الفهرس 
general introduction
methodologyOnly 14 pages are availabe for public view
 |  | from | 162 |  |  |
| | | from | 162 | | |
Abstract
Breast cancer is the second leading cause of death among women worldwide. It is considered a heterogeneous disease where magnetic resonance imaging studies have displayed that there are multiple growth patterns of breast cancer with different strategies of treatment. Among these phenotypes there are breast cancer over-expressing estrogen receptors while others with activated Ras or ErbB2 pathways are observed. Also, it has been documented that elevated levels of mevalonate synthesis lead to several types of malignancies; one of them is breast cancer. This investigation recruits the use of statins as one of the chemotherapeutic agents to treat breast cancer.
Statins are 3-hydroxy-3-methylglutaryl-CoA (HMG CoA) reductase inhibitors. Statins inhibit HMG-CoA reductase which in turn inhibits the cholesterol biosynthesis. Consequently, depletion in mevalonate occurs together with its downstream products as isoprenoid intermediates leading to lower levels of lipid attachment sites for Ras, Rac and Rho proteins driving to lower cellular and subcellular pathways remarkable for cancer progression. Statins represent two classes: hydrophilic and lipophilic classes. The class responsible for inhibiting malignancies is the lipophilic one. Lipophilic statins such as simvastatin (SV) cross the cell membranes accumulating in cells and exert a pleiotropic effect such as the anti-apoptotic effect. SV, interestingly, induces cell-cycle arrest, initiating apoptosis and ameliorate treatment of breast cancer.
Treatment of breast cancer by chemotherapeutic agents is usually accompanied by side effects owing to their effect on non–cancerous cells. Accordingly, targeting the cancerous cells ameliorate the outcome of breast cancerous patients. The delivery of anticancer drugs to tumor cells is usually achieved by the enhanced permeability and retention (EPR) effect. EPR depends on the presence of fenestrations in the imperfect tumor blood vessels and to the poor lymphatic drainage in the tissue. Passive targeting of nanoparticles (NPs) can be achieved via utilizing the disruptions occurred in basal membrane of tumor vasculature.
Amongst the nanocarriers employed in cancer targeting are lipid NPs, from which lipid nanocapsules (LNCs) and nanostructured lipid carriers (NLC) are examples.
LNCs represent smart drug delivery systems enhancing targeting the anticancer drug to tumor cells via IV administration. The choice of these nano-carriers was based on several merits such as being biodegradable, biocompatible, avoidance of the use of organic solvents, stable up to one year and the ability of encapsulating hydrophilic and lipophilic drugs. Thanks to its PEGylated structure, bypassing the engulfment of LNCs by macrophages was feasible.
NLC represent the second generation form of the solid lipid nanoparticles (SLN). NLC were developed to overcome the limitations of the previous lipidic NPs; liposomes and SLN, such as the limited drug incorporation in case of liposomes and the expulsion of drug during storage as being noticed in SLN. Expulsion of the cargo entrapped in SLN usually occurs due to crystallization of the solid lipid into the more ordered β modification with the formation of a perfect crystal lattice.
NLC were prepared via mixing spatially different types of lipids, solid lipid with liquid lipid (oil), to provide imperfections in the crystal lattice of the solidified lipid of NLC enhancing incorporation of the drug and avoiding its expulsion during storage.
Hence the aim of work in this thesis was the development of nano-formulations capable of enhancing the delivery of SV to breast cancer cells, providing sustained release, augmenting cancer cytotoxicity and improving cellular uptake. This was planned to be achieved by encapsulating SV in LNCs and NLC.
So the work in this thesis is divided into two chapters:
Chapter One: Preparation and evaluation of SV loaded LNCs.
Chapter Two: Preparation and evaluation of SV loaded NLC.
Chapter I: Preparation and evaluation of SV loaded LNCs.
In this chapter, SV loaded LNCs were successfully prepared using the phase inversion method followed by shock introduced to the system by cold water to break the produced microemulsion and enhancing the formation of nanocapsules. LNCs was composed of Labrafac lipophile® (LL), Solutol®, salted aqueous medium in addition to Epikuron®. Epikuron acted as a stabilizer to the LNC shells, increasing the biocompatibility to the biological membranes. The favorable stealth properties of LNCs and its prolonged circulation were imparted by the PEGylated surfactant; Solutol. LL represented the oily phase. The salted aqueous medium enhanced the phase inversion temperature of Solutol to be easily achieved. LNCs were optimized using the D-optimal mixture design. The percentages of the three independent variables (LL, Solutol, aqueous phase) were varied to generate mathematical models for the three responses: particle size (PS), polydispersity index (PDI) and the percentage of drug released after 48 hours (% Q48h). Validation of the model was performed utilizing 4 different points and the % Bias was calculated. The model was evaluated according to its significance using ANOVA, its R-squared value, adjusted R-squared, Predicted R-squared and adequate precision. Moreover, the actual runs were compared to the predicted ones. The selected SV loaded LNCs were visualized using transmission electron microscopy (TEM). The selected formulae were then subjected to further investigations such as differential scanning calorimetry (DSC) to ensure the molecular dispersion of SV in the lipid matrix of LNCs. Effect of aging on selected SV loaded LNCs was studied after storing the formulae at 4⁰ C for 6 months. In an important encounter, sterilization of a selected SV loaded LNCs formulation was performed by exposure to gamma radiation at a dose of 25 KGy as recommended by the European pharmacopeia followed by sterility testing. After sterilization the formulations were re-characterized by measuring the PS, PDI and % Q48h.
