and the possibly inadequate intake of certain vitamins asa result of modern lifestyles. Thepresentinterest in the common roots of health,nutrition and body care is also changing consumer expectations with regard tothe performance characteristics of cosmetic products. This development hastemporarily pushed the significance of decorative cosmetics into the background.The focus is now on protection and preventive measures against harmfulenvironmental influences and biological aging processes, as we l las the possiblerepair or regeneration of unwelcome changes in the skin. One target area isoxidativestress,i.e.the consequences o fthe creation o freactive forms o foxygeninthe skin.The reactive formsthatusually undergo physiologicalmetabolizationmay, under abnormalstressconditions and in theabsenceofadequateprotectionby enzymatic and non-enzymatic mechanisms, cause pathophysiologicaldysfunctions within the skin. Oxidative stress is also an early hallmark ofaffected neurons in Alzheimer's disease (AD). 1 , 2 The neurotoxic peptideAmyloid-beta (Abeta), a neurotoxic peptide that accumulates in A D , 3 inducesl ip id peroxidation, 4 ' 5 which generates downstream cytosolic free radicals andreactive oxygen species ( ROS )

6

. Endogenous antioxidant systems normallyprovide sufficient neuroprotection, but may fail to compensate for the increasedgeneration of reactive oxygen species (ROS) that accompaniesneurodegeneration.7 One l imiting factor in dietary consumption of antioxidantsmay be the difficulty in attaining appropriate concentrations within brain,although recent studiessuggestthatsystemic antioxidant levels are helpful.8

Vitamin is known for itsabilityto capture free radicals (reactive forms ofoxygen) and therefore to help combat their potentialskindamaging effects. Theantioxidant vitamin provides limited neuroprotection in A D . 9 " 1 2 Thislimitation is l ikely to be a consequence of its l ipophi l icnature, which retardsitsability to quench cytosolic ROS, includingthose generated from antecedentmembrane oxidative damage.6 Pharmaceutical and medical research has formany years focused on vectors as ameans of transporting drugs harmlessly tospecific parts of the body.13"16 We have earlier reported a nano-micellar drugdelivery system based on the copolymer of poly(ethylene glycol)and dimethylesters of alkylated phenol, capable of encapsulating l ipophi l ic as we l l ashydrophiliccompounds.17*20 Figure 1,shows the amphiphilic polymer A basedon poly(ethylene glycol)-co-dimethylester of alkylated phenol and cartoonrepresentation of the micelle formation by the self-assembly of the amphiphilicpolymerA .

It has been shown earlierthatthe delivery systems based on the amphiphilicpolymerA are highly efficient and increase the efficacy o fthe drug by manifoldswhen encapsulated as compared to the native form of drug. For example, the use

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141

Figure 1. Cartoon representation of nano-micelle formation by theselfassemblyofamphiphilicpolymer inaqueousmedia.

Seepage11 of colorinserts.)

o fthesenano-micellar delivery systems appearspromising for anti-inflammatorydrugs.18These nano-micelles are used in particular to transport l ipophilicactiveingredients,whichare soluble ino i l . G iventhatthe interstices of the outer layer ofskin measure about 100 nanometers, nanocarriers offer the best solution to theproblem of transporting and concentrating active ingredients in the skin. In thepresent work we report a novel dual nano-delivery system (DND's) havingcovalentlyattachedvitamin as a wayo ftransporting purevitaminE.

ExperimentalMaterials andMethods

Novozyme-435,animmobilizedenzyme, was agift fromNovozymes A / S ,Denmark.A l l other chemicals and solvents were ofanalyticalgrade and wereused as received unless otherwise noted.

CharacterizationGe l permeation chromatography (GPC) was used to determine the

molecular weights and molecular weight distributions, M w / M n of polymersamples. *H N M R and 1 3 CN M R spectra were recorded on a Bruker InstrumentInc. 250 MHz A R X spectrometer equipped with Silicone Graphics station.Light scattering data was determined by a laser light scattering photometer(Wyatt technology D A W N M od e lF) equippedwith632 nmHe-Ne laser as thelightsource.

