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During the past few decades, drug delivery has been the main scope of
pharmaceutical industry and research as the majority of biologically active
molecules was found to possess poor solubility or/and poor biological membrane
permeability, instability, rapid systemic clearance and accordingly poor
bioavailability. Consequently, the duty of drug delivery systems (DDS)
development was to improve drug stability, physicochemical characteristics,
pharmacokinetic properties and the biological effects.
The use of nanotechnology as a tool in developing DDS provided suitable nanoscale
carriers to enhance drug properties and achieve improved therapeutic
Nano-based DDS were believed to be advantageous in the delivery of
anticancer chemotherapeutic agents especially when passive and active targeting
could be achieved, making use of the unique properties of cancer tissue,
thus maximizing the therapeutic efficiency and minimizing the adverse effects.
Noticeably, gelatin, which is generally regarded as safe (GRAS), has
emerged as a novel drug delivery platform owing to its unique properties, specially
biodegradability, biocompatibility, non-antigenicity, its plentiful sources and
exceptional binding ability to different drugs. The versatility of its functional
groups allows for many surface modifications to create stealth or/and targeted
Nowadays, intensive research has been conducted on the use of
nutraceuticals and functional food in prevention and management of different
diseases and to maintain good health instead of the expensive marketed
standardized synthetic chemotherapeutic agents as they are relatively cheap and
abundant. Since ancient ages crude garlic was widely used as a seasoning
condiment to impart taste and as a remedy for inflammation, diarrhea,
respiratory problems and anti-helminthic. Recently, all the biological
activities of garlic powder were attributed to its organosulfur
constituents specially allicin. Nowadays, it was reported that allicin
additionally possess antimicrobial, antiviral, antithrombotic, antioxidant and
more interestingly anticancer and anti-metastatic effects.
Unfortunately, thermolability, photodegradability and alkaline instability
limit the allicin use as promising chemotherapeutic agent.
Among different cancer types, the liver cancer was considered the
third death-causing cancer type. Incidence of the liver cancer is usually related
to the distribution of chronic hepatitis B virus (HBV) and hepatitis C virus
(HCV) infections. Therefore, the effect of allicin on hepatocellular cancer cell
line (HepG-2) cells was investigated.
The aim of this work was to incorporate allicin in gelatin nanoparticles with
optimized colloidal properties in order to mask its strong odor as well as achieving
cancer targeting and hence, improving its cytotoxicity. In a trial to promote
hepatocellular uptake, allicin-loaded gelatin particles were conjugated to
Thus, the work in this thesis was divided into two chapters:
1- Chapter I: Preparation, optimization and biological evaluation of allicin-loaded
stealth gelatin nanoparticles.
2- Chapter II: Preparation and biological evaluation of glycyrrhetinic acidconjugated
optimized allicin-loaded GNPs.
Chapter I: Preparation, optimization and biological evaluation of allicin-loaded
stealth gelatin nanoparticles.
In this chapter, the cytotoxic potential of allicin on different human cancer cell
lines namely, human hepatocellular cancer cell line HepG-2, human breast cancer cell
line MCF-7, human lung cancer cell line A-549 and human prostatic cancer cell line PC-3
was investigated where allicin scored an IC50 of 19.26 μM, 28.51 μM, 36 μM and
77.92 μM, respectively. Accordingly, it was observed that allicin exerted
the highest inhibitory effect on hepatic cancer cells making it a promising natural
substance that could be used in the treatment of liver cancer cases.
Gelatin nanoparticles were successfully prepared by adopting the double
desolvation method using acetone as the desolvating agent. Optimization of the
experimental dependent variables (responses) of interest which were the particle size (PS)
and the polydispersity index (PDI) factors was performed using D-optimal computer aided
design. The D-optimal study design produced 16 runs combining the main three
independent variables; glutaraldehyde percentage (GA%), the cross-linking time (CLT)
and the stirring speed.
The PS of the obtained GNPs ranged between 370.7 nm ± 6.78 and 786.1 ± 100.4
nm accompanied by a PDI range between 0.039 ± 0.009 and 0.535 ± 0.069. The
percentage yield of the plain GNPs relative to the initial amount of the used gelatin was
16.16% ± 0.3 and that relative to the obtained gel-like precipitate was found to be
23.488% ± 0.44. It was found that the smallest particle size was obtained at an optimum
GA% while when the cross-linking time was increased; the particle size was significantly
increased. Finally, it was found that the particle size was significantly and inversely
affected by the stirring speed. As a conclusion, the cross-linking time was proven the
most significant factor in the design.
Regarding zeta potential (ZP), the plain GNPs and A-GNPs possessed + 28.76 ±
2.03 mV and + 20.4 ± 1.25 mV, respectively, reflecting good physical stability and low
tendency for aggregation.
Allicin was entrapped in the prepared gelatin nanospheres with an average
entrapment efficiency of 39.13 % ± 2.38. TEM morphological examination for the
prepared allicin loaded GNPs revealed that the particles were spherical and homogenous
with the absence of any aggregates with a PS range from 207 to 309 nm.
