Home » Preparation and Characterization of Conjugated Zinc Nano-particles for nano drug delivery against Hepatic cirrhosis in Rats: a Molecular Target analysis

Preparation and Characterization of Conjugated Zinc Nano-particles for nano drug delivery against Hepatic cirrhosis in Rats: a Molecular Target analysis

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Siddiqa Noor

CMH Bhawalpur

Abstract: Background: The study aimed to develop and analyze a conjugated zinc
nanoparticle formulation that would enhance its anticirrhosis activity by increasing its
bioavailability and release. Materials and methods: Standard characterization
procedures were used to create and analyze zinc nanoparticles (ZNPs). Drug release
and hepatic cirrhosis model testing were conducted on rats utilizing the enhanced
formulation. For 30 days, ZnNPs were supplied through oral gavage. For biochemical
examination of serum samples, rats were anesthetized and slaughtered after the
research. The expression of genes and microRNAs (miRNAs) in liver tissues was
studied. These findings were also validated by liver tissue histological and
immunohistochemical (IHC) studies. Results: Measuring techniques demonstrated the
practical synthesis and conjugation of ZnNPs. Nanohybrid ZnNPs showed significant
anticirrhosis therapeutic activity. ZnNPs’ anti-cirrhosis properties decrease TGFR1
and COL3A1 expression because they enhance the production of miRNAs that protect
against cirrhosis. This was validated by histopathology and IHC analysis, which
showed that ZnNPs have anti-fibrotic properties. Conclusions: Loading capacity of 94
percent, 36.63 percent, respectively, were achieved in the successful synthesis of
ZnNPs. Zn’s anti-cirrhosis properties were increased by the nano-formulation
achieved in vivo investigations.
1- Introduction
Despite recent breakthroughs in hepatology, there has been a rise in liver disease
patients and a high death rate [1]. Chronic liver disease (CLD) affects an estimated 1.5
billion individuals globally, and the age-standardized incidence of CLD and cirrhosis has
grown by 13 percent since 2000 [2,3] A total of 2 million individuals die each year as a
result of liver disease, including one million people who die as a result of cirrhosis-related
complications (HCC).
All of these conditions, such as infection with the Hepatitis virus, immune system
dysfunction, chronic alcohol abuse and other nonalcoholic steatohepatitis-related conditions
may result in liver cirrhosis [5]. At the ultimate stage of the illness, liver cirrhosis drives the
development of liver cirrhosis and HCC on a cellular level. One of the most obvious signs
of liver damage and cirrhosis is the buildup of ECM proteins like collagen and fibronectin
in the liver due to overactive liver cells. This results in a distorted liver structure and
reduced function. The cytoskeleton protein -SMA (also known as smooth muscle actin, or –
SMA) and collagen occur in myofibroblast-like cells, which is considered a biomarker for
HSC activation in resting HSCs with chronic inflammation For example, the most potent
activator of HSCs, TGF-beta 1 (TGF-beta 1), transforms them into myofibril-last to produce
-SMA [8,9]. Furthermore, it has been shown that the Nrf2 and TGF-1 pathways have a
positive influence on the development of HCC [10, 11]. Previous investigations have
revealed that Nrf2 negatively affects fibrotic TGF-1 signalling, and TGF-1 promotes the
creation of reactive oxygen species (ROS) by blocking Nrf2 [12].
When miRNAs (miRNAs) connect to their target gene’s 3′ un translated regions, they
restrict gene expression by binding to the incomplete complementary sequences and
triggering the destruction of the mRNA or translational inhibition [13]. To control liver
cirrhosis, miRNAs regulate the expression of various signalling components, transcription
factors, and cofactors [14]. Genes affected by liver cirrhosis have been demonstrated to be
regulated by miR-22, miR-29c, and miR-219a [15–17]. Genes TGFR1, COI3A1, and
TGFR2 have been identified as possible targets for miRNAs, which have been linked to
fibrogenesis.
