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INNOVATORS 2010 Preparation and Evaluation of Synergistic Anticancer Effects of Simvastatin/Tocotrienols Lipid Nanoparticles Lipid-Based INNOVATORS 2010 Preparation and Evaluation of Synergistic Anticancer Effects of Simvastatin/Tocotrienols Lipid Nanoparticles Lipid-Based Drug Delivery Graduate Student Award Sponsored by Gattefossé Hazem Ali 1, 2, Amit Shirode 1, Paul Sylvester 1, and Sami Nazzal 1 1 Department of Basic Pharmaceutical Sciences, University of Louisiana at Monroe 2 Department of Obstetrics & Gynecology, University of Texas Medical Branch

ABSTRACT Purpose (1) To prepare and characterize the TRF-NLCs, SIM-TRF NLCs and SIM-αT NLCs ABSTRACT Purpose (1) To prepare and characterize the TRF-NLCs, SIM-TRF NLCs and SIM-αT NLCs with regard to their average particle size, polydispersity index (PI), and entrapment efficiency (EE), (2) To evaluate the synergistic anticancer activity of SIM and either TRF (or αT) in NLCs, and (3) To study the in vitro dynamic digestion of SIM/TRF- NLCs to evaluate the impact of the lipid core on retrieval of SIM/TRF in the aqueous phase post lipolysis. Methodology SIM-TRF NLCs and SIM-αT NLCs were prepared by dissolving SIM (1 m. M) in either TRF (or αT)/COMP blend at 1: 1 ratio (TRF (or αT)): lipid core. COMP was used at 0. 25% (w/v), and Lutrol® F 68 was added as a surfactant at 0. 25% (w/v). Formulations were prepared by melt emulsification followed by ultrasonication for 10 minutes. The resulting nanoemulsion was annealed to 4 o. C. The average particle size and PI were measured by photon correlation spectroscopy (PCS). The entrapment efficiency (EE) of NLCs was determined by measuring the free SIM in the aqueous phase using a validated HPLC method. In vitro dynamic digestion was performed in media that mimic intestinal fasting state in presence of pancreatin as digesting enzyme. SIM/TRF concentrations in the aqueous layer were determined after digestion by HPLC. The anticancer effects against the highly malignant +SA mammary tumor cells were evaluated using MTT colorimetric assay. Young Innovators 2010

ABSTRACT (CONTINUOUS) Results SIM and TRF were successfully coencapsulated with %EE> 99. 9%. The ABSTRACT (CONTINUOUS) Results SIM and TRF were successfully coencapsulated with %EE> 99. 9%. The average particle size of SIM-TRF-COMP NLCs was 107. 5 nm; whereas, the average particle size of SIMαT-COMP NLCs was 106. 8 nm with PI values 0. 29 and 0. 25, respectively. Powder x-ray diffraction indicated presence of SIM in amorphous form. In vitro cell viability studies of αT NLCs and TRF NLCs demonstrated significant anticancer effects in the range from 8. 0 to 14 µM and from 0. 25 to 14 µM, respectively. The IC 50 values were 17. 7 µM and 1. 5 µM for αT NLCs and TRF NLCs, respectively. The anticancer effects were further potentiated when the cells were treated with SIM-αT NLCs (IC 50= 0. 76) or SIM-TRF NLCs (IC 50=0. 52). Compared to TRF-SEEDS, which attained 1. 5 m. L/min lipolysis rate, digestion of other formulations ranged from 0. 0002 to 0. 1 m. L/min for SIM-TRF-COMP NLCs and TRF-PREC NLCs, respectively. Compared to low TRF extent in the aquoeus layer (<10%), the SIM extent was higher than 60%. Conclusion Coencapsulation of SIM and either TRF (or αT) resulted in potentiated their simultaneous anticancer activity. The characterization studies suggested that stable SIM-TRF NLCs and SIM-αT NLCs could be prepared and ready for further in vivo and/or clinical investigations. Young Innovators 2010

