Synthesis and Characterization of Dual Nanodelivery Systems

Chapter 7

Synthesis and Characterization of Dual Nanodelivery Systems Containing Vitamin £ for Cosmetics and Pharmaceuticals Downloaded by PENNSYLVANIA STATE UNIV on July 3, 2012 | http://pubs.acs.org Publication Date: April 12, 2007 | doi: 10.1021/bk-2007-0961.ch007

1,2,

1

1

Rajesh Kumar *, Rahul Tyagi , Arthur C. Watterson , Virinder S. Parmar , and Jayant Kumar 1,2

2

1

Institute for Nano-Science Engineering and Technology and Center for Advanced Materials, Departments of Chemistry and Physics, University of Massachusetts, Lowell, M A 01854 Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi 110 007, India *Corresponding author: [email protected] 2

Vitamin Ε is known for its ability to capture free radicals and therefore to help combat their potential skin damaging effects. Unfortunately it is difficult for it to penetrate and spread through the outer layers of the skin on its own. To get around this problem, dual nano-delivery systems (DND's) having covalently attached vitamin Ε have been designed and synthesized using novel chemo-enzymatic routes. These synthetic nanocarriers are unique as they have the ability to further encapsulate vitamin Ε and thus offer high loading and transport capability for vitamin E. Further, these nanocarriers offer both fast and slow release of vitamin E. These nanocarriers also convert highly lipophilic vitamin Ε to watersoluble form and thus can be used in aqueous preparations for cosmetics and pharmaceutical applications.

© 2007 American Chemical Society

In Cosmetic Nanotechnology; Morgan, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

139

Downloaded by PENNSYLVANIA STATE UNIV on July 3, 2012 | http://pubs.acs.org Publication Date: April 12, 2007 | doi: 10.1021/bk-2007-0961.ch007

140 There has been a major change in the development of cosmetic products in the recent years. Vitamins and other bioactive substances are increasingly being used alongside conventional ingredients. The immediate cause is increasing knowledge of the 'stress environment' to which the skin, as the human body's largest organ, is exposed. Stress factors include environmental influences such as ultraviolet radiation and the possibly inadequate intake of certain vitamins as a result of modern lifestyles. The present interest in the common roots of health, nutrition and body care is also changing consumer expectations with regard to the performance characteristics of cosmetic products. This development has temporarily pushed the significance of decorative cosmetics into the background. The focus is now on protection and preventive measures against harmful environmental influences and biological aging processes, as well as the possible repair or regeneration of unwelcome changes in the skin. One target area is oxidative stress, i.e. the consequences of the creation of reactive forms of oxygen in the skin. The reactive forms that usually undergo physiological metabolization may, under abnormal stress conditions and in the absence of adequate protection by enzymatic and non-enzymatic mechanisms, cause pathophysiological dysfunctions within the skin. Oxidative stress is also an early hallmark of affected neurons in Alzheimer's disease (AD). The neurotoxic peptide Amyloid-beta (Abeta), a neurotoxic peptide that accumulates in A D , induces lipid peroxidation, ' which generates downstream cytosolic free radicals and reactive oxygen species (ROS) . Endogenous antioxidant systems normally provide sufficient neuroprotection, but may fail to compensate for the increased generation of reactive oxygen species (ROS) that accompanies neurodegeneration. One limiting factor in dietary consumption of antioxidants may be the difficulty in attaining appropriate concentrations within brain, although recent studies suggest that systemic antioxidant levels are helpful. 1,2

3

4

5

6

7

8

Vitamin Ε is known for its ability to capture free radicals (reactive forms of oxygen) and therefore to help combat their potential skin damaging effects. The antioxidant vitamin Ε provides limited neuroprotection in A D . " This limitation is likely to be a consequence of its lipophilic nature, which retards its ability to quench cytosolic ROS, including those generated from antecedent membrane oxidative damage. Pharmaceutical and medical research has for many years focused on vectors as a means of transporting drugs harmlessly to specific parts of the body. " We have earlier reported a nano-micellar drug delivery system based on the copolymer of poly(ethylene glycol) and dimethyl esters of alkylated phenol, capable of encapsulating lipophilic as well as hydrophilic compounds. * Figure 1, shows the amphiphilic polymer A based on poly(ethylene glycol)-co-dimethylester of alkylated phenol and cartoon representation of the micelle formation by the self-assembly of the amphiphilic polymer A . It has been shown earlier that the delivery systems based on the amphiphilic polymer A are highly efficient and increase the efficacy of the drug by manifolds when encapsulated as compared to the native form of drug. For example, the use 9

