Doxorubicin-loaded bacterial ghosts of <i>Streptococcus pneumoniae and Klebsiella pneumoniae</i> : An In-vitro in-silico lung cancer cells study

Lung cancer is the leading cause of cancer-related death in numerous countries worldwide. Given their severe side effects that can impact the patients’ quality of life, the current treatment approaches need more specific targeting to reduce chemotherapeutic toxicities. Cell-based targeted delivery has recently gained momentum as a promising platform for the delivery of anticancer drugs. As a new biotechnology platform, bacterial ghosts (BGs) have novel biomedical applications, including targeted drug delivery systems. In the current work, the BG of
Streptococcus pneumoniae
and
Klebsiella pneumoniae
ghosts were used as potential targeted-delivery agents for treating lung cancer cells.


Bacterial ghosts of
S. pneumoniae
and
K. pneumoniae
were prepared using the most recent protocol (tween/acid). The doxorubicin (DOX) uploading was optimized by applying the required conditions to maximize loading capacity. We evaluated the DOX-loaded BGs’ antiproliferative and apoptotic effects on two lung cancer cell lines (H69A, A549) and one normal human embryonic kidney cell line (HEK293) in comparison to free BGs and DOX alone. We also tested the sustained release patterns for each formula. We predicted the mechanism of augmenting the actions of both systems by a protein-protein docking study.


The preparation of well-formulated ghosts of both bacteria was successful and confirmed. The loading capacity was maximized to 31.2 ± 3.1% (n=3) for
S. pneumoniae
ghosts (SPG) and 28.8± 2.6% (n=3) for
K. pneumoniae
ghosts (KPG). The sustained release pattern of both SPG and KPG followed the Higuchi kinetic model, while Free DOX exhibited a rapid release pattern consistent with the first-order kinetic model. The antiproliferative activity using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay revealed the superiority of SPG loaded with DOX (SPG-DOX) over all other tested formulae (10.8% viability) on multi-drug-resistant lung cancer cells (H69A). The IC50s of SPG-Dox loaded were one-sixth and one-fifth of the IC50 of the Free DOX in H69A and A549, respectively, indicating more potency. The cell viability was 46.6% upon using Free DOX. In non-small cell lung cancer cells (A549), the SPG-DOX formula also showed superior antiproliferative activity (23.1% viability), as compared to control vehicles. SPG and KPG showed the highest safety toward the normal cell lines (HEK293), with more than 80% of cells surviving. Using H69A cells, KPG-DOX showed superior apoptotic activity (58.2%) over Free DOX, which achieved less than 20% cellular apoptosis. In A549 cells, SPG-DOX showed apoptotic activity of 57.7% compared to Free DOX. (32.5%). The docking study showed a strong binding interaction between a KPG surface protein (4HWM) and the overexpressed non-small cell lung cancer carcinoma receptor, epidermal growth factor receptor kinase domain (EGFR 2ITO), with a binding score (S) for the interaction of -63.94 kcal/mol. The interaction was -61.26 of SPG surface protein (3ZPP) with EGFR. The application of
S. pneumoniae
and
K. pneumoniae
BGs loaded with DOX represents a promising targeted drug delivery system for the treatment of lung cancer. The superior potency and safety of both systems warrant further investigation into their preclinical and clinical activities.