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Jamonline / 2(2); 2012 / 165–175 Research Article

Venkateswarlu G et al

Journal of Atoms and Molecules
An International Online Journal
ISSN – 2277 – 1247

TARGETING EFFICIENCY EVALUATION OF FLUOROURACIL IN PLGA MICROSPHERES CONJUGATED WITH HUMAN MONOCLONAL ANTIBODIES Venkateshwarlu Goli1*, Sravan Prasad Macharala2, Gowrishankar NL3, Manoranjan sabat1
1

Venkateshwara Institute of Pharmaceutical Sciences, Nalgonda, Andhra Pradesh, India-508001. 2 GBN Institute of Pharmacy, Ghatkesar, RR District, Andhra Pradesh, India-501301 3 Swami Vivekananda Institute of Pharmacy, Buvanagiri, Andhra Pradesh. India – 508001 Revised on: 10-04-2012 Accepted on: 19–04–2012

Received on: 17-03-2012 Abstract:

In the advent of producing an effective tumour targeting drug system, Poly lactic acid-co-glycolic acid microspheres were formulated using Double (multiple) emulsion technique with and without human Ig G. The ideal ratio of drug to albumin was attained by varying the concentration of drug and this ideal batch of drug loaded microspheres was selected and subjected to physical characteristics such as size, shape and uniformity by size distribution analysis. All the test batches of microspheres were subjected to in-vitro evaluation and ideal batch of 5-Fluorouracil microspheres with and without monoclonal antibodies were subjected in-vivo evaluation for drug distribution and targeting efficiency by comparing with free 5-Fluorouracil. The results helped to arrive for a conclusion that the Fluorouracil immune-microspheres group was more effective than Fluorouracil microspheres and Fluorouracil groups but it will be worthwhile to investigate the anticancer activity of active targeting system by conjugating specific antibodies against to specific tumour antigen. Key Words: Tumour, fluorouracil, microsphere, immunoglobulin G Introduction: * Corresponding author Venkateswarlu Goli, Email: pharmgroup2011@gmail.com Tel: +91 9985189859 Cancer is due to failures of the mechanisms that usually control the growth and

proliferation of cells. Scientific evidence suggested that about one third of 5,55,500 cancer deaths were expected to occur in 2007 will be related to nutrition, physical inactivity, obesity and other lifestyle factors and could

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Jamonline / 2(2); 2012 / 165–175 also be prevented. Cancer is related to infectious exposure e.g. Hepatitis B Virus (HBV) human papilloma virus (HPV), human immuno deficiency virus (HIV), helicobacter and other, and could be prevented through behavioral changes, vaccines or antibiotics. In addition many of the more than 1 million skin cancers that were diagnosed in 2007 could have been prevented by protection from sun’s rays. Until the 19th century, the only specific method of treatment for cancer was surgical removal, sometimes followed by cauterization of the wound. However, this approach can only cure the disease if the malignant tissue is removed completely and the tumor is at such an early stage of development that no secondary’s – termed metastases – have formed. The introduction of chemotherapy by Paul Ehrlich in 1909 started a new chapter, and since then chemically produced or modified substances have been used which act specifically against pathogens or cancer cells. The current trio of standard anticancer modalities – chemotherapy, radiation therapy and surgery are the foundations of modern oncology practice. When compared to other drug therapy, adverse effects are severe and may necessitate supportive drug therapy and intensive nursing care. Their appearance may be life threatening and may limit the further use of the drugs. Majority of the cytotoxic drugs have more profound effect on rapidly multiplying cells, because the most important target of action are the nucleic acids and their

Venkateswarlu G et al precursors; rapid nucleic acid synthesis occurs during cell division. Many cancers (especially large tumours) have a lower growth fraction (lower percentage of cells are in division) than normal bone marrow, epithelial linings, reticulo endothelial system and gonads. These tissues are particularly affected in a dose dependent manner by majority of drugs, though there are differences in susceptibility to individual members (1). Selective targeting of anticancer drugs with concomitant

elimination of toxic effects has been a goal in cancer chemotherapy (2). Drug delivery systems for cancer therapeutics have now been used by millions of patients and have resulted in the creation of new therapies as well as significantly improving existing ones. Monoclonal Antibody drug delivery systems that have been approved by regulatory authorities and that are currently in clinical use, such as controlled delivery of cancer therapeutics, local chemotherapy, polymer drug conjugates, liposomal systems, and transdermal drug delivery patches. In recent years, scientific progress has yielded a number of promising cancer treatment

approaches. These approaches are designed to enhance the specificity and potency of cancer therapeutics, improve all efficacies and reduce side effects. It is now believed that one of the most promising approaches is the monoclonal antibodies technologies in the development of anti-tumour targeting therapy. At first agents has for cancer

