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One of the approaches is the use of Nano compounds as a drug carrier because they can provide site specific or targeted drug delivery combined with optimal drug release profiles with minimal cellular side effects. Thus preventing cellular and systemic side effects.
This study aims to evaluate the therapeutic effectiveness of loading dexamethasone on Nano chitosan particles as a novel Nano-targeted treatment for bleomycin-induced pulmonary fibrosis in male C57BL/6 mice model. This is achieved with minimal systemic and cellular side effects and effective drug release of dexamethasone to the lung tissue. Dexamethasone loaded on Nano chitosan treatment was further compared with the widely used treatment dexamethasone either alone or combined with the improved form of curcumin which is Nano curcumin.
Nano chitosan (NCH) particles were chemically analyzed by scaning electron microscope (SEM) Transmission electron microscope (TEM), Energy dispersive X-ray analysis (EDXA) and Fourier transform infrared spectroscopy (FTIR). These chemical analysis were carried out for Nano-chitosan particles before and after loading with dexamethasone. Chemical characterization of the novel synthesized nano particle confirmed that dexamethasone was effectively loaded onto NCH-particles.
1-Scanning electron microscopy (SEM) of chitosan nanoparticle showd that NCH appeared like particles at 3000x with diameter ranging from 2.05 up to 5.07 µm with irregular spherical structure at the cross-section. This makes chitosan nanoparticles absorb the moisture, forming hydrogen bonds with water and slowly losing it .This behavior is most interesting in drug release. Scanning elaectron microscopy of dexamethasone-Nano chitosan at higher power of 30000x appeared like particles where the drug was adsorbed on the surface and absorbed inside Nano-chitosan particles .
2-Transmission electron microscopy (TEM) of nano-chitosan before and after loading with dexamethasone nanoparticles with particle size of 200nm.TEM of Nano-chitosan alone appeared as irregular shaped and the particles of DEX were absorbed of the surface of nano-chitosan particles .
3- Energy disperse X-ray analysis (EDXA) of DEX-NCH particle illustrated the presence of Florin content which is one of the drug’s components (DEX )with a weight % 0.55 alone while weight % of DEX- Nano-chitosan particles recorded a weight % of 0.42 indicating that the particles of the drug were adsorbed on the surface of nanoparticles and absorbed into the NCH-particles. On the other hand NCH particles showed no florin in its structure.
Also, the weight % of carbon content was elevated from 13.73 in NCH to 19.27 in Dex-NCH –particles which proved that the drug was loaded onto nano- chitosan Particles .After loading the drug,(DEX) onto NCH particles , the weight % of nitrogen content of NCH particles was increase from 0.28 to 0.39 in DEX-NCH particles, this ratifies the presence of DEX component onto NCH particles
4- Fourier transform infrared spectroscopy (FTIR) of DEX showed difference variation in bands than nano- chitosan and dexa-nanochitosan particles which combine in one band this proved that the interaction occuredbetween dexamethasone and chitosan nanoparticle.
The previous chemical analyzes that were carried out on the DEX-NCH particles and proved that DEXwas loaded onto Nanochitosan particles.
Furthermore, DEX-NCH particles were used on experimental animals as a novel treatment for PF and compared to other experimental tretments for PF.
For this experiment a total number of 128 male C57BL/6 mice weighing about 18-33gm were used. Animals were divided into eight groups.
The first group served as normal control group, the second group represented the dexamethasone group, the third group represented the nano curcumin group, the fourth group represented the bleomycin group, the fifth group represented the bleomycin treated dexamethasone group, the sixth group represented the bleomycin treated dexamethasone nano-chitosan particle, the seventh group represented the bleomycin and nano curcumin group , the eighth group represented the bleomycin treated with both dexamethasone and nano curcumin .Eight animals from each group were dissected after 14 days and 28 days.
The present study elucidated a decrease in the body weight of C57BL/6 mice after intratracheal BLM instillation for 28 consecutive days. This was accompanied with a significant (p < 0.05) elevation in lung weight and lung index in this group either after14 or 28 days of experimental study. The treatment with DEX alone or DEX loaded on NCH particles or under DEX and Nano CURC treatments produced a significant decline (p< 0.05) in lung weight, lung indices either after 14 or 28 days .
