الفهرس 
General IntroductionOnly 14 pages are availabe for public view
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Abstract
Nanoparticulate delivery systems for liver targeting
The enormous vital functions performed by the liver make it one of the major crucial organs in the body. Liver is responsible for blood detoxification, secreting bile, metabolism of carbohydrates, fats and proteins and storing vitamins and other essential materials. These tasks expose the liver to microbes, drugs and other toxic material which could subject liver to many diseases. Liver cancer is the sixth most common malignancy and the third leading cause of cancer related death. Hepatocellular carcinoma (HCC) is the major pathological type of liver cancer and accounts for approximately 80% of cases. As a treatment of this disease, chemotherapy is the second line treatment after surgery but it is limited due to chemo-resistance, inadequate specificity and safety. Nanomaterial based medicine can offer a great potential in delivering drugs to the liver either passively or actively. Passive targeting can be mediated by the production of nanoparticles of sizes below 200 nm that can penetrate 100-200 nm fenestrations in the endothelial wall of the liver. On the other hand, active targeting depends on attaching the delivery system to a moiety that can interact with liver associated receptors and internalize into cells. Many studies have proven the presence of glycyrrhizin (GL) receptors on the surface of the main liver cell, the hepatocytes. The natural origin of GL, extracted from the root of Glycyrrhiza glabra (licorice), encouraged many researchers to use this molecule and its aglycone derivative as liver targeting moiety.
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Over the years, chitosan (Cs) has been extensively used as promising drug carrier in the biomedical field. The great attention granted to this biopolymer is related to its interesting characteristics. Many studies reported that grafting a hydrophobic side chain on the chitosan backbone showed improved in vivo haemocompatability. This strategy is of great importance especially with growing attention to the use of chitosan-based nanomaterials for intravenous administration.
Ferulic acid belongs to the group of phenolic phytochemicals, widely abundant in foods as grain, bran, citrus fruits, banana, coffee and broccoli. It possesses a well-documented antioxidant activity that can be used in treating various disorders such as Alzheimer’s disease, cardiovascular diseases, diabetes and cancer. Its anticancer effect against breast cancer, colon cancer, skin cancer and liver cancer was previously reported in the literature. This may be explained by the ability of ferulic acid to modulate cell growth and proliferation, scavenge reactive oxygen species (ROS) and stimulate cytoprotective enzymes.
To this end, the current work aimed at developing modified chitosan nanoparticles (NPs) capable of targeting hepatocytes passively and actively. Chitosan was hydrophobically modified with a five-carbon chain length (valeric) moiety. Ferulic acid was physically loaded into the newly synthesized valerate chitosan nanoparticles where different NPs fabrication variables were studied. Also, polymer drug conjugates between valerate chitosan nanoparticles and ferulic acid were formulated with the aim of increasing the drug load. The passive and active targeting methods were achieved by size adjustment or by using glycyrrhizin (GL) as a targeting ligand, respectively. Moreover, the in vitro cytotoxic effect of selected
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formulae as well as the in vivo drug biodistribution in mice were assessed. Accordingly, the work in this thesis was divided into four chapters as follows:
Chapter One: Preparation and characterization of Chemically Modified Low Molecular Weight Chitosan
Chapter Two: Formulation of Optimized Ferulic acid Loaded Valerate Chitosan Nanoparticles
Chapter Three: Formulation of Optimized Ferulic acid Conjugated Valerate Chitosan Nanoparticles
Chapter Four: Cytotoxicity and In vivo Biodistribution Study of selected Ferulic acid Conjugated Nanoparticles
Chapter One: Preparation and characterization of Chemically Modified Low Molecular Weight Chitosan
Low molecular weigh chitosan was modified with a 5-carbon aliphatic chain, valeric moiety, to enhance the chitosan hemocompatibility. The reaction was carried out for either 40 minutes (MC) or for 4 hours (HC). The degree of substitution in each polymer was assessed by the ninhydrin assay. The two newly synthesized polymers were characterized by 1H NMR spectroscopy, FTIR spectroscopy, X-ray diffraction, differential scanning colorimetry (DSC) techniques and measurement of the critical aggregation concentration. Plain modified chitosan nanoparticles were prepared and compared in terms of particle size, polydispersity index and the zeta potential. The serum stability of the unmodified and the modified chitosan nanopartilcles was evaluated.