FDA

approved

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Jamonline / 2(2); 2012 / 165–175 adriamycin antibody conjugate as a tumour targeting therapeutic system. Since then many drugs have been approved and now in the market. The amount of antibody used to produce a conjugate directly with the drug is more and advantages of using drug carrier antibody conjugate system is explored conjugated with

Venkateswarlu G et al Human Monoclonal

Antibodies and to evaluate its targeting efficiency by in-vitro and in-vivo methods. Materials and methods: Preparation of Poly lactic acid-co-glycolic acid microspheres - Double (multiple) emulsion technique: Required amount of 5-Fluorouracil was weighed accurately and was dissolved in pH 6.8 phosphate buffer (which act as internal aqueous phase (W1)). 6 % (W/V) Poly lactic acid-co-glycolic acid (molecular weight;

extensively, it is not far for such system to emerge as better tumour targeting system than the drug antibody conjugates. Fluorouracil (5FU) requires enzymatic conversion to the nucleotide (ribosylation and phosphorylation) in order to exert its cytotoxic activity. 5-FU may be converted to fluorouridine by uridine phosphorylase and then to floxuridine

50,000) in ethyl acetate with 1 % surfactant (which will be an oil phase (O)) was prepared. This was emulsified by using a homogenizer at 13,000 rpm in ice bath for 5 min. The resulting water-in-oil emulsion was then emulsified applying magnetic stirrer at 600 rpm with a 2.5 % (W/V) polyvinyl alcohol solution (which will be an outer aqueous phase (W2)) to produce a W/O/W emulsion. That emulsion was agitated for 10min and the solvent was rapidly eliminated by extraction with 200 ml of an aqueous isopropyl alcohol solution (5 % V/V). Microspheres were then collected by centrifugation (3-5). The resulted microspheres were washed and dried. The process variables on 5-Fluorouracil loading into PLGA microspheres were investigated by each time one variable was varied keeping the others constant. From the results optimum level of the particular variable was selected and the subsequent evaluation of effect of

monophosphate (FUMP) by uridine kinase, or it may react directly with 5-phosphoribosyl-1pyrophosphate (PRPP), in a reaction catalyzed by orotate phosphoribosyl transferase, to form FUMP. In addition to leucovorin, a number of other agents have been combined with 5-FU in attempts to enhance the cytotoxic activity through biochemical modulation. 5-

Fluorouracil produces partial responses in 10% to 20% of patients with metastatic colon carcinomas, upper gastrointestinal tract

carcinomas, and breast carcinomas. The administration of 5-FU in combination with leucovorin in the adjuvant setting is

associated with a survival advantage for patients with colorectal cancers and gastric cancers. In this context and scope, this work is designed to target the tumour with Poly lactic acid-co-glycolic acid (PLGA) microspheres

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Jamonline / 2(2); 2012 / 165–175 drug to PLGA was observed. The ratio of lactic and glycolic acid was also included in the study i.e., with every process variable the ratio of lactic acid and glycolic acid in the PLGA was varied keeping the other

Venkateswarlu G et al In vitro release study: Accurately weighed 50 mg of the standard drug was dissolved in 100 ml of 7.4 pH phosphate buffer to give a solution of 500 µg/ml concentration and this was served as a standard stock solution. From this 10 ml solution was taken and made up to 100 ml to give a concentration of 50µg/ml and this was served as standard solution. Into a series of 10 ml volumetric flasks 1 ml, 2 ml, 3 ml, 4 ml and 5 ml of standard solution was added and the volume made up by the same solvent. The absorbance of these solutions was measured against reagent blank by U.V

parameters as constant. Estimation of amount of 5-Fluorouracil incorporated Accurately weighed 25 mg standard 5Fluorouracil was dissolved in 50 ml of mixture of acetic acid and methanol in the ratio of 1:1 to give a solution of 500 µg/ml concentration and this was served as standard stock solution. Form this 10 ml of the solution was taken and the volume was made up to 50ml with the same solvent to give a concentration of 100 µg/ml and this was served as standard solution. Into three 25 ml volumetric flasks 2.5 ml, 7.5 ml and 12.5 ml of standard solution were added and the volume was made up by using the same solvent. The absorbance of this solution was measured against reagent blank by U.V spectrophotometer. The standard curve