This study confirmed that BLM-induced lung ﬁbrosis rat model displayed a significant increase in MDA levels when compared with normal control group, also an increase in serum LDH activity. LDH levels were increased by 3.6 folds at 14 days post-BLM instillation, which is known as the inflammatory phase, and decreased to 2.4 fold increase at 28 days which is regarded as the fibrotic phase when compared to NC groups.
After either 14 or 28 of the current experimental study period to BLM treated groups with DEX alone or DEX loaded on NHC particles or under DEX and Nano CURC treatments, there was a significant decline (p< 0.05) in serum LDH and MDA.
The present results elucidate the elevations in lung tissue hydroxyproline and collagen 1 α content with subsequent significant elevations of TNF-α, TGF-β, INF-γ, NFkB and MMP2 lung tissue after 14 days of intratreacheal BLM instillation. The increase in these lung parameters progressed until 28 days of experimental study period in BLM group when compared to NC group. After either 14 or 28 of the current experimental study period to BLM treated groups with DEX alone or DEX loaded on NHC particles or under DEX and Nano CURC treatments, there was a significant decline (p< 0.05) in lung tissue hydroxyproline and collagen1α. This was also accompanied by a decrease in the inflammatory mediators including TNFα, TGF-β1, INF-γ, NF κB and lung tissue inflammatory marker MMP2.
The results of the present study validated the significant increase of total leucocytes thus increased inflammatory cell count in BAL fluid of neutrophils lymphocytes and macrophages after both 14 and 28 days of BLM induction. In addition, the microscopical investigation and the total leucocytic count and the differential counts of macrophage, lymphocytes and neutrophils in BAL fluid revealed likewise a significant decrease in the same mentioned group, After either 14 or 28 of the current experimental study period to BLM treated groups with DEX alone or DEX loaded on NHC particles or under DEX and Nano CURC treatments.
The current study revealed that BLM instillation was associated with a significant increase in Caspase-3 gene expression and Mucin (Muc 5ac) in lung tissue homogenates. This was accompanied by a profound decrease in BCL-2 gene expression in lung tissue after 14 and 28 days of study in BLM group when compared to the NC group. These results were along with a decrease in the gene expression of Caspase-3 and MUC5ac genes and a significant increase in BCL2 gene in lung tissue in the above mentioned groups when compared to BLM group, After either 14 or 28 of the current experimental study period to BLM treated groups with DEX alone or DEX loaded on NHC particles or under DEX and Nano CURC treatments.
Additionally, the histological study and the grades of lung fibrosis of lung tissue sections stained by H&E and Masson Trichrome stain aimed to support the biochemical investigations.
Furthermore, the histological examination of lung sections of BLM group of the present study revealed various changes such as many collapsed alveoli, while other alveoli were dilated and ruptured. Some bronchiole was lined by epithelial cells with deeply stained nuclei and its lumen was full of exfoliated epithelial cells. Also, heavy mononuclear cellular infiltration surrounding the bronchioles and in the interalveolar septa were present. The dilated and congested blood vessels and interstitial hemorrhage in the alveolar spaces were seen. Moreover, a significant increase in the amount of collagen and elastic fibers around bronchiole and the walls of the alveoli. The results of this study revealed that treatment by dexamethasone alone or dexamethasone loaded on Nano curcumin or dexamethasone accompanied with Nanocurcumin attenuated pulmonary fibrosis with varying extents.
However, loading dexamethasone onto Nanochitosan revealed the superlative ameliorated results in all experimented parameters of pulmonary fibrosis mice model.
In conclusion, this study confirmed that Nano chitosan is an effective drug carrier for dexamethasone that can provide drug delivery and optimal drug release to the lungs with minimal cellular side effects. Moreover, the use of the improved form of curcumin which is Nanao curcumin combined with dexamethasone is an effective treatment for PF used to overcome the disadvantages of the use of dexamethasone alone in bleomycin induced lung inflammation mice model.