In-vitro release study was conducted in the presence and the absence of
collagenase enzyme revealing that the release profile is biphasic where drug release was
hastened in the presence of collagenase. After 24 h incubation in PBS pH 7.4 in the
presence of collagenase, GNPs showed a cumulative percentage released for allicin
of 97.71% ± 2.02; while, after the same time interval in the absence of collagenase,
GNPs released only 51.74% ± 1.27 of cumulative allicin percentage.
To impart stealth properties and increase the circulation time of the prepared
nanoparticles, Poloxamer 188 was physically adsorbed on the surface of the prepared
allicin-loaded GNPs. The results of particle size and PDI measurements of these stealth
particles were 714 ± 25.21 nm and 0.663 ± 0.143, respectively. These data reflect the
presence of adequate poloxamer coat. Additionally, TEM images for the coated GNPs
(GNP/P188) revealed a particle size ranging from 280-580 nm and the amount of P188
adsorbed onto the nanoparticles (Ng/g) was determined using 1H-NMR spectroscopy
and was found to be 0.18 g/g allicin-loaded GNPs.
The zeta potential (ZP) of stealth A-GNPs was found to be +11.79 ± 3.3 mV. The
physical stability of the coated particles was preserved by the steric hinderance applied
by the hydrophilic polymeric coat.
The release of allicin from coated allicin-loaded GNPs (GNPs/P188) after 24 h
incubation in PBS pH 7.4 in the presence of collagenase showed a cumulative percentage
released for allicin of only 32.87% ± 0.54. These results reflected GNPs with a more
retarded release behavior due to the steric cloud caused by the surface adsorbed
Gamma-irradiation was employed as an efficient and acceptable method to sterilize
the lyophilized formulations without any significant effect on PS, PDI, ZP and %EE.
MTT assay of both coated and uncoated allicin-loaded GNPs showed significant
enhancement of the cytotoxic effect of allicin where the IC50 of the uncoated GNPs was
10.95 μM whereas that of the coated particles was 6.736 μM. Compared to aqueous
allicin solution (IC50 of 19.29μM), the cytotoxic potential was improved by
approximately 2-folds for the uncoated allicin loaded GNPs and 3-folds for the coated
allicin loaded GNPs. This improvement can be ascribed to the encouraged passive
endocytosis that occurred due to the formulation of allicin in a nano-carrier and also due
to the increased accumulation at the site of action due to EPR effect. A-GNPs showed
considerable physical stability over a course of 12 months.
Chapter II: Preparation and biological evaluation of glycyrrhetinic acid- conjugated
optimized allicin-loaded GNPs.
The aim of this chapter was to prepare glycyrrhetinic acid conjugated optimized
allicin-loaded GNPs to achieve effective delivery of the drug to the hepatic cancer cells
that could be administered via intratumoral injection. Ligand conjugation could promote
receptor-mediated endocytosis. Successful conjugation between glycyrrhetinic acid and
optimized allicin-loaded GNPs was confirmed using 1H-NMR spectroscopy where the
peak of the carboxylic OH at 12.3 ppm disappeared.
The average PS of the conjugated GNPs was found to be 398 ± 16.71 nm, while
the average PDI was 0.0556 ± 0.0125 showing significant but small increase in size
compared to the unconjugated particles (P < 0.05). Moreover, TEM images
revealed homogeneous and spherical particles with no aggregations. The ZP was
found to be + 18.87 ± 2.29 mV revealing considerable physical stability and the %
EE of allicin in conjugated A-GNPs was found to be 36.04 % ± 1.39.
The biphasic release pattern of the conjugated particles was closely similar to
that of the unconjugated counterparts with a cumulative percentage released for
allicin of 96.04 % ± 2.2 in the presence of collagenase enzyme and of only 46.61 % ±
3.55 in the absence of the enzyme at the end of the 24 h study. The prepared
conjugated A-GNPs was efficiently sterilized using gamma-irradiation without
affecting PS, PDI, ZP and %EE.
Regarding the cytotoxicity study on HepG-2 cell line, the IC50 of the conjugated
particles was found to be 5.046 μM. Compared to the allicin solution (IC50 of
19.29μM), the cytotoxic potential was improved by nearly 4
folds. This observed improvement in the cytotoxicity of the conjugated GNPs
was attributed to the ability of glycyrrhetinic acid to increase the internalization of
particles inside hepatic cells due to its direct interaction with the asialoglycoprotein
(ASGPR) receptor, displayed on the surface of hepatic cells. This effect could be
augmented upon the intratumoral injection of the conjugated allicin-loaded GNPs.
This favorable interaction between glycyrrhetinic acid as a ligand and the
asialoglycoprotein (ASGPR) receptor was investigated using molecular docking where
the interaction scored a binding energy of -11.53 ± 0.50 kcal / mole indicating a good
The conjugated A-GNPs showed acceptable stability in terms of PS, PDI, ZP and %EE
over 12-month storage at ambient conditions