INTRODUCTION • Recent literature reports suggest that simvastatin (SIM) has shown its ability to INTRODUCTION • Recent literature reports suggest that simvastatin (SIM) has shown its ability to suppress the growth of breast adenocarcinoma cells in vitro. • The antitumor effect of SIM has been associated with their activity as potent inhibitors of 3 hydroxy-3 -methylglutaryl-coenzyme A (HMGCo. A) reductase, an enzyme catalyzing the conversion of HMGCo. A to mevalonate, the rate limiting step in cholesterol biosynthesis. Young Innovators 2010

INTRODUCTION • HMGCo. A inhibitor blockade of mevalonate synthesis induced cell cycle arrest in INTRODUCTION • HMGCo. A inhibitor blockade of mevalonate synthesis induced cell cycle arrest in vitro and inhibited tumor growth in vivo. • Likewise, vitamin E extract of palm oil, which is commonly referred to as tocotrienol-rich-fraction or TRF, was shown to reduce HMGCo. A reductase activity by causing post-transcriptional down regulation of the enzyme. Young Innovators 2010

INTRODUCTION • Previous reports suggest that a combined low dose treatment with gamma tocotrienol INTRODUCTION • Previous reports suggest that a combined low dose treatment with gamma tocotrienol and SIM synergistically inhibited the growth of highly malignant +SA mammary epithelial cells in culture. • These findings suggested that combined treatment of SIM with tocotrienols may provide significant health benefits in the prevention and/or treatment of breast cancer, while avoiding myotoxicity associated with high dose SIM monotherapy. Young Innovators 2010

INTRODUCTION • However, successful delivery of therapeutic concentration of the drugs to the tumor INTRODUCTION • However, successful delivery of therapeutic concentration of the drugs to the tumor cells is considered a challenge. This could be circumvented by passively targeting SIM and TRF coencapsulated in nanoparticles to tumor cells. • In this report, we hypothesized that coencapsulation of SIM/TRF in lipid nanoparticles may potentiate their antitumor activity, and simultaneously minimizing the side effects associated with SIM systemic administration. Young Innovators 2010

INTRODUCTION • To test this hypothesis, we aimed to prepare and characterize the TRF-NLCs, INTRODUCTION • To test this hypothesis, we aimed to prepare and characterize the TRF-NLCs, SIM-TRF NLCs and SIM-α tocopherol (αT) NLCs with regard to their average particle size, polydispersity index (PI), and entrapment efficiency (EE). • The second aim was to evaluate the synergistic anticancer activity of SIM and either TRF (or αT) in NLCs. Young Innovators 2010

INTRODUCTION • The third aim was to study the potential for oral administration of INTRODUCTION • The third aim was to study the potential for oral administration of lipid nanoparticles by performing in vitro dynamic lipolysis experiments and evaluating the impact of lipid core on retrieval of SIM/TRF in the aqueous phase post digestion. Young Innovators 2010

MATERIALS AND METHODS Materials Bile salts; Calcium chloride dehydrate; Pancreatin; Sodium chloride; Sodium hydroxide; MATERIALS AND METHODS Materials Bile salts; Calcium chloride dehydrate; Pancreatin; Sodium chloride; Sodium hydroxide; Trizma® maleate; Methylthiazolyldiphenyl tetrazolium bromide (MTT); Bovine erum albumin (BSA); (±)-α-Tocopherol; Captex® 355 (CAPTEX, triglycerides of caprylic/capric acid); Compritol® 888 ATO (COMP, glyceryl behenate); Labrasol; Lutrol® F 68 NF (POLOX, Poloxamer 188); Precirol® ATO 5 (PREC, glyceryl palmitostearate); Simvastatin; Tocotrienol-rich-fraction of palm oil (TRF), which contains 20. 2% α-tocopherol, 16. 8% α-tocotrienol, 44. 9% γ- tocotrienol, 14. 8% δ- tocotrienol, and 3. 2% of a nonvitamin E lipid soluble contaminants; Tween® 80 ; and Lecithin (having a trade name Alcolec® FF 100). Young Innovators 2010