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The results revealed that the reaction exhibited a time dependent behavior where the 40 minutes (MC) reaction showed a degree of substitution of 53% compared to 43% for the 4 hours reaction (HC). Both 1H NMR spectroscopy and FTIR spectroscopy confirmed the successful substitution of chitosan by the valeric molecule. X-ray diffraction study showed that increasing the degree of substitution resulted in the reduction in chitosan crystallinity. The DSC thermogram denoted a shift in the exothermic peak of native chitosan from 282.35 ºC to 217.7 ºC and 265.77 ºC for MC and HC, respectively. The critical aggregation concentration for MC polymer was found to be 0.1mg/ml. The particle size of the plain nanoparticles decreased significantly with the increase in valeric content. The zeta potential values of MC NPs and HC NPs decreased compared to the nanoparticles prepared with unmodified chitosan (PC) which can be attributed to decreased -NH2 groups available on the surface of nanoparticles due to substitution. The PDI value for MC NPs was significantly lower compared to their corresponding HC NPs.
Consequently, the polymer that was prepared through the reaction condition that lasted for 40 minutes was chosen for the subsequent studies. The serum stability study revealed that, after 2 hours incubation, the unmodified chitosan (PC) nanoparticles showed a significant reduction in size while valerate chitosan nanoparticles (MC NPs) exhibited minimal increase in particle size. After 24 hours incubation, the PDI values of PC NPs reached 0.725 compared to 0.442 MC NPs. This indicated the minimal particle aggregation of MC NPs and better ex-vivo stability hence the used modified polymer was considered a promising platform for the formulation of ferulic acid loaded nanoparticles.
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Chapter Two: Formulation of Optimized Ferulic acid Loaded Valerate Chitosan Nanoparticles
In this chapter, the D-optimal design was adopted to optimize the formulation of the ferulic acid loaded valerate chitosan nanoparticles aiming at increasing the drug entrapment efficiency and decreasing the particle size below 200 nm. The independent variables and their levels were: MC concentration (1, 2 and 3 mg/ml), MC/ TPP mass ratio (1:1, 3:1 and 5:1), MC solution pH was (4.5, 5 and 5.5) and the preparation temperature (4, 22 and 40 ºC). The measured responses were the EE%, PS, PDI and ZP.
The EE% of the prepared nanoparticles ranged from 3.15% to 26.65%. The prepared formulations had a minimal particle diameter of 140.45 nm and a maximum diameter of 261.4 with polydispersity indices ranging from 0.140 to 0.477. The ZP varied between +3.8 mV and +15.2 mV.
Statistical modeling generated three successful models for EE%, PDI and ZP responses. The particles size was excluded as the proposed model showed significant lack of fit. The other three successful models were significant with an acceptable R2 ranging from 0.7746 to 0.8855. The adequate precision of the three models was sufficiently high (greater than 4) indicating their abilities to navigate the design space.
The best fitting model for EE% was the reduced two factor interaction (2FI) model. The EE% was significantly affected by the valerate chitosan solution pH and the preparation temperature. In another encounter, MC concentration, MC:TPP mass ratio and MC solution pH had the same significant influence on both PDI and ZP responses. The model fitted the
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PDI response was the reduced quadratic model while that of ZP was the linear model. The TEM photomicrographs of the selected formulation revealed that the prepared ferulic acid loaded valerate chitosan nanoparticles were almost spherical with no observed aggregation. To this end, although the prepared formulation showed a particle size below 200 nm yet the maximum drug EE% achieved was only 26.65%, a case which warranted more trials using a different loading method.
Chapter Three: Formulation of Optimized Ferulic acid Conjugated Valerate Chitosan Nanoparticles
In this chapter, a polymer drug conjugate was formulated between valerate chitosan and ferulic acid to increase the drug loading. First, plain nanoparticles were formulated and optimized at different pHs of chitosan solution and variable preparation temperatures. Ferulic acid was added to the optimized nanoparticles then the formulations were characterized. Glycyrrhizin was oxidized prior to its attachment to the surface of nanoparticles at different GL:MC ratios. The selected nanoparticles were stored at 4 ºC in the refrigerator for 6 months and the PS, PDI and ZP were studied during the storage period. Furthermore, freeze drying was carried out to enhance formulation stability. Finally, these characterizations were followed by gamma sterilization of the selected formulae.
from the results obtained it was found that:
Changing the pH of the valerate chitosan solution resulted in a significant increase in plain particle diameter when the pH of the polymer solution was adjusted to 4.5; while the nanoparticles size did not significantly change when the preparation temperature was altered. Results
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revealed that the maximum conjugation occurred at pH 5 reaching a value of 69.1 %. Addition of poloxamer 407 (0.3%w/v) resulted in a significant reduction in particles size and zeta potential. Both 1H NMR and ninhydrin assay confirmed the successful attachment of GL to the surface of nanoparticles. It is worth to mention, that GL decorated nanoparticles had smaller particle diameters and smaller PDI values compared to their respective nanoparticles without GL. TEM photomicrographs indicated that both nanoparticles (M1-P and M1-PG) were well dispersed and spherical in shape. The percentage of drug release after 24 hours was 3.143% ± 0.29. This is considered advantageous for targeting purposes to guarantee that the conjugated amount will reach the liver without any loss of the drug in the blood stream.