Evaluation of cytotoxicity of selected SV loaded LNCs were conducted on MCF-7 cell lines using crystal violet assay. SV, plain LNCs, SV-LNCs were compared according to their IC50 scores.
from the obtained results, it was found that:
1) All the prepared SV loaded LNCs had sizes between 20 - 100 nm which was significantly affected by the ratio of Solutol to oil. Increasing this ratio greatly resulted in reduction of particle size (PS) owing to its effect on interfacial tension of the oily droplets of nanocapsules.
2) All the prepared SV loaded LNCs were characterized by a mono-modal particle size distribution less than 0.126. This could be attributed to the insertion of Solutol at the water–oil interface enhancing the incorporation of SV in the oily core and reducing the interfacial tension of oily droplets. These results also reflect the high efficiency of the adopted method; phase-inversion method in producing the lipid nanocapsules.
3) The release studies showed the sustained release behavior which was enhanced by increasing the Solutol amount. Being a surface active agent, Solutol was inserted at the water-oil interface forming a coherent shell completely entrapping SV at the oily core hindering its release from oily core owing to its bulky structure. Its solubilizing nature as well may have entrapped the drug at the interface retarding its release.
4) Based on the D-optimal mixture design results:
a. The suggested model for PS response was quadratic while for PDI and % Q48 h responses linear models were proposed.
b. Analysis of the models was performed using ANOVA where p-values less than 0.5 were produced indicating the significance of the models. The p-values for PS and %Q48h responses were less than 0.0001 while for PDI p-value was equal to 0.0015.
c. High correlation was indicated between the actual and predicted runs for the three investigated models. This was explicated in the graphical presentation of the actual versus the predicted runs. This also coincides with the R-squared values for the three obtained responses; 1, 0.76 and 0.95 for PS, PDI and %Q48h, respectively.
d. Adequate precision measuring the signal to noise ratio greater than 4 was desirable. The ratio was found to be 524.32, 9.30 and 19.68 for the three models; PS, PDI and %Q48h, respectively indicating highadequacy of the model.
e. Experimental validation of the model and its feasibility for navigation through the model was investigated by comparing other 4 actual runs vs their predicted values to calculate the % Bias for the three responses. The three responses scored less than 7 % confirming the model validation and its sufficiency to navigate the experimental spaces.
5) TEM micrographs confirmed that all the particles were spherical in shape possessing smooth surface with no obvious particle aggregation.
Breast cancer is the second leading cause of death among women worldwide. It is considered a heterogeneous disease where magnetic resonance imaging studies have displayed that there are multiple growth patterns of breast cancer with different strategies of treatment. Among these phenotypes there are breast cancer over-expressing estrogen receptors while others with activated Ras or ErbB2 pathways are observed. Also, it has been documented that elevated levels of mevalonate synthesis lead to several types of malignancies; one of them is breast cancer. This investigation recruits the use of statins as one of the chemotherapeutic agents to treat breast cancer.
Statins are 3-hydroxy-3-methylglutaryl-CoA (HMG CoA) reductase inhibitors. Statins inhibit HMG-CoA reductase which in turn inhibits the cholesterol biosynthesis. Consequently, depletion in mevalonate occurs together with its downstream products as isoprenoid intermediates leading to lower levels of lipid attachment sites for Ras, Rac and Rho proteins driving to lower cellular and subcellular pathways remarkable for cancer progression. Statins represent two classes: hydrophilic and lipophilic classes. The class responsible for inhibiting malignancies is the lipophilic one. Lipophilic statins such as simvastatin (SV) cross the cell membranes accumulating in cells and exert a pleiotropic effect such as the anti-apoptotic effect. SV, interestingly, induces cell-cycle arrest, initiating apoptosis and ameliorate treatment of breast cancer.
Treatment of breast cancer by chemotherapeutic agents is usually accompanied by side effects owing to their effect on non–cancerous cells. Accordingly, targeting the cancerous cells ameliorate the outcome of breast cancerous patients. The delivery of anticancer drugs to tumor cells is usually achieved by the enhanced permeability and retention (EPR) effect. EPR depends on the presence of fenestrations in the imperfect tumor blood vessels and to the poor lymphatic drainage in the tissue. Passive targeting of nanoparticles (NPs) can be achieved via utilizing the disruptions occurred in basal membrane of tumor vasculature.