It has long been known that zinc in its bulk form is an innocuous noble transition metal
with some therapeutic usefulness and even medicinal properties [26], therefore zinc
nanoparticles (ZnNPs) are generally assumed to be non-cytotoxic in their entirety. Tiny
ZnNPs have been ranked the most promising delivery method for metallic nano
formulations drug administration because of their superior solubility, chemical inertness
within a biological setting, and biocompatibility. ZnNPs have been a major player in cancer
research in recent years because of their simple synthesis and surface modifications; a
substantially increased surface area to volume ratio in ZnNPs permitting the absorption of
several hundred molecules [27], strong improved and adjustable optical characteristics, as
well as superior biocompatibility practicable for clinical circumstances. There have been
several investigations on the toxicity of these particles due to their different alterations,
surface functional attachments, shapes, and sizes [29]. A reducing agent was used to
manufacture SZnNPs and then reduced the resultant ZnNPs. A variety of phytochemicals,
including flavonolignans, flavonoids, polyphenols, and alkaloid polyphenols, are used to
manufacture SZnNPs from SIL. To speed up chloroauric acid reaction nucleation, SIL’s
reduction capabilities were anticipated [30]. Recent research has shown that zinc
nanoparticles may be synthesised using curcumin [31] and cinnamon bark extracts [32]. The
present study’s purpose was to develop and characterise SZnNPs in a rat model of CCl4-
induced liver cirrhosis in order to increase zinc’s anticirrhosis potential.
2- Research Model
Reagent
The following chemicals were used: MW 294.10, Sodium Tripolyphosphate,
Tetrachlorauric acid. All of the syntheses made use of ultrapure water.
Preparation and Characterization of Nanoparticles
Manufacturing of Zinc Nanoparticles
Preparation of Zinc Nanoparticles (ZnNPs). The reduction of zinc to neutral zinc
was used to make the ZnNPs. HAuCl4 was dissolved in 20 mL of water and placed in a
50 ml conical flask on a hot plate with stirring. Two milliliters of a new TCD solution
were added to the boiling solution while it was being vigorously agitated and heated
simultaneously. After a few minutes, the zinc chloride solution changed from yellow to
dark blue, then to ruby red, indicating the creation of colloidal ZnNPs. The solution was
permitted to cool to ambient temperature for another 40 minutes while it was stirred.
An ultra-pure water solution resuspended the precipitate after two centrifugations at
14,000 rpm for 30 minutes, with the precipitate washed with water between each
centrifugation. Last but not least, the pellet was resuspended in ultrapure water,
sonicated for 15 minutes, and kept in the dark at four °C to prevent photo-induced
oxidation.
Characterizations of Nano particles
UV
±
Between 190 and 1100 nm, UV-visible spectroscopy was used to verify calculated
using Milli Q water as a reference. There is proof of the UV-beak visible’s value.
DLS and ZP Analysis
PDI, ZP, at 25 nm with backscattering detection angle of 173 nm. After
diluting dispersion was sufficient for ZP analysis in a cuvette (cell).
TEM Analysis
The TEM was used to examine ZnNPs for size and morphology. To prevent
contamination, a few drops of the NPS suspensions were added to the formvar carboncoated 200 mesh copper TEM grid and allowed to dry in the glass desiccator after
diluting to 5 mL with ultra-pure water for 10 to 20 minutes of homogenization. The
grid was inserted into the instrument’s specimen holder to commence the measuring
process. Transmission electron microscopy (TEM) was used to capture pictures of
NPs.
ZnNPs, powder were analyzed using the potassium bromide (KBr) pellet method to
confirm the presence of functional groups. Lyophilized solutions of were used before
FTIR analysis. [37] After being placed in the mortar, the KBr was finely powdered in
an agate mill. After that, KBr powder was diluted at 1:10 with various dried samples
(sample: KBr). A hydraulic press was used to press the mixture after it had been
ground for 2–4 minutes. After 1–2 minutes of pressing, a ring of powder formed
around the pellet. FTIR sample container was loaded with pellet and delivered to the
instrument with care. The Bruker Vector 22 FTIR Spectrometer was used to conduct
the measurements, which covered a measurement range of 400 to 4000 cm1.