6

13

17

16

20


12


141

Figure 1. Cartoon representation of nano-micelle formation by the self assembly of amphiphilic polymer A in aqueous media. (See page 11 of color inserts.)

of these nano-micellar delivery systems appears promising for anti-inflammatory drugs. These nano-micelles are used in particular to transport lipophilic active ingredients, which are soluble in oil. Given that the interstices of the outer layer of skin measure about 100 nanometers, nanocarriers offer the best solution to the problem of transporting and concentrating active ingredients in the skin. In the present work we report a novel dual nano-delivery system (DND's) having covalently attached vitamin Ε as a way of transporting pure vitamin E. 18

Experimental Materials and Methods Novozyme-435, an immobilized enzyme, was a gift from Novozymes A/S, Denmark. A l l other chemicals and solvents were of analytical grade and were used as received unless otherwise noted. Characterization Gel permeation chromatography (GPC) was used to determine the molecular weights and molecular weight distributions, M / M of polymer samples. *H N M R and C N M R spectra were recorded on a Bruker Instrument Inc. 250 M H z 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 Model F) equipped with 632 nm He-Ne laser as the light source. w

n

1 3


142


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

Coupling of α-tocopherol bromoacetate (2) with Poly [(poly (oxyethylene)oxy-5-hydroxyisophthaloyl] (3) Compound 2 (0.86 g, 1.55 mmol) and 3 (1.83 g, 2.32 mmol) were dissolved in dry acetone (20 ml). To this solution was added anhydrous potassium carbonate (0.32 g, 2.32 mmol). The reaction mixture was then refluxed and monitored by T L C in 10 % methanol/chloroform. After completion, the potassium carbonate was removed by simple filtration and solvent was removed under vacuum to obtain the desired product 4.

Synthesis of the amphiphilic polymer 5 To a solution of 4 (1.5 g, 1.89 mmol) and bromodecane (220 mg, 1.0 mmol) in dry acetone was added anhydrous potassium carbonate and the resulting solution was refluxed in an oil bath maintained at 60 °C. After completion of the reaction (checked by TLC), potassium carbonate was filtered off and acetone was removed to give the amphiphilic polymer 5 as a low melting solid.

Results and Discussion We have previously reported nano-micelles based on copolymer of poly(ethylene glycol) and dimethyl esters of alkylated phenol A , that facilitate intracellular delivery of otherwise nonpermeable molecules. This was accomplished by exposing differentiated SH-SY-5Y cultures to rhodamine either aliquoted directly into culture medium or to an equivalent final concentration of rhodamine that had been encapsulated into nano-micelles described above. No detectable rhodamine fluorescence was observed above background following incubation of cultures with non-encapsulated rhodamine. By contrast, prominent rhodamine fluorescence was observed within the cytosolic compartment of cells incubated with nano-micelle encapsulated rhodamine. 21

21


143 These nano-micelles also found to increase the efficacy of the lipophilic antioxidant vitamin Ε against Abeta-induced ROS when used in encapsulated form. In continuation of our earlier work and interest on the encapsulation and transport of vitamin Ε using nano-miceller delivery systems, we report the novel D N D ' s having covalently attached vitamin Ε as a way of transporting pure vitamin E . The synthesized nano-carriers are unique due to the flexibility of both bound as well as encapsulated vitamin E. The attachment of the hydrophobic vitamin Ε in the side chain will help in the micellization in aqueous media and also increase the vitamin Ε content in the nano-carriers. This also allows a better control over the release and bioavailability of vitamin Ε and can be used in combination of other bioactive agents. The amphiphilic nature of these nano carriers make them soluble in both hydrophilic and lipophilic medium. The synthetic approach for these nano-carrriers is shown in Scheme 1 and Scheme 2. The α-tocopherol bromoacetate 2 was synthesized by the acylation of atocopherol with bromoacetyl bromide. The H - N M R spectrum of compound 2 showed the signal at δ 4.13 for the O C O C H B r confirming the acylation of the phenolic hydroxyl of Vitamin Ε (Figure 2). The α-tocopherol bromoacetate 2 was attached to dimethylisophthalate-co-PEG-600 copolymer 3, by the alkylation of phenolic hydroxyl in the presence of potassium carbonate to give vitamin Ε conjugated copolymer 4. The copolymer 3 was in turn synthesized by enzyme (immobilized Candida antarctica lipase Β commercially known as Novozyme 435) catalyzed condensation polymerization reaction of dimethyl 5hydroxy isophthalate with polyethylene glycol (600 g/mol) under solvent less conditions. The alkylation of the phenolic hydroxyl group of 3 by 2 was confirmed by the appearance of a signal at δ 5.06 for the O C O C H 0 protons and disappearance of the signal at δ 4.13 for O C O C H B r (Figure 2).


