Syntheses of Curcumin Bioconjugates and Study of Their Antibacterial

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Bioconjugate Chem. 2001, 12, 464−469

ARTICLES Syntheses of Curcumin Bioconjugates and Study of Their Antibacterial Activities against β-Lactamase-Producing Microorganisms Sanjay Kumar, Upma Narain,† Snehlata Tripathi, and Krishna Misra* Nucleic Acids Research Laboratory, Department of Chemistry, University of Allahabad, Allahabad-211 002, India. Received May 9, 2000; Revised Manuscript Received September 18, 2000

In the present study curcumin bioconjugates, viz. di-O-glycinoylcurcumin (I), di-O-glycinoyl-C4glycylcurcumin (II), 5′-deoxy-5′-curcuminylthymidine (5′-cur-T) (IV), and 2′-deoxy-2′-curcuminyluridine (2′-cur-U) (V) have been synthesized and characterized by elemental analysis and 1H NMR. The turmeric peptide (Tp) was isolated from the aqueous turmeric extract of the turmeric rhizome. The antibacterial activity of these four bioconjugates and also of the turmeric peptide and sodium salt of curcumin (III) have been tested particularly for β-lactamase-producing microorganisms.

INTRODUCTION

Most novel drug-delivery systems have stemmed from work on new polymers, lipid vesicles, cyclodextrins, prodrugs, and viral vectors. The rationale behind systemic drug delivery is to achieve greater efficacy with lower toxicity. Enhancing the specificity of therapeutic drugs and thereby improving their site-specific delivery is the primary goal of today’s pharmaceutical industries. The past decade has seen extensive use of liposomes and lipid-based delivery systems to improve the pharmacological properties of a variety of drugs (1-7). The principal benefits afforded by therapeutic agents through their liposomal encapsulation were enhanced plasma circulation lifetimes, increased delivery to specific sites, and changes in tissue distribution, which can result in reduced toxic effects. Lipid-oligonucleotide complexes can provide levels of encapsulation, but their size and charge severely impair their ability to distribute systemically after parenteral administration and subsequently extravasate to the disease site. As a result, this type of liposomal carrier has a limited utility for the treatment of systemic diseases (8). Keeping this rationale in mind, a bioconjugate can be synthesized composed of a nucleoside and any suitable molecule which should itself be active against inflammatory disorders, and it must be devoid of any charge. Nucleosides and amino acids rapidly cross the plasma membrane of the cells by a facilitated transport mechanism (9), thus gaining rapid entry into the cells. The curcumin molecule, meeting such requirements, was chosen for this dual purpose. It is the main coloring component of turmeric, the rhizome of the plant Curcuma * Author for correspondence. Phone no. +91-532-465462/ 467154. Fax no. +91-532-623221/607367. E-mail: krishnamisra@ hotmail.com/[email protected]. † Department of Pathology, Kamala Nehru Memorial Hospital, Allahabad- 211 002, India.

longa linn. Curcumin (1,7-bis(4-hydroxy-5-methoxyphenyl)-1,6-heptadiene-3,5-dione/diferuloyl methane) has been used for centuries as a traditional medicine for external/internal wounds, liver diseases (particularly jaundice), blood purification, and inflamed joints (rheumatoid arthritis) (10-13). It has been reported to have antioxidant properties (14, 15). Turmeric and curcumin have been shown to inhibit carcinogen-induced mutation in the Ames assay and the formation of tumors in several experimental systems, suggesting antiinitiating and/or antipromoting activity against several chemical carcinogens (16-20). Recently, it has been reported to decrease total cholesterol and LDL cholesterol levels in serum and also to increase the beneficial HDL cholesterol level. Curcumin is reported to inhibit the proliferation of HUVEC (human umbilical vein endothelial cells); therefore, it could turn out to be a useful compound for the development of novel anticancer therapy (21). It has also been claimed that curcumin inhibits HIV-I integrase protein, thus potentially preventing HIV-I from infecting CD-4 and CD-8 cells (22). The other attractive features of curcumin to explore as a vulnerary agent is that, despite being eaten daily for centuries in Asian countries, curcumin has not been reported to be toxic (11). Curcumin has an interesting structure with two phenolic groups and one active methylene function, which are potential sites for attaching biomolecules. There is not much scope for structural variation in the curcumin molecule in order to enhance its activity, since it is reported that blocking the phenolic groups decreases its antioxidant activity; therefore, phenolic groups appear to be the sites involved in enzymatic activity at receptor sites. The double bonds are essential for proper conformational flexibility of the molecule. However, its meager absorption through the intestinal wall on oral intake needs to be improved in order to achieve significant concentration inside the cells for appropriate activity. One of the most easily accessible approaches is to make bioconjugates of this molecule by