Amongst the nanocarriers employed in cancer targeting are lipid NPs, from which lipid nanocapsules (LNCs) and nanostructured lipid carriers (NLC) are examples.
LNCs represent smart drug delivery systems enhancing targeting the anticancer drug to tumor cells via IV administration. The choice of these nano-carriers was based on several merits such as being biodegradable, biocompatible, avoidance of the use of organic solvents, stable up to one year and the ability of encapsulating hydrophilic and lipophilic drugs. Thanks to its PEGylated structure, bypassing the engulfment of LNCs by macrophages was feasible.
NLC represent the second generation form of the solid lipid nanoparticles (SLN). NLC were developed to overcome the limitations of the previous lipidic NPs; liposomes and SLN, such as the limited drug incorporation in case of liposomes and the expulsion of drug during storage as being noticed in SLN. Expulsion of the cargo entrapped in SLN usually occurs due to crystallization of the solid lipid into the more ordered β modification with the formation of a perfect crystal lattice.
NLC were prepared via mixing spatially different types of lipids, solid lipid with liquid lipid (oil), to provide imperfections in the crystal lattice of the solidified lipid of NLC enhancing incorporation of the drug and avoiding its expulsion during storage.
Hence the aim of work in this thesis was the development of nano-formulations capable of enhancing the delivery of SV to breast cancer cells, providing sustained release, augmenting cancer cytotoxicity and improving cellular uptake. This was planned to be achieved by encapsulating SV in LNCs and NLC.
So the work in this thesis is divided into two chapters:
Chapter One: Preparation and evaluation of SV loaded LNCs.
Chapter Two: Preparation and evaluation of SV loaded NLC.
Chapter I: Preparation and evaluation of SV loaded LNCs.
In this chapter, SV loaded LNCs were successfully prepared using the phase inversion method followed by shock introduced to the system by cold water to break the produced microemulsion and enhancing the formation of nanocapsules. LNCs was composed of Labrafac lipophile® (LL), Solutol®, salted aqueous medium in addition to Epikuron®. Epikuron acted as a stabilizer to the LNC shells, increasing the biocompatibility to the biological membranes. The favorable stealth properties of LNCs and its prolonged circulation were imparted by the PEGylated surfactant; Solutol. LL represented the oily phase. The salted aqueous medium enhanced the phase inversion temperature of Solutol to be easily achieved. LNCs were optimized using the D-optimal mixture design. The percentages of the three independent variables (LL, Solutol, aqueous phase) were varied to generate mathematical models for the three responses: particle size (PS), polydispersity index (PDI) and the percentage of drug released after 48 hours (% Q48h). Validation of the model was performed utilizing 4 different points and the % Bias was calculated. The model was evaluated according to its significance using ANOVA, its R-squared value, adjusted R-squared, Predicted R-squared and adequate precision. Moreover, the actual runs were compared to the predicted ones. The selected SV loaded LNCs were visualized using transmission electron microscopy (TEM). The selected formulae were then subjected to further investigations such as differential scanning calorimetry (DSC) to ensure the molecular dispersion of SV in the lipid matrix of LNCs. Effect of aging on selected SV loaded LNCs was studied after storing the formulae at 4⁰ C for 6 months. In an important encounter, sterilization of a selected SV loaded LNCs formulation was performed by exposure to gamma radiation at a dose of 25 KGy as recommended by the European pharmacopeia followed by sterility testing. After sterilization the formulations were re-characterized by measuring the PS, PDI and % Q48h.
Evaluation of cytotoxicity of selected SV loaded LNCs were conducted on MCF-7 cell lines using crystal violet assay. SV, plain LNCs, SV-LNCs were compared according to their IC50 scores.
from the obtained results, it was found that:
1) All the prepared SV loaded LNCs had sizes between 20 - 100 nm which was significantly affected by the ratio of Solutol to oil. Increasing this ratio greatly resulted in reduction of particle size (PS) owing to its effect on interfacial tension of the oily droplets of nanocapsules.
2) All the prepared SV loaded LNCs were characterized by a mono-modal particle size distribution less than 0.126. This could be attributed to the insertion of Solutol at the water–oil interface enhancing the incorporation of SV in the oily core and reducing the interfacial tension of oily droplets. These results also reflect the high efficiency of the adopted method; phase-inversion method in producing the lipid nanocapsules.
3) The release studies showed the sustained release behavior which was enhanced by increasing the Solutol amount. Being a surface active agent, Solutol was inserted at the water-oil interface forming a coherent shell completely entrapping SV at the oily core hindering its release from oily core owing to its bulky structure. Its solubilizing nature as well may have entrapped the drug at the interface retarding its release.