21

22

!

2

17

2

2

The degree of functionalization was determined by comparing the integration of main chain proton at δ 4.50 (for 4 protons) to that at δ 5.06 (for 2 protons) for side chain proton and found to be 30 %. The partial derivatization of phenolic hydroxyl groups was further evident from the two sets of aromatic signals due to substituted and non-substituted aromatic rings. The signal for the substituted aromatic rings appeared lower field as compared to non-substituted aromatic rings. Interestingly, the methylene protons due to the main chain at δ 4.50 also showed splitting, giving two different overlapping triplets (Figure 2). These may be due to the different chemical environment of the methylene protons. The Vitamin Ε conjugated copolymer 4, is highly soluble in water but does not aggregate to form nano-micelles as observed by static light scattering technique. This may be due to the improper balance of hydrophilic to hydrophobic segments. Therefore, the partially (30 %) Vitamin Ε conjugated copolymer 4, was further reacted with excess of bromodecane to completely alkylate the phenolic hydroxyl group of polymer 4 as shown in Scheme 2.



Scheme 1. Synthesis of Vitamin Ε conjugated copolymer of dimethylisophthalate and PEG600, 4.

Br





146

1

' I ' ' ' ' I ' ' ' ' I ' ' ' ' I ' ' ' I ' ' ' ' I ' ' ' ' I ' ' 8 5 8.0 7.5 7.0 6.5 6.0 S.S

1

1

1

1

1

' I ' ' I ' ' ' ' I ' ' I ' ' ' ' I ' ' 5.0 4.5 4.0 3.5 3.0

1

1

' I ' ' ' ' I ' ' ' ' I ' ' ' ' I ' ' ' I ' ' ' ' f ' ' ' ' I ' 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, copolymer 3, and vitamin Ε conjugated copolymer 4.


147 l

The H N M R spectrum of the amphiphilic polymer showed a triplet at δ 4.0 and assigned to methylene protons (e) (Figure 3). This confirmed the attachment of the bromodecane chain to the phenolic hydroxyl group. The signal at δ 5.06 assigned to methylene protons (d) confirmed die presence of the vitamin E, suggesting that there was no side chain hydrolysis. The composition of the side chains in the polymer 5 were determined by comparing the signal at δ 5.06 for the vitamin Ε side chain to the signal at δ 4.0 for the decane side chain and found to be 1:2. The aromatic region in the H N M R spectrum of the copolymer 5 also showed distribution of the protons due to chemically different environment of the phenyl ring attached to Vitamin Ε and decane chains substantiating the presence of both side chains, i.e. Vitamin Ε and decane. Surprisingly, the two overlapping sets of triplet at δ 4.50 disappeared and only as expected only one triplet appeared, suggesting the complete substitution of the phenolic hydroxyl groups.


!

Self-assembly of the synthesized vitamin Ε containing polymer 5, in aqueous and organic media was studied by static light scattering. It was observed that when the polymer 5 was dissolved in water, it aggregates to form nanomicelles. The radius of gyration (Rg) and hydrodynamic radius (R ) of polymer 5 in aqueous solution were determined by static and dynamic light scattering techniques at 25 °C and found to be 57.2 and 10.5 nm. However no micellization was observed in toluene when analyzed by static light scattering. The nano-micelle formed by the polymer 5, was further used to encapsulate vitamin Ε and the encapsulation of vitamin Ε increased from 17 % to 26 % as determined by U V spectroscopy. The nano-micelle formed by the polymer 5 was stable to 65 °C versus 55 °C reported for the nano-micelle formed by the polymer of poly(ethylene glycol)-co-dimetyl ester of alkylated phenol, A . h

2 3

Conclusions In summary, polymeric nano-carriers containing the bound Vitamin Ε have been designed and synthesized. The synthesized nano-carriers have the flexibility of further encapsulating Vitamin Ε or other bioactive agents, and allow a handle for controlling the concentration of Vitamin Ε in various preparations for cosmetic and pharmaceutical applications. The synthesized Vitamin Ε containing nano-carriers are highly soluble in both water and oil, making them suitable for both aqueous and non-aqueous preparations. The evaluation of the synthesized nano-carriers in various applications is underway.