MATERIALS AND METHODS Preparation of lipid nanoparticles • SIM, TRF (or αT) were allowed MATERIALS AND METHODS Preparation of lipid nanoparticles • SIM, TRF (or αT) were allowed to dissolve in compritol® 888 ATO (COMP) or precirol ATO 5 (PREC) at 80°C. Lutrol® F 68 (POLOX) was dissolved in purified water and heated to 80°C. The hot surfactant solution was added to the molten lipid under high-shear homogenization at 20, 000 rpm. After 5 minutes, the o/w microemulsion was sonicated for 10 minutes. Nanoparticles were formed by keeping the dispersions overnight at 4 o. C. The concentrations of the lipid phase, including the drug(s) (TRF or αT), and the emulsifier in the dispersions were 0. 25% and 0. 25% (w/v), respectively. The amount of SIM and TRF (or αT) added to the lipid phase was to 1 m. M of SIM and 5 m. M of TRF (or αT). For in vitro lipolysis experiments the concentration of the lipid core, TRF, and the surfactant were 2. 5% (w/v), each. SIM concentration was kept at 10 m. M. • Self-emulsifying drug delivery systems of TRF (TRF-SEEDs) and TRF-microemulsion were prepared by the same procedure. The lipid core was captex® 355 and TRF (each 2. 5% w/v) for TRF-SEDDS and TRF alone (2. 5% w/v) for TRF-microemulsion. • For the powder x-ray diffraction (PXRD) studies both binary blends of TRF (or αT) with COMP and ternary blends of SIM, TRF (or αT) were prepared. The blends were prepared by mixing the molten ingredients with either SIM/TRF (or αT) at 85 o. C. Molten blends were allowed to re-congeal at room temperature overnight prior to analysis. Young Innovators 2010

MATERIALS AND METHODS • Determination of average particle size, polydispersity index (PI), and zeta MATERIALS AND METHODS • Determination of average particle size, polydispersity index (PI), and zeta potential They were measured by photon correlation spectroscopy (PCS) at 25 o. C by using Nicomp. TM 380 ZLS zeta potential and submicron particle size analyzer. Samples were diluted with a previously filtered deionized water. • Determination of SIM-NPs entrapment efficiency (EE) It was determined by measuring the concentration of the free unloaded SIM in the aqueous phase of the NLC dispersions by using a previously validated HPLC method. SIM entrapment was calculated from the following equation: • High performance liquid chromatographic (HPLC) analysis SIM analysis was performed by injecting the samples into a C 18 (4. 6 X 100 mm) Onyx® monolithic analytical column. SIM detection of was carried out at λmax = 238 using a 15% v/v water in methanol solution as the mobile phase. Data acquisition was performed using a chromatography software Chrom. Quest. TM version 4. 2. Young Innovators 2010

MATERIALS AND METHODS • Powder x-ray diffraction (PXRD) measurements PXRD was obtained by wide-angle MATERIALS AND METHODS • Powder x-ray diffraction (PXRD) measurements PXRD was obtained by wide-angle X-ray scattering (WARS, 2τ = 5 -50 o, step size = 0. 5) using a Philips PW 1830 X-ray generator fitted with a copper anode tube (Cu-Kα radiation, λ = 1. 5418 nm) and a Goniometer PW 18120 detector. Data of the scattered radiation were recorded at an anode voltage of 40 k. V and a current of 35 m. A. • Cell line and culture conditions Highly malignant +SA mammary epithelial cell lines were grown and maintained in serum-free Dulbecco’s modified Eagle’s medium (DMEM)/F 12 control media containing bovine serum albumin (BSA), transferrin, soybean trypsin inhibitor, penicillin-streptomycin, insulin, and EGF as a mitogen. Cells were maintained at 37 o. C in a humidified atmosphere of 95% air and 5% CO 2. Viable cell number was determined using the 3 -(4, 5 -dimethylthiazol-2 yl)-2, 5 -diphenyl tetrazolium bromide (MTT) colorimetric assay. For all experiments, freshly prepared nanoparticle dispersions were filtered through sterile syringe filters. These dispersions were added to the culture media at various concentrations to prepare treatment media supplemented with TRF or αT NLCs with or without SIM. Young Innovators 2010