Spectrophotometer. A standard curve against concentration and absorbance was plotted. Accurately weighed 50 mg of 5-Fluorouracil loaded PLGA microspheres were suspended in 100 ml of phosphate buffer solution in a 250 ml conical flask. Then the flask was kept in a shaker cum incubator which was adjusted to 80 horizontal stokes per minute at 370C. 5 ml of the drug released solution was withdrawn at various time intervals of 0.25, 0.5, 1, 2, 4, 8, 16 and 24 hours. The samples withdrawn were filtered through a membrane filter of pore size of 0.22µm by using vacuum pump (6). The drug content was estimated in the clear filtrate by U.V spectrometer. Cumulative release profiles (%) were

against concentration and absorbance was plotted. 25 mg portion of the 5-Fluorouracil containing PLGA microspheres were

incubated with 25 ml of mixture of acetic acid and methanol in the ratio of 1:1 at 40C for 24 hours. After 24 hours incubation, the

microspheres were separated by high speed centrifugation and the drug content was analyzed by using the supernatant by U.V spectrometer. All rights reserved© 2011

analyzed as a function of incubation time.

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Jamonline / 2(2); 2012 / 165–175 Preparation of 5-Fluorouracil ImmunoMicrospheres 100 mg of 5-Fluorouracil microspheres were incubated with 10 mg of Human Monoclonal antibodies (Human IgG) dissolved in 10 ml of Hanks Balanced Salt Solution(HBSS) for 30 minutes at 40C and then washed with HBSS to remove the un-conjugated monoclonal antibodies and stored at 40C (7). In-Vitro Cytotoxicity Test Aliquots of (1 ml) of cell type Hep-2 (1x105/ml) which was added to RPMI 1640 medium supplemented with 10% heat

Venkateswarlu G et al Fc = Conversion factor (104). No of viable cells is equal to difference between total number of cells present and the number of dead cells. Percentage viability = (Total number of viable cells/Total number of cells) x 100 In-Vivo Passive and Active targeting Wistar mice were fed with a standard diet and water ad libitum. The animals were housed in spacious polypropylene cages bedded with rice husk. The animal room was well ventilated and maintained under standard experimental conditions (Temperature 27°C and 12 hours light / dark cycle) throughout the experimental period. Animal experiments were carried out following the guidelines of the animal ethics committee of the institute. The efficacy of the formulation was evaluated by involving 72 mice of 2 batches containing 36 mice each. Both the batches were further divided into 6 groups, each containing 6 mice. All the mice were induced cancer with EAC and were allowed to grow with tumor. The treatment was started from the 2nd & 11th day for the batch I and II respectively. Group I of both the batches was not treated with any drug and allowed to grow with cancer. This group served as control. Group II received the solvent HBSS which was administered by intravenous through tail vein. This group served as solvent control. Group III received the empty microspheres dispersed in HBSS. Group IV received the www.jamonline.in 169

inactivated foetal calf serum was seeded in to 24 well micro titre plate. Subsequently varying 25µg/ml, Fluorouracil amount of 5-Fluorouracil and 75µg/ml, (FU) 5-

50µg/ml

microspheres

(FU-MS)

equivalent to 25µg/ml, 50µg/ml and 75µg/ml of 5-Fluorouracil per well and 5-Fluorouracil immune-microspheres (FU-IMS) equivalent to 25µg/ml, 50µg/ml and 75µg/ml of 5Fluorouracil per well were added and incubated at 370C under 5% atmospheric CO2 for three days. Along with the different samples of anticancer drug a control was also performed to check the effect of auto lyse of cells. Number of viable cells remaining in each of the wells was determined by trypan blue dye exclusion technique. Total number of viable cells = Nv x Df x Fc, where Nv = Number of viable cells, Df = Dilution factor, All rights reserved© 2011

Jamonline / 2(2); 2012 / 165–175 drug (FU) 1.4mg/kg and group V & VI received the FU-MS & FU-IMS equivalent to 1.4mg 5-Fluorouracil/kg respectively.