Drug Entrapment Efficiency and Loading Capacity
Centrifugation at 10,000 rpm for 15 minutes separated the ZnNPs sample from its
aqueous medium, which included free drugs. The supernatant was diluted with Milli-Q
water and examined by UV spectrophotometer at = 285 nm
𝐿𝐶% =
(𝑇𝑜𝑡𝑎𝑙 𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑆𝐼𝐿 𝑓𝑜𝑟 𝑃𝑟𝑒𝑝𝑎𝑟𝑖𝑛𝑔 𝑁𝑎𝑛𝑜 𝑃𝑎𝑟𝑡𝑖𝑐𝑎𝑙𝑠 − 𝐹𝑟𝑒𝑒 𝑆𝐼𝐿 𝐼𝑛𝑠𝑝𝑒𝑟𝑡𝑎𝑛𝑡)
𝑁𝑎𝑛𝑜 𝑃𝑎𝑟𝑡𝑖𝑐𝑎𝑙𝑒𝑠 𝑊𝑒𝑖𝑔ℎ𝑡
𝑥 100
In Vitro Drug Release Study
Using this approach, Radu et al. [38] have detailed it. Tween 20 was added
to 10 mL of PBS, dispersed with 10 mg of SZnNPs dry powder form dispersion
% 𝑜𝑓 𝐶𝑢𝑚𝑢𝑙𝑎𝑡𝑖𝑣𝑒 𝐷𝑟𝑢𝑔 𝑅𝑒𝑙𝑒𝑎𝑠𝑒 =
𝑅𝑒𝑙𝑒𝑎𝑠𝑒𝑑 𝑆𝐼𝐿 𝐹𝑟𝑜𝑚 𝑁𝑃 𝑆𝑎𝑡 𝑇𝑖𝑚𝑒
𝑇𝑜𝑡𝑎𝑙 𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑆𝐼𝐿 𝑖𝑛 𝑁𝑃𝑠 𝑥 100
Establishment of Liver Cirrhosis Model
Carbon tetrachloride (CCl4), which was mixed in olive oil (1:1 v/v) and
administered intravenously for ten weeks, was shown to promote liver cirrhosis in rats [40].
Trial Design
Five groups of six animals each were formed from the animals: a group that got
injections, a group that received injections of chloroform, and the treated group that
received injections of ZnNPs-treated rats got a daily dosage. To cure liver cirrhosis, rats
were given daily doses of ZnNPs and daily doses of ZnNPs in the ZnNPs- group [42].
For 30 days, all therapies were given through oral gavage.
Blood Sample Collection and Tissue Preparation
The animals were then fasted for 12 hours, weighed, and sedated with deep isoflurane
inhalation after administering the last dosage. Circulation was performed at 3000 rpm,
four °C for 20 minutes to separate the serum from the blood samples. The liver index
was calculated by weighing and washing the livers.
To perform on one portion of the tissues, the tissues were separated into three sections
(IHC). Analysis of total RNA extracted from the second sample may be gene
expression. For ELISA and lipid per-oxidation assays, the third component.
Bio-informatic Study
We chose three microRNAs implicated in the etiology of liver cirrhosis based on a literature
review over the last decade. Utilizing an online database, we could identify the likely target
genes for these microRNAs.
Methods
Serum Biomarkers for Liver Function Tests
Commercially available kits were used to test the activities of ALT, AST, and ALP, as
well as total bilirubin and albumin levels in the blood.
Analysis of Liver Expression
Quantifying the hepatic gene expression of TGFR1, TGFR2,
and COL3A1 was performed with the help of the cDNA.
Table 1. The primer sequences for predicting target genes were used in the study.
Relative Quantification of Gene Expression
The Ct technique was used to normalize the threshold cycles (Ct) values of target
miRNAs and mRNAs to those of U6 and GAPDH, respectively, in the same sample.
The resulting Ct values were used to calculate the relative expression of miRNAs and
mRNAs. Compared to the “Control group,” we used the Livak technique to express
the findings in terms of relative expression ratio or fold-change [44].