10.1021/bc0000482 CCC: $20.00 © 2001 American Chemical Society Published on Web 06/15/2001

Syntheses of Antibacterial Curcumin Bioconjugates

covalent attachment with ligands to promote entry into the pathogen cell. Since glycine and uridine are essential components of bacterial cell wall, when linked covalently to curcumin they may act as carrier molecules and thereby increase the intracellular delivery of curcumin. Henceforth, the nucleoside-curcumin bioconjugates and amino acid-curcumin bioconjugates can serve a dual purpose of systemic delivery as well as a therapeutic agent against viral diseases. The size of these molecules as compared to liposomes (>1-2 µm) is small enough to get thorough passage in pulmonary capillaries, and the approach for improving the uptake involves such a design that would allow enzyme-mediated transformation of the bioconjugate within the target organ. The conjugate bonds reported herein in the case of amino acidcurcumin are exclusively enzyme sensitive to cause a positive systemic delivery. During the present work, a number of such conjugates have been synthesized, e.g., di-O-glycinoylcurcumin (I), di-O-glycinoyl-C4-glycylcurcumin (II), 5′-deoxy-5′-curcuminylthymidine (5′-curT) (IV), and 2′-deoxy-2′-curcuminyluridine (2′-cur-U) (V), and tested against a number of Gram-positive and Gramnegative bacteria isolated from clinical specimens. MATERIALS AND METHODS

All solvents used were thoroughly dried and distilled prior to use. Curcumin, glycine, allyl bromide, and triphenyl phosphite were purchased from Merck-Schuchardt, Germany. Nucleosides were purchased from Sigma Chemical Company, St. Louis, MO. MuellerHinton broth, agar, and sterile disks were purchased from Hi Media Laboratory Ltd, Mumbai, India. The water-soluble turmeric peptide (Tp) was purified from aqueous turmeric extract, lyophilized, and stored at -30 °C. Mueller-Hinton broth and agar have been selected for testing aerobic and facultative anaerobic bacterial isolates for fastidious organisms such as Streptococci and Peptococci. The agar was supplemented with 5% defibrinated blood. The microsusceptibility tests were standardized at pH 7.4, and agar and broth were incubated in an ambient air incubator at 37 °C. The inoculum was prepared from broth culture that had been incubated for 4-6 h, when growth was considered in the logarithmic phase. Amoxyclave, taken as standard drug, is a combination of amoxicillin and clavulanate. Experimental Guidelines To Determine Zone of Inhibition by Kirby-Baur’s Method (24). 100 mg each of I, II, III, IV, V, curcumin, and peptide were weighed and dissolved in 10 mL of acetone. From this stock solution, serial dilutions were performed to get 30, 15, 7.5, 3.75, 1.88, and 0.94 µmol/mL with acetone in sterile test tubes. Sterilized filter disks were dipped in these solutions and subsequently dried to remove acetone. Nutrient agar medium plates were prepared using Mueller-Hinton agar and allowed to solidify. Seven different bacterias were selected, viz. Enterobacter cloaceae, Peptococcus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Micrococcus cocci, Staphylococcus aureus, and Streptococcus pyogenes and 1 mL each of the bacteria’s culture broth was added to the plates and spread with the help of a sterile spreader. The filter paper disks soaked in bacterial strain were placed asceptically over the inoculated plates using sterile forceps. The plates were incubated at 37 °C for 18 h in upright position. The zone of inhibition was measured using scale. Experimental Guidelines To Determine Mic by the Micro Dilution Broth Susceptibility Test. Dif-