Acknowledgements The authors gratefully acknowledge financial support to RT from CFCI, University of Massachusetts Lowell.


148

References 1. 2. 3.


4. 5. 6. 7. 8. 9. 10. 11. 12.

13. 14. 15. 16. 17. 18.

19. 20.

21. 22. 23.

Markesbery, W. R. and Carney, J.M. 1999, 9, 133. Zhu, X.; Raina, A . K . ; Lee, H.G.; Casadesus, G.; Smith, M . A . ; Perry, G . Brain Res. 2004, 1000, 32. Huang, X . ; Moir, R.D.; Tanzi, R.E.; Bush, A.I.; Rogers, J.T. Ann Ν Y Acad Sci 2004, 1012, 153 Koppal, T.; Drake, J.; Butterfield, D.A. Biochim Biophys Acta 1999,1453,407. Butterfield, D.A.; Koppal, T.; Subramaniam, R.; Yatin, S. Rev Neurosci. 1999, 10, 141. Subramaniam, R.; Koppal, T.; Green, M . ; Yatin, S.; Jordan, B . ; Drake, J.; Butterfield, D.A Neurochem Res. 1998, 23, 1403. Rottkamp, C.A.; Nunomura, Α.; Raina, A . K . ; Sayre, L . M . ; Perry, G.; Smith, M . A . Alzheimer Dis Assoc Disord. 2000, 14 Suppl 1:S62. Polidori, M.C.; Mattioli, P.; Aldred, S.; Cecchetti, R.; Stahl, W.; Griffiths, H . ; Senin, U.; Sies, H.; Mecocci, P. Dement Geriatr Cogn Disord. 2004, 18, 265. Berman, K.; Brodaty, H . CNS Drugs. 2004, 18, 807. Growdon, J. J Alz Dis.2001, 3, 287. Vina, J.; Lloret, Α.; Orti, R.; Alonso, D. Mol Aspects Med. 2004, 25, 117. Zandi, P.P.; Anthony, J.C.; Khachaturian, A.S.; Stone, S.V.; Gustafson, D.; Tschanz, J.T.; Norton, M.C.; Welsh-Bohmer, K . A . ; Breitner, J.C. Arc Neurol. 2004, 61, 82. Zulli, F.; Suter, F.; Mibelle, A . G.; Buchs, S. SOFW Journal. 1997, 123, 883. Hildebrand, G.; Niehus, H . ; Muller, R. H . Proc Int Symp Control Rel of Bio Mater. 1998, 25,433. M u , L.; Feng, S.S. J Control Rel. 2003, 86, 33. M u , L.; Feng, S.S. J Control Rel. 2003, 80, 129. Kumar, R.; Shakil, N . A . ; Chen, M . H . ; Parmar, V.S.; Samuelson, L . A . ; Kumar, J.; Watterson, A . C . J Macromol Sci-Pure Appl Chem 2002, 39, 1137. Kumar, R.; Chen, M . H . ; Parmar, V . S.; Samuelson, L . Α.; Kumar, J.; Nicolosi, R.; Yoganathan, S.; Watterson, A . C. J Am Chem Soc. 2004, 126, 10640. Kumar, R.; Bruno, F.; Parmar, V . S.; Kumar, J.; Watterson, A . C.; Chittibabu, K . G.; Samuelson, L. A . Chem Commun. 2004, 862. Danprasert, K.; Kumar, R.; Cheng, M . H . ; Gupta, P.; Shakil, Ν. Α.; Prasad, A . K.; Parmar, V . S.; Kumar, J.; Samuelson, L . Α.; Watterson, A.C. Eur Poly J. 2003, 39, 1983. Shea, T.B.; Ortiz, D.; Nicolosi, R.J.; Kumar, R.; Watterson, A . C . J Alz Dis. 2005, 7, 297. Kim, Y.D.; Lee, J.N.; Kim, W.C.; Kim, Y . H . ; Kim, M . K . ; Ku, M.S.; Cho, I.H. Ger Offen. 1999, D E 19747600. Kumar, R.; Tyagi, R.; Shakil, N.A.; Parmar, V.S.; Kumar, J.; Watterson, A . C . J Macro Sci-Pure Appl Chem. 2005, 42, 1523.


Synthesis and Characterization of Dual Nanodelivery Systems

Recommend Documents