4) Based on the D-optimal mixture design results:
a. The suggested model for PS response was quadratic while for PDI and % Q48 h responses linear models were proposed.
b. Analysis of the models was performed using ANOVA where p-values less than 0.5 were produced indicating the significance of the models. The p-values for PS and %Q48h responses were less than 0.0001 while for PDI p-value was equal to 0.0015.
c. High correlation was indicated between the actual and predicted runs for the three investigated models. This was explicated in the graphical presentation of the actual versus the predicted runs. This also coincides with the R-squared values for the three obtained responses; 1, 0.76 and 0.95 for PS, PDI and %Q48h, respectively.
d. Adequate precision measuring the signal to noise ratio greater than 4 was desirable. The ratio was found to be 524.32, 9.30 and 19.68 for the three models; PS, PDI and %Q48h, respectively indicating highadequacy of the model.
e. Experimental validation of the model and its feasibility for navigation through the model was investigated by comparing other 4 actual runs vs their predicted values to calculate the % Bias for the three responses. The three responses scored less than 7 % confirming the model validation and its sufficiency to navigate the experimental spaces.
5) TEM micrographs confirmed that all the particles were spherical in shape possessing smooth surface with no obvious particle aggregation.
Breast cancer is the second leading cause of death among women worldwide. It is considered a heterogeneous disease where magnetic resonance imaging studies have displayed that there are multiple growth patterns of breast cancer with different strategies of treatment. Among these phenotypes there are breast cancer over-expressing estrogen receptors while others with activated Ras or ErbB2 pathways are observed. Also, it has been documented that elevated levels of mevalonate synthesis lead to several types of malignancies; one of them is breast cancer. This investigation recruits the use of statins as one of the chemotherapeutic agents to treat breast cancer.
Statins are 3-hydroxy-3-methylglutaryl-CoA (HMG CoA) reductase inhibitors. Statins inhibit HMG-CoA reductase which in turn inhibits the cholesterol biosynthesis. Consequently, depletion in mevalonate occurs together with its downstream products as isoprenoid intermediates leading to lower levels of lipid attachment sites for Ras, Rac and Rho proteins driving to lower cellular and subcellular pathways remarkable for cancer progression. Statins represent two classes: hydrophilic and lipophilic classes. The class responsible for inhibiting malignancies is the lipophilic one. Lipophilic statins such as simvastatin (SV) cross the cell membranes accumulating in cells and exert a pleiotropic effect such as the anti-apoptotic effect. SV, interestingly, induces cell-cycle arrest, initiating apoptosis and ameliorate treatment of breast cancer.
Treatment of breast cancer by chemotherapeutic agents is usually accompanied by side effects owing to their effect on non–cancerous cells. Accordingly, targeting the cancerous cells ameliorate the outcome of breast cancerous patients. The delivery of anticancer drugs to tumor cells is usually achieved by the enhanced permeability and retention (EPR) effect. EPR depends on the presence of fenestrations in the imperfect tumor blood vessels and to the poor lymphatic drainage in the tissue. Passive targeting of nanoparticles (NPs) can be achieved via utilizing the disruptions occurred in basal membrane of tumor vasculature.
Amongst the nanocarriers employed in cancer targeting are lipid NPs, from which lipid nanocapsules (LNCs) and nanostructured lipid carriers (NLC) are examples.
LNCs represent smart drug delivery systems enhancing targeting the anticancer drug to tumor cells via IV administration. The choice of these nano-carriers was based on several merits such as being biodegradable, biocompatible, avoidance of the use of organic solvents, stable up to one year and the ability of encapsulating hydrophilic and lipophilic drugs. Thanks to its PEGylated structure, bypassing the engulfment of LNCs by macrophages was feasible.
NLC represent the second generation form of the solid lipid nanoparticles (SLN). NLC were developed to overcome the limitations of the previous lipidic NPs; liposomes and SLN, such as the limited drug incorporation in case of liposomes and the expulsion of drug during storage as being noticed in SLN. Expulsion of the cargo entrapped in SLN usually occurs due to crystallization of the solid lipid into the more ordered β modification with the formation of a perfect crystal lattice.
NLC were prepared via mixing spatially different types of lipids, solid lipid with liquid lipid (oil), to provide imperfections in the crystal lattice of the solidified lipid of NLC enhancing incorporation of the drug and avoiding its expulsion during storage.
Hence the aim of work in this thesis was the development of nano-formulations capable of enhancing the delivery of SV to breast cancer cells, providing sustained release, augmenting cancer cytotoxicity and improving cellular uptake. This was planned to be achieved by encapsulating SV in LNCs and NLC.
So the work in this thesis is divided into two chapters:
Chapter One: Preparation and evaluation of SV loaded LNCs.
Chapter Two: Preparation and evaluation of SV loaded NLC.
Chapter I: Preparation and evaluation of SV loaded LNCs.