MATERIALS AND METHODS • Statistical analysis IC 50 values (dose resulting in 50% cell MATERIALS AND METHODS • Statistical analysis IC 50 values (dose resulting in 50% cell growth inhibition) were determined by non-linear regression curve fit analysis using Graph. Pad Prism 5. Differences among the various treatment groups in cell growth and viability studies were determined by analysis of variance (ANOVA) followed by Duncan’s t-test. A difference of p<0. 05 was considered to be significant as compared to vehicle-treated controls. • Preparation of lipolysis reagents A p. H 6. 5 buffer was prepared by adding the following ingredients to sufficient deionized water to prepare one liter of the buffer; Ca. Cl 2. 2 H 2 O (5 m. M), Na. Cl (150 m. M), tri-maleate (50 m. M), and Na. OH (39. 75 m. M). Bile salts (5 m. M) and lecithin (1. 25 m. M) were added to each 100 m. L of this buffer. Young Innovators 2010

MATERIALS AND METHODS • In vitro lipolysis experiments using bio-relevant media Lipolysis experiments were MATERIALS AND METHODS • In vitro lipolysis experiments using bio-relevant media Lipolysis experiments were performed in a water-jacketed reaction vessel (37 o. C). One milliliter of the formulation was added to the bio-relevant medium. The p. H- stat autotitrator was set to maintain the p. H at 6. 5. After 10 minutes from the beginning of each experiment, one milliliter of preincubated (37 o. C for 20 minutes) pancreatin suspension (250 mg/m. L) was added and the experiments were allowed to proceed for 120 minutes. The volume of sodium hydroxide consumed during the experiment was recorded analyzed by the autotitrator using a Titra. Master 85 software version 3. 1. 0. Every 30 minutes, an aliquot of the digestion media was taken, centrifuged, and finally analyzed for SIM and/or TRF content by using HPLC. Young Innovators 2010

RESULTS • As shown in Table 1, the average particle size of unloaded SLN RESULTS • As shown in Table 1, the average particle size of unloaded SLN was 152 nm. The size of the nanoparticles decreased significantly to ~100 nm for TRF- and αT-based NLCs with or without SIM. All nanoparticle dispersions had PI values in the range from 0. 20 to 0. 29 and had ZP values in the range from -9. 0 to -21. 0 m. V. The EE% of SIM in SIM-TRF NLCs and SIM- αT NLCs was 99. 9 ± 1. 3 and 99. 9 ± 0. 8, respectively. Young Innovators 2010

RESULTS Table 1: Composition, average particle size, and zeta potential (ZP) of unloaded SLNs RESULTS Table 1: Composition, average particle size, and zeta potential (ZP) of unloaded SLNs and TRF or αT NLCs with or without SIM. Each point represents mean ± SD. Average Polox. 188 particle size (% w/v) (nm) COMP (% w/v) αT (m. M) TRF (m. M) SIM (m. M) Unloaded SLNa 0. 50 0 0. 25 SIM-TRF NLCb 0. 25 0 5 1 SIM- αT NLCc 0. 25 5 0 TRF-NLCd 0. 25 0 αT-NLCse 0. 25 5 Formulation PI ZP (m. V) 151. 7 ± 1. 2 0. 28 ± 0. 00 - 21. 7 ± 0. 8 0. 25 107. 5 ± 0. 73 0. 29 ± 0. 01 - 13. 4 ± 0. 4 1 0. 25 106. 8 ± 2. 54 0. 25 ± 0. 01 -9. 0 ± 0. 8 5 0 0. 25 100. 3 ± 0. 91 0. 23 ± 0. 01 - 13. 5 ± 0. 4 0 0 0. 25 101. 2 ± 1. 27 0. 20 ± 0. 02 - 10. 5 ± 1. 2 a Unloaded SLNs, blank solid lipid nanoparticles made from compritol® 888 ATO (COMP) only as a lipid phase. b SIM-TRF-COMP NLCs, nanostructure lipid carriers with simvastatin and tocotrienol rich fraction (TRF). c SIM- αT-COMP NLCs, nanostructure lipid carriers with simvastatin and α-tocopherol (α T). d TRF-NLCs, nanostructure lipid carriers with TRF. e αT-NLCs, nanostructure lipid carriers with αT. Young Innovators 2010