Bioconjugate Chem., Vol. 12, No. 4, 2001 465

ferent concentrations 30, 15, 7.5, 3.75, 1.88, and 0.94 µmol/mL of I, II, III, IV, V, curcumin, and turmeric peptide were prepared in sterile dry test tubes. Nutrient broth was prepared using Mueller-Hinton broth (M391), and 4.9 mL of it was added to each test tube and then sterilized after plugging. After cooling, 0.1 mL of each of the dilutions was added to the test tubes, and final volume was brought to 5.0 mL. To each of the test tubes was added 0.1 mL of bacterial culture broth. The test tubes were shaken to uniformly mix the inoculum with the broth. The tubes were incubated at 37 °C for 18 h. Appearance of any turbidity shows that the compound is not able to inhibit the growth of bacteria, while no turbidity indicates the inhibition of microorganism by the sample. 1,7-Bis(4-O-glycinoyl-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (I). Curcumin (368 mg; 1 mmol) was dissolved in dry pyridine, mixed with N-phthaloylglycinoyl chloride (536 mg; 2.4 mmol), and stirred at room temperature for 6 h. After the completion of reaction, the reaction mixture was poured into crushed ice and thoroughly extracted with EtOAc. The organic layer was concentrated and treated with ammonia: pyridine (9:1 v/v) for 1 min at room temperature. The organic layer was concentrated and purified by silica gel column chromatography using a DCM:methanol gradient, yield 40% (192 mg). The pure product was characterized by elemental data and 1H NMR. Anal. Found: C, 62.05; H, 5.56; N, 5.61%. Calcd for C25H26O8N2: C, 62.17; H, 5.39; N, 5.80%. 1H NMR (CDCl3) δ ) 3.70 (s, 6H, OCH3), 4.13 (s, 2H, C4-H), 4.49-4.61 (m, 4H, CH2NH2), 6.53 (d, 2H, C2-H and C6-H), 6.85-7.08 (m, 6H, Ar-H), 7.53 (d, 2H, C1-H and C7-H). 1,7-Bis(4-O-glycinoyl-3-methoxyphenyl)-1,6-heptadiene-C4-glycinoyl-3,5-dione (II). Curcumin (368 mg; 1 mmol) was dissoved in EtOH, NaOEt (containing 83 mg of metallic sodium; 3.6 mmol) was added dropwise for 10 min, and the reaction mixture was stirred at room temperature for 30 min. The resulting sodium salt was concentrated under vacuo, thoroughly washed with EtOH, dissolved in dry pyridine, mixed with N-phthaloylglycinoyl chloride (804 mg; 3.6 mmol), added to the reaction mixture, and stirred at room temperature for 6 h. After completion of reaction, the reaction mixture was poured into crushed ice and thoroughly extracted with EtOAc. The organic layer was concentrated and treated with ammonia:pyridine (9:1 v/v) for 1 min at room temperature. The reaction mixture was poured into crushed ice and extracted again with EtOAc. The organic layer was concentrated and purified by silica gel column chromatography using a DCM:methanol gradient, yield 38% (205 mg). The pure product was characterized by elemental data and 1H NMR. Anal. Found: C, 59.88; H, 5.60; N, 7.51%. Calcd for C27H29O9N3: C, 60.04; H, 5.48; N, 7.78%. 1H NMR (CDCl ) δ ) 3.73 (s, 6H, OCH ), 4.05 (s, 1H, 3 3 C4-H), 4.43-4.79 (m, 6H, CH2NH2), 6.58 (d, 2H, C2-H and C6-H), 6.81-7.03 (m, 6H, Ar-H), 7.56 (d, 2H, C1-H and C7-H). 5′-Deoxy-5′-curcuminylthymidine (IV). 5′-Deoxy-5′bromothymidine (305 mg, 1 mmol), synthesized by a procedure developed in our laboratory (23), was dissolved in dry pyridine and mixed with III (627 mg, 1.3 mmol). The reaction mixture was stirred at room temperature for 8 h. The product was treated with 2 N alcoholic NaOH at room temperature for 45 min to afford 5′-deoxy-5′-curcuminylthymidine by the usual workup procedure as described above, yield 40% (237 mg). The pure product was characterized by elemental data and 1H NMR. Anal. Found: C, 62.59; H, 5.56; N, 4.54%.