In this chapter, SV loaded LNCs were successfully prepared using the phase inversion method followed by shock introduced to the system by cold water to break the produced microemulsion and enhancing the formation of nanocapsules. LNCs was composed of Labrafac lipophile® (LL), Solutol®, salted aqueous medium in addition to Epikuron®. Epikuron acted as a stabilizer to the LNC shells, increasing the biocompatibility to the biological membranes. The favorable stealth properties of LNCs and its prolonged circulation were imparted by the PEGylated surfactant; Solutol. LL represented the oily phase. The salted aqueous medium enhanced the phase inversion temperature of Solutol to be easily achieved. LNCs were optimized using the D-optimal mixture design. The percentages of the three independent variables (LL, Solutol, aqueous phase) were varied to generate mathematical models for the three responses: particle size (PS), polydispersity index (PDI) and the percentage of drug released after 48 hours (% Q48h). Validation of the model was performed utilizing 4 different points and the % Bias was calculated. The model was evaluated according to its significance using ANOVA, its R-squared value, adjusted R-squared, Predicted R-squared and adequate precision. Moreover, the actual runs were compared to the predicted ones. The selected SV loaded LNCs were visualized using transmission electron microscopy (TEM). The selected formulae were then subjected to further investigations such as differential scanning calorimetry (DSC) to ensure the molecular dispersion of SV in the lipid matrix of LNCs. Effect of aging on selected SV loaded LNCs was studied after storing the formulae at 4⁰ C for 6 months. In an important encounter, sterilization of a selected SV loaded LNCs formulation was performed by exposure to gamma radiation at a dose of 25 KGy as recommended by the European pharmacopeia followed by sterility testing. After sterilization the formulations were re-characterized by measuring the PS, PDI and % Q48h.
Evaluation of cytotoxicity of selected SV loaded LNCs were conducted on MCF-7 cell lines using crystal violet assay. SV, plain LNCs, SV-LNCs were compared according to their IC50 scores.
from the obtained results, it was found that:
1) All the prepared SV loaded LNCs had sizes between 20 - 100 nm which was significantly affected by the ratio of Solutol to oil. Increasing this ratio greatly resulted in reduction of particle size (PS) owing to its effect on interfacial tension of the oily droplets of nanocapsules.
2) All the prepared SV loaded LNCs were characterized by a mono-modal particle size distribution less than 0.126. This could be attributed to the insertion of Solutol at the water–oil interface enhancing the incorporation of SV in the oily core and reducing the interfacial tension of oily droplets. These results also reflect the high efficiency of the adopted method; phase-inversion method in producing the lipid nanocapsules.
3) The release studies showed the sustained release behavior which was enhanced by increasing the Solutol amount. Being a surface active agent, Solutol was inserted at the water-oil interface forming a coherent shell completely entrapping SV at the oily core hindering its release from oily core owing to its bulky structure. Its solubilizing nature as well may have entrapped the drug at the interface retarding its release.
4) Based on the D-optimal mixture design results:
a. The suggested model for PS response was quadratic while for PDI and % Q48 h responses linear models were proposed.
b. Analysis of the models was performed using ANOVA where p-values less than 0.5 were produced indicating the significance of the models. The p-values for PS and %Q48h responses were less than 0.0001 while for PDI p-value was equal to 0.0015.
c. High correlation was indicated between the actual and predicted runs for the three investigated models. This was explicated in the graphical presentation of the actual versus the predicted runs. This also coincides with the R-squared values for the three obtained responses; 1, 0.76 and 0.95 for PS, PDI and %Q48h, respectively.
d. Adequate precision measuring the signal to noise ratio greater than 4 was desirable. The ratio was found to be 524.32, 9.30 and 19.68 for the three models; PS, PDI and %Q48h, respectively indicating highadequacy of the model.
e. Experimental validation of the model and its feasibility for navigation through the model was investigated by comparing other 4 actual runs vs their predicted values to calculate the % Bias for the three responses. The three responses scored less than 7 % confirming the model validation and its sufficiency to navigate the experimental spaces.
5) TEM micrographs confirmed that all the particles were spherical in shape possessing smooth surface with no obvious particle aggregation.
Breast cancer is the second leading cause of death among women worldwide. It is considered a heterogeneous disease where magnetic resonance imaging studies have displayed that there are multiple growth patterns of breast cancer with different strategies of treatment. Among these phenotypes there are breast cancer over-expressing estrogen receptors while others with activated Ras or ErbB2 pathways are observed. Also, it has been documented that elevated levels of mevalonate synthesis lead to several types of malignancies; one of them is breast cancer. This investigation recruits the use of statins as one of the chemotherapeutic agents to treat breast cancer.