RESULTS • As shown in Fig. 1, The PXRD for bulk SIM revealed major RESULTS • As shown in Fig. 1, The PXRD for bulk SIM revealed major peaks at 2θ = 9. 25, 16. 75, 17. 25, 18. 75, 22. 75, 25. 25, 28. 25, and 31. 75. Nonetheless, the apparent peaks at 2θ = 16. 75, 17. 25, and 18. 75 in the physical mixture indicate the presence of SIM in crystalline form. These peaks disappeared in the solidified ternary blends, and only COMP-derived peaks appeared (2θ = 20. 75, 21. 25). No changes in the crystallinity of the ternary blends were seen after six month of storage. • As shown in Fig. 2, αT based NLCs, at 0. 25 -8. 0 µM, had not effect on cellular viability (IC 50=17. 7 µM). Addition of SIM to the αT significantly inhibited +SA cell growth (IC 50=0. 76 µM). Similarly, TRF based NLCs showed a decrease in the cellular viability (IC 50=1. 5 µM). Addition of SIM to TRF significantly inhibited +SA cell growth (IC 50=0. 52 µM). Young Innovators 2010

RESULTS (After 6 months of storage) SIM-αT-COMP ternary blend (Fresh sample) (After 6 months RESULTS (After 6 months of storage) SIM-αT-COMP ternary blend (Fresh sample) (After 6 months of storage) SIM-TRF-COMP ternary blend (Fresh sample) SIM- αT-COMP phys. Mix. SIM-TRF-COMP phys. Mix. Bulk COMP Bulk SIM Figure 1: Powder x-ray diffraction patterns for bulk simvastatin (SIM), SIM in physical mixture with TRF and COMP, SIM in physical mixture with αT and COMP, SIM blend with TRF and COMP freshly prepared and after 6 month of storage at controlled room temperature, SIM blend with αT and COMP freshly prepared and after 6 months of storage. Curves were displaced along the ordinate for better visualization. Young Innovators 2010

RESULTS 80 * 70 60 Viability (%) A 90 80 * 100 90 Viability RESULTS 80 * 70 60 Viability (%) A 90 80 * 100 90 Viability (%) 100 50 40 70 B 60 50 40 30 30 20 20 10 10 0 0 0. 25 0. 75 1 1. 25 1. 5 2 4 6 8 10 12 0 14 0 0. 25 0. 75 αT concentration (µM) 1 100 * 70 90 Viability (%) C Viability (%) 80 2 4 6 8 10 12 14 * 100 90 1. 25 1. 5 TRF concentration (µM) 60 50 40 80 70 D 60 50 40 30 30 20 20 10 10 0 0. 25 0. 75 1 1. 25 1. 5 2 4 6 8 10 12 14 0 0. 25 0. 75 0. 05 0. 15 0. 25 0. 3 0. 4 0. 8 1. 2 1. 6 2 1. 25 1. 5 2 4 6 8 10 12 14 TRF concentration (µM) αT concentration (µM) 0 1 2. 4 2. 8 SIM concentration (µM) 0 0. 05 0. 15 0. 25 0. 3 0. 4 0. 8 1. 2 1. 6 2 2. 4 2. 8 SIM concentration (µM) Figure 2: Anticancer effects of αT NLC (A), TRF NLCs (B), SIM/ αT NLCs (C), and SIM/TRF NLCs (D) on neoplastic +SA mammary epithelial cells. Vertical bars indicate the mean cell count + SEM (n=6). *P< 0. 05 as compared to the vehicle-treated control group. Young Innovators 2010