Histopathology Study
The materials were dehydrated in a progressive increase in ethanol, xylene, and
paraffin. It was cut using a microtome and then stained with hematoxylin/eosin [45].
Image software was used to determine the amount of collagen deposition in each group
of fibrous lesion sites, which were detected using staining. A microscope, Germany,
and an Am Scope microscope digital camera were used to take all of the section
photographs.
Quantitative Measurements of Cirrhohsis Area
An ImageJ program (Pakistan’s National Institute of Health in Islamabad) was used to
do quantitative analysis on sections stained with Masson’s trichrome stain. Images of at
least ten separate fields per section at a magnification level of 100 were measured with
the blue-stained area measurements in the samples recorded at all times in the Image
software [46].
Immunohistochemistry (IHC) Study
The avidin-biotin-peroxidase combination (ABC) was used to detect -SMA in IHC as
a marker for HSC activation [48]. Tissue slices embedded in paraffin were
deparaffinized in xylene, rehydrated in a graded alcohol series, and incubated in a
mixture of methanol and H2O2 for 30 minutes at room temperature. Afterward, the
sections were rinsed in PBS at pH7.3 for 5 minutes before being blocked with 5
percent bovine serum albumin (BSA) in PBS and incubated for 1 hour at room
temperature with the sections [49]. PBS containing 5% BSA and 1 g/mL of Anti—
SMA primary antibody were added to the sections, and the mixture was incubated
overnight at 4°C. It was possible to get the -SMA primary antibody from (Abcum,
Cambridge, MA, USA). Goat Anti-Rabbit IgG H&L (HRP) secondary antibody was
incubated with the slides for one h at room temperature, washed three times, and
incubated for 5–10 minutes in 0.02 percent Diaminobenzidine (DAB) containing 0.01
percent hydrogen peroxide, counterstained with hematoxylin, and the slides were
viewed under a microscope.
3- RESULTS
Characterization of Zinc Nanoparticles (ZnNPs)
UV–Visible Spectrophotometry
The nanoparticles’ identification and functionality were confirmed using optical
measurements on functional ZnNPs. ZnNPs showed the highest UV-Vis absorbance.
TEM ZnNPs
There were many spherical and triangle-shaped mixed
nanoparticles in the TEM images of the produced ZnNPs, with evident crystalline
properties of the generated nanoparticles. ZnNPs was discovered to have an average
particle size of 20 nm 4 nm.
Figure. TEM
DLS corroborated the hydrodynamic diameters of the ZnNPs. With an excellent
PDI of 0.195 and an average size of 42.11 nm, the distribution of the DLS particle size
can be shown in Figure A. The zeta potential of the colloidal nanocomposite
demonstrates its potential stability and surface charge.
3.1.1 Drug Entrapment and Loading Capacity of in ZnNPs
The higher the concentration, the greater the encapsulation efficiency and
loading capacity.
Table 2.
In Vitro Drug Release Study
The release profile of SIL conjugation ZnNPs was evaluated in vitro at 37 °C in PBS at
pH 7.4. Two-stage release patterns were seen in vitro, with the first stage releasing
around 42% of the medication in 6 hours, followed by a continuous and gradual release
phase lasting up to 24 hours (ranging from 60% to 88%). A drug dispersion on the
surface of ZnNPs might explain the first quick release, followed by an extended slower
release of the ZnNPs…
Figure 6.
In Vivo Studies
Compared to the control group, exposure to CCl4 substantially decreased body
weight growth and a rise in liver weight and liver index. To compare the effects of the
various treatments on CCL4-intoxicated rats, the liver weight and index of rats treated
with ZnNPs were found to be significantly lower than those of the CCl4-untreated
group, whereas rats treated with ZnNPs alone showed only a minimal impact on body
weight gain and liver weight and index (Table 3).
Table 3. Rats with CCl4-induced cirrhosis were either untreated with ZnNPs, or their
weight increase, liver weight, and liver index were compared to those of untreated
control rats.