Statins are 3-hydroxy-3-methylglutaryl-CoA (HMG CoA) reductase inhibitors. Statins inhibit HMG-CoA reductase which in turn inhibits the cholesterol biosynthesis. Consequently, depletion in mevalonate occurs together with its downstream products as isoprenoid intermediates leading to lower levels of lipid attachment sites for Ras, Rac and Rho proteins driving to lower cellular and subcellular pathways remarkable for cancer progression. Statins represent two classes: hydrophilic and lipophilic classes. The class responsible for inhibiting malignancies is the lipophilic one. Lipophilic statins such as simvastatin (SV) cross the cell membranes accumulating in cells and exert a pleiotropic effect such as the anti-apoptotic effect. SV, interestingly, induces cell-cycle arrest, initiating apoptosis and ameliorate treatment of breast cancer.
Treatment of breast cancer by chemotherapeutic agents is usually accompanied by side effects owing to their effect on non–cancerous cells. Accordingly, targeting the cancerous cells ameliorate the outcome of breast cancerous patients. The delivery of anticancer drugs to tumor cells is usually achieved by the enhanced permeability and retention (EPR) effect. EPR depends on the presence of fenestrations in the imperfect tumor blood vessels and to the poor lymphatic drainage in the tissue. Passive targeting of nanoparticles (NPs) can be achieved via utilizing the disruptions occurred in basal membrane of tumor vasculature.
Amongst the nanocarriers employed in cancer targeting are lipid NPs, from which lipid nanocapsules (LNCs) and nanostructured lipid carriers (NLC) are examples.
LNCs represent smart drug delivery systems enhancing targeting the anticancer drug to tumor cells via IV administration. The choice of these nano-carriers was based on several merits such as being biodegradable, biocompatible, avoidance of the use of organic solvents, stable up to one year and the ability of encapsulating hydrophilic and lipophilic drugs. Thanks to its PEGylated structure, bypassing the engulfment of LNCs by macrophages was feasible.
NLC represent the second generation form of the solid lipid nanoparticles (SLN). NLC were developed to overcome the limitations of the previous lipidic NPs; liposomes and SLN, such as the limited drug incorporation in case of liposomes and the expulsion of drug during storage as being noticed in SLN. Expulsion of the cargo entrapped in SLN usually occurs due to crystallization of the solid lipid into the more ordered β modification with the formation of a perfect crystal lattice.
NLC were prepared via mixing spatially different types of lipids, solid lipid with liquid lipid (oil), to provide imperfections in the crystal lattice of the solidified lipid of NLC enhancing incorporation of the drug and avoiding its expulsion during storage.
Hence the aim of work in this thesis was the development of nano-formulations capable of enhancing the delivery of SV to breast cancer cells, providing sustained release, augmenting cancer cytotoxicity and improving cellular uptake. This was planned to be achieved by encapsulating SV in LNCs and NLC.
So the work in this thesis is divided into two chapters:
Chapter One: Preparation and evaluation of SV loaded LNCs.
Chapter Two: Preparation and evaluation of SV loaded NLC.
Chapter I: Preparation and evaluation of SV loaded LNCs.
In this chapter, SV loaded LNCs were successfully prepared using the phase inversion method followed by shock introduced to the system by cold water to break the produced microemulsion and enhancing the formation of nanocapsules. LNCs was composed of Labrafac lipophile® (LL), Solutol®, salted aqueous medium in addition to Epikuron®. Epikuron acted as a stabilizer to the LNC shells, increasing the biocompatibility to the biological membranes. The favorable stealth properties of LNCs and its prolonged circulation were imparted by the PEGylated surfactant; Solutol. LL represented the oily phase. The salted aqueous medium enhanced the phase inversion temperature of Solutol to be easily achieved. LNCs were optimized using the D-optimal mixture design. The percentages of the three independent variables (LL, Solutol, aqueous phase) were varied to generate mathematical models for the three responses: particle size (PS), polydispersity index (PDI) and the percentage of drug released after 48 hours (% Q48h). Validation of the model was performed utilizing 4 different points and the % Bias was calculated. The model was evaluated according to its significance using ANOVA, its R-squared value, adjusted R-squared, Predicted R-squared and adequate precision. Moreover, the actual runs were compared to the predicted ones. The selected SV loaded LNCs were visualized using transmission electron microscopy (TEM). The selected formulae were then subjected to further investigations such as differential scanning calorimetry (DSC) to ensure the molecular dispersion of SV in the lipid matrix of LNCs. Effect of aging on selected SV loaded LNCs was studied after storing the formulae at 4⁰ C for 6 months. In an important encounter, sterilization of a selected SV loaded LNCs formulation was performed by exposure to gamma radiation at a dose of 25 KGy as recommended by the European pharmacopeia followed by sterility testing. After sterilization the formulations were re-characterized by measuring the PS, PDI and % Q48h.
Evaluation of cytotoxicity of selected SV loaded LNCs were conducted on MCF-7 cell lines using crystal violet assay. SV, plain LNCs, SV-LNCs were compared according to their IC50 scores.
from the obtained results, it was found that:
1) All the prepared SV loaded LNCs had sizes between 20 - 100 nm which was significantly affected by the ratio of Solutol to oil. Increasing this ratio greatly resulted in reduction of particle size (PS) owing to its effect on interfacial tension of the oily droplets of nanocapsules.