RESULTS • As shown in Fig. 3, TRF-SEDDs underwent a substantially fast lipolysis rate RESULTS • As shown in Fig. 3, TRF-SEDDs underwent a substantially fast lipolysis rate (1. 5 m. L/min), and the order of the digestion velocity was : TRF-SEDDs > TRF-PREC NLCs (0. 11 m. L/min)> > SIM-TRF -PREC NLCs (0. 08 m. L/min)> TRF microemulsion=TRF-COMP NLCs (~0. 007 m. L/min)> SIM-TRF-COMP NLCs (0. 0002 ml/min). • As shown in Fig. 4, TRF extent in the aqueous layer during lipolysis was less than 10% within the 120 -minute digestion experiments. Preparation of TRF in SEDDs, microemulsion, or nanoparticles increased the TRF extent significantly (p<0. 05) compared to TRF oil. • As shown in Fig. 5, SIM extent in the aqueous layer during lipolysis was higher than 60% within the 120 -minute digestion experiments. Young Innovators 2010

10 Sodium hydroxide (m. L) 9 8 RESULTS 100 TRF in the aqeuous layer 10 Sodium hydroxide (m. L) 9 8 RESULTS 100 TRF in the aqeuous layer after lipolysis (%) TRF-SEDDS TRF-PREC NLCs SIM-TRF-PREC NLCs 2. 5% TRF microemulsion TRF-COMP NLCs SIM-TRF-COMP NLCs 7 6 5 4 3 2 1 30 minutes 90 60 minutes 80 90 minutes 120 minutes 70 60 50 40 30 20 10 0 0 20 40 60 80 100 120 0 Time (min) Figure 3: A representative graph showing the in vitro lipolysis of TRF-NLCs and SIM-TRF NLCs compared to TRF-SEDDS and TRF-microemulsion. Young Innovators 2010 TRF oil 2. 5% TRF-SEDDs TRF-COMP TRF-PREC microemulsion NLCs Figure 4: Extent of TRF in the aqueous layer during in vitro dynamic lipolysis of TRF-NLCs compared to TRFSEDDS, TRF microemulsion, and TRF oil. Vertical bars represent the average value ± SD (n=3).

RESULTS 100 30 minutes 60 minutes 80 90 minutes 70 120 minutes SIM extent RESULTS 100 30 minutes 60 minutes 80 90 minutes 70 120 minutes SIM extent in the aqueous layer (%) 90 60 50 40 30 20 10 0 SIM-TRF-PREC NLCs SIM-TRF-COMP NLCs Figure 5: Extent of SIM in the aqueous layer during in vitro dynamic lipolysis of SIM-TRF NLCs made from COMP and PREC. Vertical bars indicate the average value ± SD (n=3). Young Innovators 2010

DISCUSSION • Lipid nanoparticles, with average particle size ~100 nm, were successfully prepared by DISCUSSION • Lipid nanoparticles, with average particle size ~100 nm, were successfully prepared by melt-emulsification method. We were able to encapsulate liquid oils (TRF or αT) without compromising their size. • SIM EE was very high due to liquid oil-solid lipid nanocmpartments. Polydispersity Index (PI) indicated that the nanoparticles were mono-disperse. • Although ZP values were low, the average particle size did not change over 6 months of storage at room temperature (data not shown) which imply the long-term stability. This might be attributed to presence of the non-ionic surfactant (poloxamer 188) which forms a hydrophilic sheath around the nanoparticle surfaces. This coat may sterically stabilize the nanoparticles. Young Innovators 2010

DISCUSSION • In PXRD, the absence of SIM diffraction pattern in the ternary blends DISCUSSION • In PXRD, the absence of SIM diffraction pattern in the ternary blends suggested the presence of SIM as a molecular dispersion in the partially crystalline COMP. from PXRD studies, there was no evidence of SIM crystallization upon storage of the nanoparticles for 6 months. Young Innovators 2010