Venkateswarlu G et al uniformity and the size distribution were not satisfactory. Since aggregation of

microspheres was noted when 5-Fluorouracil concentration increased above 7.5 mg. From the above observation, the 7.5 mg batch was found to be uniformly distributed and was selected as an ideal batch for the conjugation of monoclonal antibodies for drug targeting. All the batches of microspheres of 5Fluorouracil loaded with various

Survival time, Packed cell volume, Body weight and Hematological parameters like RBC count, WBC count, Differential count and Hemoglobin content were studied during the period of study. Result and discussion: The PLGA microspheres containing 5-

concentrations were subjected to in-vitro drug release study and were observed that there was a gradual increase in the percentage of drug release during the 24 hours study. The cumulative % release of 5-Fluorouracil for 2.5 mg, 5.0 mg, 7.5 mg, 11.0 mg and 12.5 mg were observed to be 84.76, 87.62, 92.96, 93.17 and 93.4 respectively. Since there was no significant difference in the cumulative % release of 7.5 mg, 10 mg and 12.5 mg based on other parameters such as size distribution analysis and percentage of drug incorporated 7.5 mg batch with a cumulative % release of 92.96 was selected as ideal batch for further studies. This batch can be considered suitable for the investigation as passive and active targeting system against cancer cell Dalton’s Lymphoma Ascites (EAC) induced tumour studies in mice. The 5-Fluorouracil immuno-microspheres

Fluorouracil were prepared. The average size of the microspheres consists 2.5mg, 5mg, 7.5mg, 10mg and 12.5mg of 5-Fluorouracil in 6% of PLGA possesses 2.01, 2.14, 2.38, 2.76 and 2.93 µ respectively. The increase in concentration of the drug proportionately increases in size of the microspheres range from 2.01 µ to 2.93 µ. The 7.5 mg drug loaded microspheres were selected as ideal batch considering the particle size

distribution, uniformity and the tendency to aggregate based on the results of drug loading estimation into the microspheres. The amount of 5-Fluorouracil loaded into microspheres results showed a gradual increase in drug pay load to the increase in concentration of drug. It was found that the percentage of 5Fluorouracil payload was high (i.e. 75%) for 7.5 mg batch. In the higher concentrations like 10 mg and 12.5 mg batches the percentage of drug pay load was only 72.3% and 68.7% respectively. Though the 10 mg and 12.5 mg batches of microspheres contain more amount of drug, its % drug pay load, All rights reserved© 2011

(FU-IMS) were prepared with purchased Human Ig G. Figure 1 shows the

photomicrographs of FU-IMS. To assess the affinity of the microsphere system on the cells www.jamonline.in 170

Jamonline / 2(2); 2012 / 165–175 in-vitro on HEp-2 cell line was tested with FU, FU-MS and FU-IMS in three

Venkateswarlu G et al post day infection groups, FU-IMS batch showed 67.42 % tumour inhibition, FU-MS showed 45.89 % and FU showed 40.79 %. In 11th day post infection group the % tumour inhibition was 63.70, 42.72 and 20.98 for FUIMS, FU-MS and FU respectively. The significant antitumour activity of FU-IMS is due to the efficient targeting of 5-Fluorouracil to antigen positive cells in turn to yield a high concentration of 5-Fluorouracil localized around target cells, finally interaction of 5Fluorouracil with the target sites of cells for the inactivation. The efficacy of FU-MS with and without monoclonal antibodies was also found encouraging with the results of RBC and WBC count shown in Table 2. The RBC count for FU-IMS, FU-MS and control for batch I was 9.24 x 106, 8.43 x 106 and 7.68 x 106 respectively. In batch II it was found to be in the same way. In case of WBC count batch I and batch II, FU-IMS and FU-MS showed closer values of normal mice. Similarly the results of differential count and haemoglobin estimation were found to be closer with the normal mice. In the treatment against EAC tumour FU-IMS showed better therapeutic efficacy in terms of all the parameters analyzed during the study. The results of the mean survival time and % increase in life span were calculated for both the batches and given in Table 2. It was observed that the 2nd day post infection batch showed better % increase in life span than the 11th day post infection batch. In 2nd day post infection