2) All the prepared SV loaded LNCs were characterized by a mono-modal particle size distribution less than 0.126. This could be attributed to the insertion of Solutol at the water–oil interface enhancing the incorporation of SV in the oily core and reducing the interfacial tension of oily droplets. These results also reflect the high efficiency of the adopted method; phase-inversion method in producing the lipid nanocapsules.
3) The release studies showed the sustained release behavior which was enhanced by increasing the Solutol amount. Being a surface active agent, Solutol was inserted at the water-oil interface forming a coherent shell completely entrapping SV at the oily core hindering its release from oily core owing to its bulky structure. Its solubilizing nature as well may have entrapped the drug at the interface retarding its release.
4) Based on the D-optimal mixture design results:
a. The suggested model for PS response was quadratic while for PDI and % Q48 h responses linear models were proposed.
b. Analysis of the models was performed using ANOVA where p-values less than 0.5 were produced indicating the significance of the models. The p-values for PS and %Q48h responses were less than 0.0001 while for PDI p-value was equal to 0.0015.
c. High correlation was indicated between the actual and predicted runs for the three investigated models. This was explicated in the graphical presentation of the actual versus the predicted runs. This also coincides with the R-squared values for the three obtained responses; 1, 0.76 and 0.95 for PS, PDI and %Q48h, respectively.
d. Adequate precision measuring the signal to noise ratio greater than 4 was desirable. The ratio was found to be 524.32, 9.30 and 19.68 for the three models; PS, PDI and %Q48h, respectively indicating highadequacy of the model.
e. Experimental validation of the model and its feasibility for navigation through the model was investigated by comparing other 4 actual runs vs their predicted values to calculate the % Bias for the three responses. The three responses scored less than 7 % confirming the model validation and its sufficiency to navigate the experimental spaces.
5) TEM micrographs confirmed that all the particles were spherical in shape possessing smooth surface with no obvious particle aggregation.
Breast cancer is the second leading cause of death among women worldwide. It is considered a heterogeneous disease where magnetic resonance imaging studies have displayed that there are multiple growth patterns of breast cancer with different strategies of treatment. Among these phenotypes there are breast cancer over-expressing estrogen receptors while others with activated Ras or ErbB2 pathways are observed. Also, it has been documented that elevated levels of mevalonate synthesis lead to several types of malignancies; one of them is breast cancer. This investigation recruits the use of statins as one of the chemotherapeutic agents to treat breast cancer.
Statins are 3-hydroxy-3-methylglutaryl-CoA (HMG CoA) reductase inhibitors. Statins inhibit HMG-CoA reductase which in turn inhibits the cholesterol biosynthesis. Consequently, depletion in mevalonate occurs together with its downstream products as isoprenoid intermediates leading to lower levels of lipid attachment sites for Ras, Rac and Rho proteins driving to lower cellular and subcellular pathways remarkable for cancer progression. Statins represent two classes: hydrophilic and lipophilic classes. The class responsible for inhibiting malignancies is the lipophilic one. Lipophilic statins such as simvastatin (SV) cross the cell membranes accumulating in cells and exert a pleiotropic effect such as the anti-apoptotic effect. SV, interestingly, induces cell-cycle arrest, initiating apoptosis and ameliorate treatment of breast cancer.
Treatment of breast cancer by chemotherapeutic agents is usually accompanied by side effects owing to their effect on non–cancerous cells. Accordingly, targeting the cancerous cells ameliorate the outcome of breast cancerous patients. The delivery of anticancer drugs to tumor cells is usually achieved by the enhanced permeability and retention (EPR) effect. EPR depends on the presence of fenestrations in the imperfect tumor blood vessels and to the poor lymphatic drainage in the tissue. Passive targeting of nanoparticles (NPs) can be achieved via utilizing the disruptions occurred in basal membrane of tumor vasculature.
Amongst the nanocarriers employed in cancer targeting are lipid NPs, from which lipid nanocapsules (LNCs) and nanostructured lipid carriers (NLC) are examples.
LNCs represent smart drug delivery systems enhancing targeting the anticancer drug to tumor cells via IV administration. The choice of these nano-carriers was based on several merits such as being biodegradable, biocompatible, avoidance of the use of organic solvents, stable up to one year and the ability of encapsulating hydrophilic and lipophilic drugs. Thanks to its PEGylated structure, bypassing the engulfment of LNCs by macrophages was feasible.
NLC represent the second generation form of the solid lipid nanoparticles (SLN). NLC were developed to overcome the limitations of the previous lipidic NPs; liposomes and SLN, such as the limited drug incorporation in case of liposomes and the expulsion of drug during storage as being noticed in SLN. Expulsion of the cargo entrapped in SLN usually occurs due to crystallization of the solid lipid into the more ordered β modification with the formation of a perfect crystal lattice.