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Chart 1. 1H NMR Assignments for I, II, IV, and V

Calcd for C31H32O10N2: C, 62.77; H, 5.40; N, 4.72%. 1H NMR (CDCl3) δ ) 2.08 (s, 3H, C5-CH3 of T); 2.41-2.48, 2.62-2.67 (m, 2H, 2′-H); 3.37-3.41 (m, 1H, C4-H of Cur); 3.59 (s, 6H. OCH3); 3.91 (m, 2H, 5′-H); 4.06-4.09 (m, 1H, 4′-H); 5.41-5.49 (m, 1H, 3′-H); 6.39-6.43 (m, 1H, 1′-H); 6.51 (d, 2H, C2-H and C6-H of Cur); 6.79-7.13 (m, 6H, Ar-H); 7.54 (d, 2H, C1-H and C7-H); 7.73 (s, 1H, C6-H of T). 2′-Deoxy-2′-curcuminyluridine (V). 2′-Deoxy-2′chlorouridine (263 mg, 1 mmol), synthesized by the known procedure (22), was dissolved in dry pyridine and mixed with Ia (627 mg, 1.3 mmol). The reaction mixture was stirred at room temperature for 8 h. The product was treated with 2 N alc NaOH to get 2′-deoxy-2′-curcuminyluridine by the usual workup procedure as describe above, yield 40% (237 mg). The pure product was characterized by elemental data and 1H NMR. Anal. Found: C, 60.37; H, 5.19; N, 4.66%. Calcd for C30H30O11N2: C, 60.55; H, 5.05; N, 4.71%. 1H NMR (CDCl3) δ ) 2.462.59 (m, 1H, 2′-H); 3.34 (d, 1H, C4-H of Cur); 3.62 (s, 6H, OCH3); 3.69 (d, 2H, 5′-H); 4.11-4.16 (m, 1H, 4′-H); 5.135.15 (m, 1H, 3′-H); 5.81 (d, 1H, C5-H of uridine); 6.34 (d, 1H, 1′-H); 6.59 (d, 2H, C2-H and C6-H of Cur); 6.98-7.23 (m, 6H, Ar-H); 7.51 (d, 2H, C1-H and C7-H of Cur); 7.78 (d, 1H, C6-H of uridine).

RESULTS AND DISCUSSION

Schemes 1 and 2 show the experimental steps involved in the preparation of I and II, and IV and V, respectively. In the case of II, a strong base, NaOEt, was used whereas in the case of I, curcumin itself was treated with N-phthaloylglycinoyl chloride (Scheme 1). 5′-Deoxy-5′-curcuminylthymidine (5′-cur-T) (IV) and 2′deoxy-2′-curcuminyluridine (2′-cur-U) (V) were synthesized by adding 5′-deoxy-5′-bromothymidine and 2′-deoxy2′-chlorouridine (23) to pyridine mixed with III as shown in (Scheme 2). The antibacterial activity of bioconjugates I, II, III, IV, V, and Tp were compared with curcumin from a known microdilution broth susceptibility test method. These were serially diluted (30, 15, 7.50, 3.75, 1.88, 0.94 µmol/ mL) and added to Mueller-Hinton broth, after which a standardized bacterial suspension was added. One of the disks was kept free of antibiotic and served as growth control, and the other disks were inoculated with a calibrated suspension of microorganism to be tested and incubated at 37 °C for 18 h. The lowest concentration of curcumin bioconjugates in µmol/mL that prevents the in vitro growth of microorganism is represented as MIC (minimum inhibitory concentration) (Table 1) and correlated with the zone of inhibition (Table 2).

Syntheses of Antibacterial Curcumin Bioconjugates

Bioconjugate Chem., Vol. 12, No. 4, 2001 467

Scheme 1

Scheme 2

Each test was performed in triplicate, and the MICs reported represent the result of at least two repetitions. Out of four, three bioconjugates (I, II, and V) show a positive result on multiresistant microorganisms. The

most encouraging result was found against Streptococcus pyogenes, with I having an MIC of 1.88 µmol/mL, compared with one of the most widely marketed antibiotics, viz. Amoxyclav, that has an MIC of 7 µmol/mL (6

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Table 1. MIC Correlation Diagram (in µmol/mL)a,b no. 1 2 3 4 5 6 7

name of bacteria

I

E. cloacae Peptococcus Staphylococcus epidermidis Staphylococcus saprophyticus Micrococcus cocci Staphylococcus aureus Streptococcus pyogenes

II

3.75 3.75 15.00 3.75 3.75 15.00 1.88d

7.50 7.50 15.00 30.00 15.00 30.00 3.75

V

Cs

15.00 30.00 7.50 15.00 3.75 7.50 15.00

30 R 7.50 7.50 7.50 15.00 R

Cur Rc R R R R R 15.00

T R 3.75 R R R R R

a I: Di-O-glycinoylcurcumin, II: di-O-glycinoyl-C4-glycylcurcumin, V: 2′-deoxy-2′-curcuminyluridine (2′-cur-U), Cs: sodium salt of curcumin, Cur: curcumin, T: turmeric. b Results with 5′-deoxy-5′-curcuminylthymidine (5′-cur-T) are not included. c R: resistant (below 10 mm). d I shows the best result against Streptococcus pyogenes (1.88 µmol/mL), and it was compared with Amoxyclav (7 µmol/mL).