DISCUSSION • From the cell culture experiments, it was evident that the lower IC DISCUSSION • From the cell culture experiments, it was evident that the lower IC 50 values of the TRF NLCs might be due to improved internalization of the NLCs by endocytosis into the cells. Similarly, when SIM was added to either TRF or αT based NLCs, the IC 50 decreased reflecting the cytotoxic effect of SIM against +SA mammary epithelial cells. Of most significance was the observed decrease in cell viability when SIM and TRF were coencapsulated into nanoparticles, which demonstrated the potential therapeutic benefits of a combined SIM and TRF treatment. Young Innovators 2010

DISCUSSION • Dynamic lipolysis commenced after 10 minutes had been elapsed and pancreatin suspension DISCUSSION • Dynamic lipolysis commenced after 10 minutes had been elapsed and pancreatin suspension was added indicating dependence of lipolysis on existence of the digesting enzymes. The in vitro lipolysis rate was affected by the lipid composition. It is mainly dependent on the mobitlity of the system (SEDDs versus solid lipids) and the triglyceride chain length. Young Innovators 2010

DISCUSSION • Throughout 120 -minute lipolysis experiments, TRFSEDDS underwent a substantially fast lipolysis rate, DISCUSSION • Throughout 120 -minute lipolysis experiments, TRFSEDDS underwent a substantially fast lipolysis rate, and the degradation velocity. The fastest degradation rate of TRF-SEDDS was attributed to the nature of the lipid which is a medium chain triglyceride (Captex® 355, triglycerides of capric/caprylic acid (C 8 -10)). However, TRF-COMP NLC formulations made from long chain pure triglycerides (COMP (C 22)) attained low degradation rate. These findings suggest preferential anchorage of the digesting enzymes with the medium chain triglycerides with a subsequent increase in lipolysis rate. Young Innovators 2010

DISCUSSION • SIM/TRF extent in the aqueous layer during digestion of lipid nanoparticles was DISCUSSION • SIM/TRF extent in the aqueous layer during digestion of lipid nanoparticles was comparable (p>0. 05) to those of either SEDDs or TRF microemulsion, implying dependence of digestion on the physicochemical properties of the drugs rather than lipid composition. Young Innovators 2010

CONCLUSION • Coencapsulation of SIM and either TRF (or αT) resulted in potentiated their CONCLUSION • Coencapsulation of SIM and either TRF (or αT) resulted in potentiated their simultaneous anticancer activity. The characterization studies suggested that stable SIM-TRF NLCs and SIMαT NLCs could be prepared and ready for further in vivo and/or clinical investigations. Young Innovators 2010

CONCLUSION • Although lipolysis rate is a measure of the lipid chain length, it CONCLUSION • Although lipolysis rate is a measure of the lipid chain length, it did not influence the SIM/TRF partitioning into the aqueous layer post lipolysis. Young Innovators 2010

ACKNOWLEDGMENTS • This work was partially supported by a grant from First Tech International ACKNOWLEDGMENTS • This work was partially supported by a grant from First Tech International Ltd. • The authors would like to acknowledge Dr. John Anderson from the Math and Physics Department for his assistance with the PXRD studies. Young Innovators 2010

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BIO/CONTACT INFO Dr. Hazem Ali, Ph. D. , is a post-doctoral fellow in the BIO/CONTACT INFO Dr. Hazem Ali, Ph. D. , is a post-doctoral fellow in the department of Obstetrics & Gynecology, University of Texas Medical Branch at Galveston, United States. He graduated from University of Louisiana at Monroe with Ph. D. degree in pharmacy (Major: pharmaceutics). Current address: Clinical Sciences building 301 Univesrity Blvd. Galveston, Texas 77555 -0587 E-mail: [email protected] edu Work phone: (409) 747 -4983 Fax: (409) 747 -0266 Young Innovators 2010