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142Ac y l a t i o nofV i tam i n with bromoacetyl bromide

To a solution of-tocopherol (2 g, 4.64 mmol) and triethyl amine (0.7 g,6.93 mmol) in dichloromethane was added bromoacetyl bromide (1 g, 4.95mmol) drop by drop at room temperature. The reaction mixture was stirred atroom temperatureand monitored by thin layer chromatorgraphy ( T L C ) in 50%hexane/chloroform. After completion of the reaction, the solvent was removedon rotavapour and the residue obtained was redissolved in hexane to remove thesalt. The insoluble salt in hexane was filtered off and the filtrate obtained wasdried by removing the hexane under reduced pressure, washed with methanoland dried to obtain the desired product 2.

Coupling of-tocopherol bromoacetate (2) with Po ly [(poly (oxyethylene)-oxy-5-hydroxyisophthaloyl](3)

Compound2 (0.86 g, 1.55 mmol) and 3 (1.83 g, 2.32 mmol) were dissolvedin dry acetone (20 ml). To this solution was added anhydrous potassiumcarbonate (0.32 g, 2.32 mmol). The reaction mixture was then refluxed andmonitored by T L C in 10 % methanol/chloroform. After completion, thepotassium carbonate was removed by simple filtration and solvent was removedunder vacuum to obtain the desired product 4.

Synthesis of the amphiphilic polymer 5Toa solution of4(1.5 g, 1.89 mmol) and bromodecane (220 mg, 1.0 mmol)

in dry acetone was added anhydrous potassium carbonate and the resultingsolutionwas refluxed in an oil bath maintained at 60 C. Aftercompletiono fthereaction (checked by T L C ) , potassium carbonate was filtered off and acetonewas removed to give theamphiphilicpolymer 5 as a low meltingsol id .

ResultsandDiscussionWe have previously reported nano-micelles based on copolymer of

poly(ethylene glycol) and dimethylestersof alkylated phenol A ,that facilitateintracellular delivery of otherwise nonpermeable molecules.21 This wasaccomplishedby exposing differentiated S H - S Y - 5 Ycultures to rhodamine eitheraliquoted directly into culture medium or to an equivalent finalconcentration ofrhodamine thathad been encapsulated into nano-micelles described above. Nodetectable rhodamine fluorescence was observed above background followingincubationof cultureswithnon-encapsulated rhodamine. By contrast, prominentrhodamine fluorescence was observedwithinthe cytosolic compartment of cellsincubatedwithnano-micelle encapsulated rhodamine.21

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143These nano-micelles also found to increase the efficacy of the l ipophi l ic

antioxidant vitamin against Abeta-induced ROS when used in encapsulatedform.21 In continuation of our earlier work and interest on the encapsulation andtransportofvitamin using nano-miceller delivery systems, we report the novelD N D ' s having covalently attached vitamin as a way of transporting purevitaminE.

The synthesized nano-carriers are unique due to the f lexibi l i tyof both boundas we l las encapsulated vitamin E. Theattachmentof the hydrophobic vitamin in the side chain w i l l help in the micellization in aqueous media and alsoincrease the vitamin content in the nano-carriers. This also allows a bettercontrol over the release and bioavailability of vitamin and can be used incombination of other bioactiveagents. The amphiphilicnature ofthese nanocarriers make them soluble in both hydrophilic and l ipophi l ic medium. Thesynthetic approach forthesenano-carrriers is shown inScheme1andScheme2.

The -tocopherol bromoacetate 2 was synthesized by the acylation of a-tocopherol with bromoacetyl bromide.22 The H - N M R spectrum of compound2showed the signal at 4.13 for the O C O C H 2B r confirming the acylation of thephenolic hydroxyl ofV i t amin Figure2). The-tocopherolbromoacetate2was attached to dimethylisophthalate-co-PEG-600 copolymer 3, by thealkylationof phenolic hydroxyl in the presenceof potassium carbonate to givevitamin conjugated copolymer4. The copolymer3was in turn synthesized byenzyme (immobilized Candida antarctica lipase commercially known asNovozyme435) catalyzed condensation polymerization reaction17ofdimethyl5-hydroxy isophthalate with polyethylene g lyco l (600 g/mol) under solvent lessconditions. The alkylation of the phenolic hydroxyl group of 3 by 2 wasconfirmedby theappearanceof a signal at 5.06 for the O C O C H 2 0 protons anddisappearance o fthe signal at 4.13 forO C O C H 2B r Figure2).