Table 2. Antibacterial Activity of Curcumin Bioconjugates (zone of inhibition in mm)a,b no. 1 2 3 4 5 6 7

name of bacteria

I

II

V

Cs

Cur

T

E. cloacae Peptococcus Staphylococcus epidermidis Staphylococcus saprophyticus Micrococcus cocci Staphylococcus aureus Streptococcus pyogenes

24, 22, 20, 16 18, 16, 14, 12 14, 12 18, 16, 14, 12 16, 15, 13, 11 14, 12 28, 24, 20, 18, 16

20, 18, 16 16, 12, 11 15,13 12 15, 12 12 22, 24, 18, 16

14, 12 13 16, 15, 14 13, 12 18, 16, 14, 12 13, 12, 11 14, 12

13 R 15,12, 11 20, 16, 14 16, 13, 11 14, 12 R

Rc R R R R R 20, 16

R 22, 20 18, 12 R R R R R

a I: Di-O-glycinoylcurcumin, II: di-O-glycinoyl-C4-glycylcurcumin, V: 2′-deoxy-2′-curcuminyluridine (2′-cur-U), Cs: sodium salt of curcumin, Cur: curcumin, T: turmeric. b Zone of inhibition is reported with respect to serial dilution (30, 15, 7.5, 3.75, 1.88, 0.94 µmol/ mL) c R: resistant (below 10 mm).

µmol/mL reported). This shows that I is 3.7 times more effective than Amoxyclav. The result of zone of inhibition is also encouraging, as the disk containing 30 µg of Amoxyclav was purchased and same amount of I was loaded on a separate disk. The zone of inhibition of Amoxyclav was found to be 20 mm while I showed 28 mm zone of inhibition. The MICs of II, V, and III were found between 4 and 8 µmol/mL in most cases as compared to available antibiotics on the market having an MIC between 6 and 10 µmol/mL. Tp has a significant result against Peptococcus, MIC 3.75 µmol/mL In comparison to the above results, curcumin and turmeric were found to be resistant in most cases except against Streptococcus pyogenes where curcumin shows MIC 15 µmol/mL. The results with 5′-deoxy-5′curcuminylthymidine (IV) were far from satisfactory against the selected bacterial strains, which may be due to the fact that thymidine is not a natural component of bacterial genome and hence unable to internalize in the bacterial cell wall (data not shown). The results suggest that amino acids glycine and D-alanine are natural components of the bacterial cell wall, thus the positive results of I and II, whereas the positive result of V is due to uridine, which is an essential component of the bacterial cell wall and thus well recognized by bacteria. However, the amino acid bioconjugates have been found to be bacteriologically more active (approximately two times) than nucleoside bioconjugates, since the former, in addition to being internalized like the latter, are also more water soluble. The hydrophilic nature of these bioconjugates may also help in active transport across cellular membrane. It is therefore anticipated that bioconjugates composed of D-alanine would give the best results. This work is in progress and will be published elsewhere. CONCLUSION

Conventionally, antibiotics are used to kill bacteria or to halt their division with the intention of preventing replication of the bacterial genome, but overuse of antibiotics in humans and livestock has led to the rapid evolution of bacteria that are resistant to multiple drugs (25), and recently there has been a call for worldwide use