concentrations (25 µg/ml, 50 µg/ml and 75µg/ml). The study was carried out in the expectation of preferable affinity of FU-IMS with the cells and release of the drug from the microsphere to the cells thereby exhibiting cytotoxicity. The results were as expected that the FU-MS and FU-IMS have shown

sufficient cytotoxicity with trypan blue dye exclusion technique as shown in Table 1 and figure 2. The efficacy of the formulation was studied and the results shown that the FUIMS group was found to be more effective in drug targeting and in exhibiting the

antitumour activity against EAC. The % decrease in body weight after treatment was calculated by observing the weight gain on 20th day after cancer induction. In both the treated batches (treatment after 2nd and 11th day post infection) of FU-IMS showed % weight decrease 66.66 and 53.33 respectively. Whereas in FU-MS the % weight decrease was 53.33 and 40.00. In FU it was only 40.00 and 20.00. Based on these results we can say that FU-IMS has exhibited active targeting of the drug against EAC and similar type of results were observed in the case of % tumour inhibition and increase in life span. The tumour inhibition was calculated by

estimating the packed cell volume of EAC. It was observed that the 2nd day post infection group showed better tumour inhibition than the 11th day post infection groups. In 2nd day

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Jamonline / 2(2); 2012 / 165–175 batch FU-IMS group showed 95.56 % increase in life span, FU-MS group showed 74.54 % increase in life span and FU group showed 47.78 % increase in life span, compared to the control group. In 11th day post infection batch FU-IMS group showed 71.36 %, FU-MS group showed 47.17 % and FU group showed 19.76 % compared to the control group. It may be noted that the efficacy of the FU-IMS system was 47.78 % more than that of the FU treated batch in 2nd day post infection batch and 51.6% for 11th day post infection batch with respect to the % increase in life span. Andrew M. Scott et al., (1997) reported that the range of possible therapeutic agents with monoclonal antibody targeting therapy was impressive with the selective delivery of toxins and other References:

Venkateswarlu G et al

1. Foster RW. Basic Pharmacology, 4th ed. London: Butter Worth Heineman; 1996; 319-322. 2. Kenneth JW, Andren ES. Magnetic microspheres: A vehicle for selective targeting of drugs, Methods of Drug Del. In: Garret MI editor. Encycl. of

Pharmacology and therapeutics, Section 120, Permamom Press; 1986; 39-57. 3. Mehta RC, Jeyanthi R, Calis S, Thanoo BC, Burton KW, Deluca PP.

Biodegradable microspheres as depot system for parental delivery of peptide drugs. Journal of Controlled Release, 1994; 29: 375-384. 4. Watts PJ, Davis MC, Melia CD. using evaporation; an

cytotoxic materials to tumours, possible by killing these agents with monoclonal

Microencapsulation emulsification/solvent

antibodies (8). Kang Choon Lee et al., (1990) produced monoclonal antibodies against acute lymphoblastic leukemia antigen by mouse tumour induction and the methotrexate loaded immune-microspheres were prepared by the glutaraldehyde activation method (9). The above results helped to arrive for a conclusion that the FU-IMS group was more effective than FU-MS and FU groups but it will be worthwhile to investigate the anticancer activity of active targeting system by

overview of techniques and applications. Critical reviews in therapeutic drug carrier systems, 1990; 7: 235-259. 5. Benoit JP, Marchais H, Rolland H, Velde VV. Biodegradable microspheres:

advances in production technology. In: Benita S, editors. Microencapsulation: Methods and Industrial Applications. New York; Marcel Dekker: 1996; 35-72. 6. Narayani R, Panduranga Rao K.

Preparation, Characterization and in-vitro stability of hydrophilic gelatin

conjugating specific antibodies against to specific tumour antigen.

microspheres using a gelatin-methotrexate conjugate, Int. J. Pharm., 1993; 59: 85-91.