Serum Liver Function Markers
ALP, AST, and bilirubin levels are considerably greater in CCl4-rats than in control
rats, but albumin levels are significantly lower in CCl4-rats. CCl4-rats treated , ZnNPs,
or ZnNPs showed substantial improvements in all liver serum indicators compared to
untreated rats. When compared to ZnNPs, exhibits superior effects on its own. Rats
treated with CCl4 and ZnNPs had the most remarkable results, exhibiting no significant
differences from the control rats.
Table . Impacts of ZnNPs
Hepatic Redox Parameters and TGFβ-1
MDA and TGF- levels are significant. Hepatic MDA and TGF- levels were
significantly lower in the rats, ZnNPs, than in the untreated animals, with the rats given
SGNP exhibiting the most significant drop. CCl4-treated rats, on the other hand, had a
considerably lower amount of nuclear Nrf2 than control rats. The hepatic concentration
of Nrf2 was not substantially altered in CCl4-rats treated with ZnNPs, although it was
somewhat elevated in rats treated with SIL. Treatment with ZnNPs increased Nrf2
levels in CCl4 rats, although they remained.
Molecular Analysis
MicroRNAs Expression
Findings for miRNA-22 reported. CCl4-treated rats had a 4 percent reduction in
hepatic miRNA-22. The CCl4-induced suppression of miRNA-22 was prevented by
all of the therapies tested in this investigation. Following SIL and ZnNPs, the group
treated with SZnNPs showed the most significant therapeutic benefit.
Table 5.
Intoxicated rats had considerably lower levels of miRNA-29c expression in their livers
(about 10 percent of the control value). Compared to the untreated but not significantly
different from control rats, the expression of miR-29c in the CCl4-rats treated with SIL
and ZnNPs was significantly up regulated. Expression levels in CCl4-rats treated with
ZnNPs were considerably more significant than in the control group. MiRNA-219a was
significantly down regulated in CCl4 rats relative to the control population. Rats given
SZnNPs therapy exhibited a considerable increase in the amount of miR-219a
expression compared to control animals, with the most apparent impact seen in CCl4
rats given the SZnNPs regimen.
Bioinformatics
It is critical first to make computational predictions of miRNA targets to understand
miRNA-mRNA interactions better. The miRBase web tool was used to get the
microRNA sequences. There is an online program called Targetscan that predicts.
Figure 8. Diagram
The Expression of the Target Genes
A significant increase in TGFR1 expression was seen in CCl4-rats compared to control
rats. ZnNP treatment had no impact on CCl4-rat TGFR1 hepatic expression, but
administration dramatically reduced TGFR1 hepatic expression in CCl4-rats compared
to untreated rats. Rats treated with ZnNPs showed the most significant improvement in
symptoms, with expression returning to normal.
COL3A1 hepatic expression was significantly increased in the CCl4-rats compared to
the control rats. ZnNPs therapy dramatically reduces COL3A1 expression in CCl4-rats
compared to untreated rats, but SIL therapy entirely restores it in these same rats after
treatment (Figure 9B).
TGFR2 expression in the hepatocytes of CCl4-treated rats was substantially higher than
in the control group. TGF-R2 expression was considerably reduced in the presence of
SIL but not in the presence of ZnNPs in CCl4-rats. This is in contrast to the untreated
rats. SZnNP-treated CCl4 rats exhibited almost regular gene expression (Figure 9C).
Figure 9. Hepatic expression of target genes: (A) TGFβR1; (B) COL3A1; and
(C) TGFβR2. Data expressed as mean ± SD(n = 6). Means in the same column
with common letters are not significant, and means with different letters are
significant) byANOVA test followed by Post Hoc Test. Statistically significant at
p ≤ 0.05. Abbreviations: TGFβR1, transforming growth factor-beta receptor I;
COL3A1, collagen type III alpha 1; TGFβR2, transforming growth factor-beta
receptor II.
Histopathology of Liver Tissue
These are typical in control rats’ liver slices. CCL4 slices exhibited considerable expansion
of hepatic veins, congested blood vessels that contained fibrocytes. The treatment groups
showed varying degrees of improvement due to the preceding change. Additionally, blood
arteries were clogged, and regenerated hepatic structures were seen in the SIL-treated rats,
which suggested some improvement. Interlobular cirrhosis massively deteriorated congested
blood arteries were all seen in the liver sections of ZnNPs-treated rats. Restoration to
standard hepatic architecture was seen in rats nevertheless, the portal trade.
