Autophagic blockage by metformin-loaded PLGA nanoparticles causes cell cycle arrest of HepG2 cells - supplementary figure

Aim: To fabricate and characterize metformin-loaded PLGA nanoparticles and investigate their inhibitory effect on HepG2 cells. Materials & methods: The nanoparticles were prepared using a double emulsification method, then characterized and subjected to a series of in vitro assays on HepG2 cells. Results: The nanoparticles were ∼277.9 nm in size, and the entrapment efficiency and drug loading of metformin were 31.3 and 14.4%, respectively. In vitro studies suggested that the nanoparticles showed a higher inhibitory effect on HepG2 cells compared with metformin alone, mainly attributed to its blockage of autophagy, and ultimately result in cell cycle inhibition. Conclusion: The metformin-loaded PLGA nanoparticles could inhibit mTOR activity, increase p53 levels and decrease HIF1A levels, which ultimately caused HepG2 cell cycle arrest.Plain language summary: Metformin, a well-known drug for the treatment of diabetes, has potential anticancer effects. Our experiment is fabricating metformin into nanoformulations (tiny particles) to enhance its anticancer effect. Cancer cells respond to nutrient-deficient environments by autophagy, this involves breaking down internal structures to scavenge for nutrients, which is one of the reasons why cancer cells are so resilient. If we can interfere with this autophagy of cancer cells, we can reduce the viability of cancer cells. Speaking of autophagy,we have tomention lysosomes, which are acidic organelles within the cell that are the end point of autophagy. Lysosomes need to maintain an acidic environment to ensure the activity of various enzymes within them. These enzymes break down a variety of biological components into ‘building blocks’ which can be used to make other structures. Our study found that the nanoformulation disrupts the lysosomal acidic environment and thus causes autophagy blockage. As a result, cancer cells are unable to cope with nutrient deficiencies through autophagy and suffer the negative effects of autophagy blockage, such as the inability to degrade damaged organelles and proteins within the cancer cell. This causes the growth and proliferation of cancer cells to slow down and results in the death of the cancer cells.</p

Antifibrogenic effect of cell transplantation measured by immunohistology and qRT-PCR analyses.

<p>(A) Immunohistochemical staining of collagen type I (Col I) and α-smooth muscle actin (α-SMA) before (CCl₄ 6w) and after (CCl₄ 10w) cell transplantation (Scale bars: 100 µm). (B) Quantitative RT-PCR analysis of fibrosis related markers α₂-procollagen, fibronectin, α-SMA, TGF-β,PDGF-B, PDGFR, MMP-2 and TIMP-2. Each sample was repeated three times with three samples from each group. *p<0.05; n.s: p>0.05.</p

Protective effect of cell transplantation in liver function.

<p>Blood samples of normal and experimental rats were collected and serum levels of albumin (ALB), glutamic oxalacetic transaminase (AST) and glutamic pyruvic transaminase (ALT) were tested before (CCl₄ 6w) and after (CCl₄ 10w) cell transplantation. (n = 6/group; *p<0.05).</p

Increased sinusoidal blood vessel density by transplanted cells.

<p>(A) Representative views of immunohistological staining of CD31 in HD group, RD group and PBS group (Scale bars: 150 µm). Number of CD31 positive vessels counted from immunohistological staining. Five views in each sample with three samples in each group were analyzed. (B) Expression of Ang-1, Tie-2, VEGF-A and VEGFR-2 analyzed by qRT-PCR. Each sample was repeated three times with three samples from each group. *p<0.05.</p

Promotion of liver regeneration by transplanted cells.

<p>(A) The liver weights and liver/body weight ratios at 4 weeks post-treatment. (B) Representative views of immunohistological staining of Ki-67 in HD group, RD group and PBS group (Scale bars: 150 µm). Percentage of Ki-67 positive cells calculated from immunohistological staining. Five views from each sample with three samples in each group were analyzed. (C) Expression of hepatocyte growth factor (HGF) analyzed by qRT-PCR. Each sample was repeated three times with three samples from each group. *p<0.05.</p

Antifibrogenic effect of cell transplantation in liver fibrosis.

<p>(A) Representative structural changes in livers were detected by hematoxylin & eosin (HE), picric acid-sirius red (PSR) and Masson staining (Scale bars: 100 µm). (B) Quantitative analyses of liver fibrosis were performed from PSR and Masson staining. Five views from each sample with three samples in each group were analyzed. *p<0.05.</p

Primers used in qRT-PCR analyses.

<p>Primers used in qRT-PCR analyses.</p

Endothelial progenitor cells enriched in HD cultured bone marrow cells.

<p>(A) Morphology of rat bone marrow cells in HD and RD culture. (Scale bars:100 µm). (B) Flow cytometry analyses of HD and RD cultured cells after 15 days of expansion. (C) Tube formation ability of RD and RD cultured cells on matrigel. (Scale bars:200 µm).</p

Biomechanical properties of repaired cornea postoperatively at 6 months (n = 6 in each group).

<p>*<i>P</i> < 0.05 compared with normal cornea.</p

Expansion of Endothelial Progenitor Cells in High Density Dot Culture of Rat Bone Marrow Cells

<div><p>In vitro expansion of endothelial progenitor cells (EPCs) remains a challenge in stem cell research and its application. We hypothesize that high density culture is able to expand EPCs from bone marrow by mimicking cell-cell interactions of the bone marrow niche. To test the hypothesis, rat bone marrow cells were either cultured in high density (2×10⁵ cells/cm²) by seeding total 9×10⁵ cells into six high density dots or cultured in regular density (1.6×10⁴ cells/cm²) with the same total number of cells. Flow cytometric analyses of the cells cultured for 15 days showed that high density cells exhibited smaller cell size and higher levels of marker expression related to EPCs when compared to regular density cultured cells. Functionally, these cells exhibited strong angiogenic potentials with better tubal formation in vitro and potent rescue of mouse ischemic limbs in vivo with their integration into neo-capillary structure. Global gene chip and ELISA analyses revealed up-regulated gene expression of adhesion molecules and enhanced protein release of pro-angiogenic growth factors in high density cultured cells. In summary, high density cell culture promotes expansion of bone marrow contained EPCs that are able to enhance tissue angiogenesis via paracrine growth factors and direct differentiation into endothelial cells.</p></div