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Jamonline / 2(2); 2012 / 165–175 7. Kang F, Singh J. Conformational stability of a model protein (bovine serum

Venkateswarlu G et al 9. Kang Choon Lee, Yoon Joong Lee, Won Bae Kim and Chang Yong Cha.

albumin) during primary emulsification process of PLGA microspheres synthesis, Int. J. Pharm. 2003; 260 (1): 149-156. 8. Andrew MS, Sydney W. Antibody based immunological therapies: Current opinion in immunology 1997; 9: 717-722.

Monoclonal antibody based targeting of methotrexate loaded microspheres, Int. J. of Pharm., 1990; 59: 27-33.

S.No 1 2 3 4 5 6 7 8 9 10

Group Control FU-25 µg/ml FU-50 µg/ml FU-75 µg/ml FU-MS 25 µg/ml FU-MS 50 µg/ml FU-MS 75 µg/ml FU-IMS 25 µg/ml FU-IMS 50 µg/ml FU-IMS 75 µg/ml

Total No. of viable cells present 10.76 x 104 6.70 x 104 4.68 x 104 3.54 x 104 7.76 x 104 6.42 x 104 5.38 x 104 7.71 x 104 6.70 x 104 5.14 x 104

Table 1: In-Vitro Cytotoxicity

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Jamonline / 2(2); 2012 / 165–175
Batch I Group control FU FU-MS FUIMS 5± 1.17

Venkateswarlu G et al
Batch II FUIMS 7± 1.36

control

FU

FU-MS

Body weight of mice increase (gm)

15 ± 0.15

9 ± 1.81

7 ± 1.23

15 ±1.27

12 ± 1.52

9± 1.26

% Decrease in body weight of mice WBC Average No. of cells (x 10 ) RBC Average No. of cells (x 106) Haemoglobin Estimation (%) Basophil
3

0

40.00

53.33

66.66

0

20.00

40.00

53.33

26.21 ± 0.13 7.68 ± 0.23 8.32 ± 0.67 0 65.3 ± 2.21

24.73 ± 0.16 8.28 ± 0.12 9.85 ± 0.54 0 51.5± 2.23 1.1 ± 0.14 55.7 ± 2.19 1.1 ± 0.31 7.23 ± 0.33 2.09 ± 0.22 40.79

22.57 ± 0.23 8.43 ± 0.23 11.12 ± 0.81 0 39.8 ± 2.40

19.46 ± 0.19 9.24± 0.21 12.59 ±0.71 0 21.2 ± 2.43 0.7 ± 0.12 76.2 ± 2.14 1.5 ± 0.18 3.56 ± 0.72 1.15 ± 0.23 67.42

28.19 ± 0.24 7.83 ± 0.14 8.04 ± 0.72 0 66.5 ± 2.22 1.7 ± 0.11 30.5 ± 2.26 0.8 ± 0.22 17.23 ± 0.24 5.29 ± 0.32 --

26.34 ± 0.31 8.06 ± 0.18 9.04 ± 0.32 0 55.8 ± 2.32 1.2 ± 0.11 47.3 ± 2.32 1.0 ± 0.31 13.33 ± 0.56 4.18 ± 0.13 20.98

24.34 ± 0.21 8.29 ± 0.21 9.23 ± 0.71 0 43.2± 2.02 1.0 ± 0.14 59.8 ± 2.31 1.2 ± 0.24 9.38 ± 0.13 3.03 ± 0.28 42.72

21.81 ± 0.22 9.12 ± 0.20 10.80± 0.27 0 28.6 ± 2.11 0.8 ± 0.11 72.4 ± 2.33 1.4 ± 0.10 5.92 ± 0.74 1.92 ± 0.32 63.70

Neutrophil

Eosinophil

1.6 ± 0.21

0.9 ± 0.11

Lymphocyte

32.8 ± 2.06

63.6 ± 2.04

Monocyte

0.9 ± 0.13

1.3 ± 0.20

Total volume

13.53 ± 0.62 3.53 ± 0. 23 --

5.48 ± 0.92 1.91 ± 0.22 45.89

Packed cell

% Tumour inhibition

* Mean ± SD of 6 animals

Table 2: In-Vivo Passive and Active targeting All rights reserved© 2011 www.jamonline.in 174

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Venkateswarlu G et al

Figure 1: Photomicrographs of FU-IMS

Figure 2: In-Vitro Cytotoxicity

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