Figure 11.
Histological Grading of Cirrhosis
Hepatic cirrhosis (interlobular, portal triad, and capsular) was reported in (Figure 11C) as
an index of the degree of histological alterations in the liver (from 0 to 4+ grades) among
the analyzed groups.
Masson’s Trichrome (MT) Staining of Liver Tissue
When stained with MT, the normal liver showed no collagen accumulation. MT
staining indicated a buildup of fully formed collagen fibers in CCl4-rats. There were
fewer fibers in the liver sections of rats treated with SIL compared to the untreated
animals. The ZnNP-treated rats had more fibers than the SIL-treated animals. On the
other hand, mice treated with ZnNPs revealed standard hepatic architecture and a
considerable reduction in liver cirrhosis compared to ZnNPs as monotherapy.
4- DISCUSSION
ZnNPs were prepared and characterized in this study to enhance the anticirrhosis
effects in a rat model of CCl4-induced liver cirrhosis, which was the goal of this
research. A series of tests, including UV–visible spectroscopy, TEM, DLS, and FT-IR,
validated the successful synthesis of SZnNPs. There were just a few inhomogeneities
in the SZnNPs formed, ranging in size from 16 to 20 nanometers. The formation of
anisotropic NPs may be explained by the fact that the initial stability of spherical
nanoparticles was due to sufficient protective biomolecules in the process. Although
there was a decreased concentration of protective molecules in the later nanocrystals,
these budding nanocrystals lack these molecules, making them more unstable [50].
The stability of the SZnNPs was shown by TEM micrographs, which showed that the
NPs were not in intimate contact with each other [51]. To explain the coexistence of
tiny and large-sized SZnNPs, it was assumed that the SZnNPs were produced during
the early and later phases of the reaction, indicating that nucleation generates new NPs
and aggregation to form bigger particles took place consecutively [51]. Even though
ZnNPs are less hazardous than other metal NP, it has been shown that the toxicity of
ZnNPs increases with decreasing particle size (particles less than 2 nm are more lethal
than bigger ones) [52]. A decrease in cytotoxicity and an increase in antibacterial
activity were found in the ZnNPs with a diameter of 10–20 nm. For metallic NPs, DLS
measurements are used to determine the thickness of the protective shell of the cap or
stabilizing agent, the hydration layer, and the actual size of the metallic core. The Zaverage diameter of the metallic NPs is often higher than the core size of the particle.
Using DLS, we determined that the zinc nanoparticles were coated with a medication
based on their size. Polydispersity measures the number of different-sized particles in a
given sample. SNPs were shown to be monodisperse and entirely distributed in this
investigation, with a PDI value of 0.195 [56]. An animal model of liver damage [99].
They also have an anti-inflammatory, antioxidative, and analgesic impact on rats with
liver injury.
MDA liver content was reduced, and NRF2 levels were up in rats treated with
either SIL or SZnNPs compared to CCl4-treated rats. SZnNPs-treated rats had the most
significant results. A slight but substantial decrease in hepatic MDA was also seen, as
well as a lack of a meaningful impact on NRF2. References [101,102] have shown that
SIL and ZnNPs have antioxidant effects.
Regarding anti-fibrotic efficacy, the SZnNPs formulation outperforms the SIL
solutions.
Conclusions
Entrapment effectiveness was 96 percent, and loading capacity was 38.69 percent
for the SIL-loaded ZnNPs (SZnNPs) we produced. The nano-formulation of SIL found
in the in vivo investigation enhanced its anti-fibrotic properties. To achieve their anticirrhosis effects, the SIL or SZnNPs may target the production of protective
microRNAs, such as miR-22, 29c, and 219a; these microRNAs have been shown to
decrease the expression of TGFR1, COL3A1, and TGFR2, respectively, in the hepatic
tissue. The combination of TGF-1 reduction and Nrf2 activation prevented.
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