The degree of functionalization was determined by comparing theintegration of main chain proton at 4.50 (for 4 protons) tothatat 5.06 (for2protons) for side chain proton and found to be 30 %. The partial derivatization ofphenolic hydroxyl groups was further evident from the two sets of aromaticsignals due to substituted and non-substituted aromatic rings. The signal for thesubstituted aromatic rings appeared lower field as compared to non-substitutedaromatic rings. Interestingly, the methylene protons due to the main chain at 4.50 also showed splitting,givingtwo different overlapping triplets Figure 2).These may be due to the different chemical environment of the methyleneprotons.

The V i t amin conjugated copolymer4, is highly soluble in water butdoesnot aggregate to form nano-micelles as observed by static light scatteringtechnique. This may be due to the improper balance of hydrophilic tohydrophobic segments. Therefore, the partially (30 %) Vi tamin conjugatedcopolymer 4, was further reacted with excess of bromodecane to completelyalkylatethe phenolic hydroxyl group o fpolymer4as shown inScheme2.

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r

Sm1ShsoVamncuecymodmhsohaeaPG4

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146

I ' ' ' ' I ' ' ' ' I ' ' ' ' I 1 ' ' ' I ' ' ' ' I ' ' ' ' I ' ' 1 ' I 1 ' ' 1 I ' ' ' ' I 1 1 ' ' I ' ' ' ' I ' ' 1 ' I ' ' ' ' I ' ' ' ' I ' ' ' ' I 1 ' ' ' I ' ' ' ' f ' ' ' ' I '8 5 8 .0 7.5 7.0 6.5 6.0 S.S 5.0 4.5 4.0 3.5 3.0 2 .5 2.0 1.5 1.0 0.5 0.0 -0.5

Figure 2. *HNMR spectra of a-tocopherol 1),bromoacetyl tochopherol 2,copolymer3,and vitamin conjugated copolymer 4.

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147The l HN M R spectrum of the amphiphilic polymer showed a triplet at 4.0

and assigned to methylene protons (e) (Figure3).This confirmed theattachmentofthe bromodecane chain to the phenolic hydroxyl group. The signal at 5.06assigned to methylene protons (d) confirmed die presence of the vitamin E,suggesting that therewas no side chain hydrolysis. The composition of the sidechains in the polymer5were determined by comparing the signal at 5.06 for thevitamin side chain to the signal at 4.0 for thedecaneside chain and found to be1:2. The aromatic region in the HN M R spectrum o fthe copolymer5also showeddistributionof the protons due to chemically different environment of the phenylring attached to Vi tamin anddecanechains substantiating the presence of bothside chains, i.e. Vi tamin and decane. Surprisingly, the two overlappingsetsoftriplet at 4.50 disappeared and only as expected only one triplet appeared,suggesting the complete substitutiono fthe phenolichydroxylgroups.Self-assembly of the synthesized vitamin containing polymer 5, inaqueousand organic media was studied by static light scattering. It was observedthat when the polymer 5 was dissolved in water, it aggregates to form nano-micelles.The radius ofgyration(Rg) and hydrodynamic radius (Rh) ofpolymer5in aqueous solution were determined by static and dynamic light scatteringtechniques at 25 C and found to be 57.2 and 10.5 nm. However nomicellization was observed in toluene when analyzed by static light scattering.The nano-micelle formed by the polymer 5, was further used to encapsulatevitamin and the encapsulation ofvitamin increased from 17 % to 26 % asdetermined byU V spectroscopy. The nano-micelle formed by the polymer 5 wasstable to 65 C versus 55 C reported for the nano-micelle formed by thepolymer o fpoly(ethyleneglycol)-co-dimetylesterofalkylatedphenol, A . 2 3

ConclusionsInsummary, polymeric nano-carriers containing the bound Vi tamin have

been designed and synthesized. The synthesized nano-carriers have thef lexibi l i ty of further encapsulating V i t amin or other bioactive agents, andallow a handle for controlling the concentration of V i t amin in variouspreparations for cosmetic and pharmaceutical applications. The synthesizedVi t amin containing nano-carriers are highly soluble in both water and oil ,making them suitable for both aqueous and non-aqueous preparations. Theevaluation o fthe synthesized nano-carriers in various applications is underway.

AcknowledgementsThe authors gratefully acknowledge financial support to RT from C FC I ,

Universityof Massachusetts Lowe l l .

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