NLC were prepared via mixing spatially different types of lipids, solid lipid with liquid lipid (oil), to provide imperfections in the crystal lattice of the solidified lipid of NLC enhancing incorporation of the drug and avoiding its expulsion during storage.
Hence the aim of work in this thesis was the development of nano-formulations capable of enhancing the delivery of SV to breast cancer cells, providing sustained release, augmenting cancer cytotoxicity and improving cellular uptake. This was planned to be achieved by encapsulating SV in LNCs and NLC.
So the work in this thesis is divided into two chapters:
Chapter One: Preparation and evaluation of SV loaded LNCs.
Chapter Two: Preparation and evaluation of SV loaded NLC.
Chapter I: Preparation and evaluation of SV loaded LNCs.
In this chapter, SV loaded LNCs were successfully prepared using the phase inversion method followed by shock introduced to the system by cold water to break the produced microemulsion and enhancing the formation of nanocapsules. LNCs was composed of Labrafac lipophile® (LL), Solutol®, salted aqueous medium in addition to Epikuron®. Epikuron acted as a stabilizer to the LNC shells, increasing the biocompatibility to the biological membranes. The favorable stealth properties of LNCs and its prolonged circulation were imparted by the PEGylated surfactant; Solutol. LL represented the oily phase. The salted aqueous medium enhanced the phase inversion temperature of Solutol to be easily achieved. LNCs were optimized using the D-optimal mixture design. The percentages of the three independent variables (LL, Solutol, aqueous phase) were varied to generate mathematical models for the three responses: particle size (PS), polydispersity index (PDI) and the percentage of drug released after 48 hours (% Q48h). Validation of the model was performed utilizing 4 different points and the % Bias was calculated. The model was evaluated according to its significance using ANOVA, its R-squared value, adjusted R-squared, Predicted R-squared and adequate precision. Moreover, the actual runs were compared to the predicted ones. The selected SV loaded LNCs were visualized using transmission electron microscopy (TEM). The selected formulae were then subjected to further investigations such as differential scanning calorimetry (DSC) to ensure the molecular dispersion of SV in the lipid matrix of LNCs. Effect of aging on selected SV loaded LNCs was studied after storing the formulae at 4⁰ C for 6 months. In an important encounter, sterilization of a selected SV loaded LNCs formulation was performed by exposure to gamma radiation at a dose of 25 KGy as recommended by the European pharmacopeia followed by sterility testing. After sterilization the formulations were re-characterized by measuring the PS, PDI and % Q48h.
Evaluation of cytotoxicity of selected SV loaded LNCs were conducted on MCF-7 cell lines using crystal violet assay. SV, plain LNCs, SV-LNCs were compared according to their IC50 scores.
from the obtained results, it was found that:
1) All the prepared SV loaded LNCs had sizes between 20 - 100 nm which was significantly affected by the ratio of Solutol to oil. Increasing this ratio greatly resulted in reduction of particle size (PS) owing to its effect on interfacial tension of the oily droplets of nanocapsules.
2) All the prepared SV loaded LNCs were characterized by a mono-modal particle size distribution less than 0.126. This could be attributed to the insertion of Solutol at the water–oil interface enhancing the incorporation of SV in the oily core and reducing the interfacial tension of oily droplets. These results also reflect the high efficiency of the adopted method; phase-inversion method in producing the lipid nanocapsules.
3) The release studies showed the sustained release behavior which was enhanced by increasing the Solutol amount. Being a surface active agent, Solutol was inserted at the water-oil interface forming a coherent shell completely entrapping SV at the oily core hindering its release from oily core owing to its bulky structure. Its solubilizing nature as well may have entrapped the drug at the interface retarding its release.
4) Based on the D-optimal mixture design results:
a. The suggested model for PS response was quadratic while for PDI and % Q48 h responses linear models were proposed.
b. Analysis of the models was performed using ANOVA where p-values less than 0.5 were produced indicating the significance of the models. The p-values for PS and %Q48h responses were less than 0.0001 while for PDI p-value was equal to 0.0015.
c. High correlation was indicated between the actual and predicted runs for the three investigated models. This was explicated in the graphical presentation of the actual versus the predicted runs. This also coincides with the R-squared values for the three obtained responses; 1, 0.76 and 0.95 for PS, PDI and %Q48h, respectively.
d. Adequate precision measuring the signal to noise ratio greater than 4 was desirable. The ratio was found to be 524.32, 9.30 and 19.68 for the three models; PS, PDI and %Q48h, respectively indicating highadequacy of the model.
e. Experimental validation of the model and its feasibility for navigation through the model was investigated by comparing other 4 actual runs vs their predicted values to calculate the % Bias for the three responses. The three responses scored less than 7 % confirming the model validation and its sufficiency to navigate the experimental spaces.
5) TEM micrographs confirmed that all the particles were spherical in shape possessing smooth surface with no obvious particle aggregation.