of antibiotic rotation schemes to conquer the problems of resistance (26). β-Lactamase present in bacterial cellular fluid hydrolyzes the amide bond of the β-lactam ring of penicillins and cephalosporins, producing acidic derivatives which have no antibacterial properties, thereby making the bacteria resistant to that particular antibiotic drug. To develop a new armory of agents active against antibiotic-resistant bacteria, nonconventional antibiotics could be designed that can internalize themselves in the bacterial cell wall easily. The structures should be devoid of the β-lactam ring for better efficacy. Curcumin, the main molecule studied in this paper, is reported to be capable of expanding the cell membrane in in vivo conditions in eukaryotic cells, inducing echinocytosis (27), and changes in cell shape are accompanied by transient exposure to phosphatidyl serine. Membrane asymmetry was recovered in the presence of aminophospholipid traslocase, which remains active in the presence of curcumin, thereby suggesting that curcumin is able to be transported if covalently attached to a molecule which can easily cross the cell wall both in eukaryotic and in prokaryotic cells. This fact is supported by the studies reported here, by curcumin bioconjugates, where curcumin is conjugated with a biomolecule which is the active part of either the bacterial genome, i.e., uridine (V), or of the bacterial cell wall, i.e., amino acids (I and II) gave excellent results, whereas IV, i.e., curcumin conjugated with thymidine, failed to show positive results since thymine is not a natural component of the bacterial genome, thus unable to internalize in the bacterial cell wall. More such bioconjugates may be designed by keeping one of the components common to either the bacterial cell wall or bacterial genome conjugated with curcumin, thereby improving the efficiency of curcumin comprehensively at low doses, and may prove to be better systemic drugs by concentrating mainly on and around malignant and ill-fated cells by recognizing the bacterial cells. This study proves once again the efficacy of curcumin and its bioconjugates and their antibacterial properties. The present work suggests that the need for nonantibiotic drugs (28) which may overcome antibiotic resis-

Syntheses of Antibacterial Curcumin Bioconjugates

tance may be fulfilled in the near future. A new series of inexpensive and highly bioactive drugs with no toxicity may be in the offing. ACKNOWLEDGMENT

The authors are very thankful to Department of Science and Technology (DST), New Delhi, India, for financial assistance. LITERATURE CITED (1) Perlaky, L., Saijo, Y., Busch, R. K., Bennett, C. F., Mirabelli, C. K., Crooke, S. T., and Busch, H., (1993) Growth inhibition of tumor cell line by antisense oligonucleotides designed to inhibits p120 expression. Anti-Cancer Drug Des. 8, 3-14. (2) Saijo, Y., Perlaky, L., Wang, H., and Busch, H. (1994) Pharmacokinetics, tissuedistribution, and stability of antisense oligodeoxynucleotides phosphorothioate ISIS 3466 in mice. Oncol. Res. 6, 243-249. (3) Marzo, A. L., Fitzpatrick, D. R., Robinson, B. W. S., and Scott, B. (1997) Antisense oligonucleotides specific for transforming growth factor b2 inhibit the growth of malignant mesothelioma both In vitro and In vivo. Cancer Res. 57, 32003207. (4) Sacco, M. G., Barbieri, O., Piccini, D., Noviello, E., Zoppe, M., Zucchi, I., Frattini, A., Villa, A., and Vezzoni, P. (1998) In vitro and In vivo antisense-mediated growth inhibition of a mammary adenocarcinoma from MMTV-neu transgenic mice. Gen. Ther. 5, 388-393. (5) Gokhaley, P. C., Soldatenkov, V., Wang, F. H., Rahmen, D., Drischilo, A., and Kasid, U. (1997) Antisense raf oligodeoxyribonucleotide is protected by liposomal encapsulation and inhibits Raf-1 protein expression In vitro and In vivo implication for gene therapy of radioresistant cancer. Gen. Ther. 4, 1289-1299. (6) Litzinger, D. C., Brown, J. M., Wala, I., Kaufman, S. A., Van, G. Y., Farrell, C. L., and Collin, D. (1996) Fate of cationic liposomes and their complex with oligonucleotide in vivo. Biochim. Biophys. Acta 1281, 139-149. (7) Hope, M. J., Bally, M. B., Webb, G., and Cullis, P. R. (1985) Production of large unilamellar vesicles by a rapid extrusion procedure, characterisation of size distribution, trapped volume and ability to maintain a membrane potential. Biochim. Biophys. Acta 812, 55-56. (8) Litzinger, D. C. (1997) Limitation of cationic liposomes for antisense oligonucleotide delivery in vivo. J. Liposome Res. 7, 51-61. (9) Robins, R. K and Revankar, G. R. (1988) Design of Nucleoside Analogues as Potential Anti Viral Agents, in Antiviral Drug Development: A Multidisciplinary Approach (Clercq, E. D., and Walker, R., Eds.), pp 11-36, Plenum Press, New York. (10) Govindrajan, V. S. (1979) Turmeric chemistry, technology and quality. CRC Crit. Rev. Food Sci. 12, 199-301.

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