After six month storage at 4 ºC, formula M1 did not show any significant change in PS. However, both M1-P and M1-PG showed minimal change in the PS from 118.72 nm to 139.77 nm and from 109.33nm to 97.11, respectively. Regarding the PDI values, all the formulae exhibited significant increase in the particle size distribution.
Freeze drying of the selected formulation was performed to ensure long term stability of the nanoparticles. Trehalose was utilized as a cryoprotectant at two concentrations: 5% and 7.5 % w/v. Both M1 and M1-PG formulations exhibited a significant increase in particle size at either 7.5% w/v trehalose concentration or at both concentrations, respectively. However, M1-P formulation succeeded in preserving its size at both trehalose concentrations due to poloxamer 407 inclusion. All the PDI values after nanoparticles reconstitution ranged from 0.385 to 0.51 indicating fairly homogenous dispersion. It is worth to mention that there were no significant
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recorded differences between the particle size and PDI values of the selected formulae before and after gamma irradiation.
To sum up, ferulic acid was efficiently conjugated with valerate chitosan nanoparticles with high conjugation efficiency. Furthermore, decorating the nanoparticles with GL for active targeting purpose was achieved successfully.
Chapter Four: Cytotoxicity and In vivo Biodistribution Study of selected Ferulic acid Conjugated Nanoparticles
This chapter was implemented to study the cytotoxic activity of the fabricated nanoparticles and the their biodistribution in swiss albino mice.
The cytotoxicity study was performed for the selected formulations (M1, M1-P, M1-PG) and was compared to the solution of the drug in DMSO, the solution of glycyrrhizin in water and the drug-free (plain) nanoparticles by measuring the cell viability using the sulforhodamine B (SRB) assay.
M1-PG exhibited the highest cytotoxicity with the lowest IC50 value of 60 μg/ml. This enhanced cytotoxicity confirmed that glycyrrhizin modification may induce glycyrrhizin receptor mediated internalization. Glycyrrhizin solution showed no cytotoxicity in the same concentration that is used in the nanoparticles. Poloxamer 407 modification led to an augmented cytotoxicity where the IC50 values were 90.5 μg/ml and 197 μg/ml for M1-P and M1 nanoparticles, respectively.
Accordingly, the nanoparticles that scored the highest cytotoxicity i.e. M1-P and M1-PG were selected for the in vivo biodistribution studies.
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In order to carry out the biodistribution studies, the selected nanoparticles formulations were re-prepared utilizing the radiolabeled 125 I-ferulic acid. Accordingly, thirty swiss albino mice each weighing 20-25gram were divided into three groups as follow:
group I (n=10): received M1-P formula (passive targeting).
group II (n=10): received M1-PG formulation (active targeting with glycyrrhizin).
group III (n=10): received 125I-Ferulic acid solution dissolved in ethanol: phosphate buffer mixture (control).
The radiolabeling condition succeeded in achieving a high reaction yield of about 88%. Six hours post-injection, it was obvious that the hepatic drug deposition of glycyrrhizin functionalized nanoparticles was significantly higher compared to M1-P formula and drug solution. However, the M1-P exhibited non-specific uptake by the reticulo-endothelial organs (liver, kidney and spleen). The increased liver uptake associated with the glycyrrhizin modified nanoparticles confirmed the recognition of the nanoparticles by glycyrrhizin receptors on hepatocytes. It is worth pointing out that, the drug solution showed the highest accumulation in the kidneys with respect to the nanoparticles formulation suggesting the rapid elimination of ferulic acid by the kidneys. Twenty four hours post injection, a high liver accumulation of the nanoparticles formulation was observed compared to the drug solution.
To recapitulate, the development of a serum-stable valerate chitosan platform possessing a high-loading capacity (through conjugation) was
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successful. The fabricated systems proved to be of great potential to target the liver actively using the natural glycyrrhizin and offer a novel liver cancer remedy.