Cellular Interactions with Photo-Cross-Linked and pH-Sensitive Polymersomes: Biocompatibility and Uptake Studies

Polymeric nanoparticles, specifically polymersomes, are at the leading edge of the rapidly developing field of nanotechnology. However, their use for biological applications is primarily limited by the biocompatibility of the components. Hence, optimization of polymersome synthesis protocols should carefully consider aspects of cellular toxicity. In this work, we investigate the viability of HDF and HeLa cells treated with photo-cross-linked and pH-sensitive polymersomes. We demonstrate how aspects of polymersome preparation conditions such as cross-linking density and UV irradiation time may affect their cytotoxic properties. Additionally, we also study the cellular uptake of our polymersomes into the cell types mentioned

Cell Instructive Microporous Scaffolds through Interface Engineering

The design of novel biomaterials for regenerative medicine requires incorporation of well-defined physical and chemical properties that mimic the native extracellular matrix (ECM). Here, we report the synthesis and characterization of porous foams prepared by high internal phase emulsion (HIPE) templating using amphiphilic copolymers that act as surfactants during the HIPE process. We combine different copolymers exploiting oil–water interface confined phase separation to engineer the surface topology of foam pores with nanoscopic domains of cell inert and active chemistries mimicking native matrix. We further demonstrate how proteins and hMSCs adhere in a domain specific manner

Cell Instructive Microporous Scaffolds through Interface Engineering

The design of novel biomaterials for regenerative medicine requires incorporation of well-defined physical and chemical properties that mimic the native extracellular matrix (ECM). Here, we report the synthesis and characterization of porous foams prepared by high internal phase emulsion (HIPE) templating using amphiphilic copolymers that act as surfactants during the HIPE process. We combine different copolymers exploiting oil–water interface confined phase separation to engineer the surface topology of foam pores with nanoscopic domains of cell inert and active chemistries mimicking native matrix. We further demonstrate how proteins and hMSCs adhere in a domain specific manner

The role of the two splice variants and extranuclear pathway on Ki-67 regulation in non-cancer and cancer cells

<div><p>Ki-67 is a nuclear protein that has been used in cancer diagnostic because of its specific cell-cycle dependent expression profile. After quantifying and characterising the expression level of Ki-67, as a function of the cell cycle, we found out that the two main splice variants of the protein (<i>i</i>.<i>e</i>. α and β) are differently regulated in non-cancerous and cancerous cells both at mRNA and protein level. We were able to correlate the presence of the α variant of the protein with the progression through the interphase of cell cycle. We also observed that the different expression profiles correspond to different degradation pathways for non-cancerous and cancerous cells. Furthermore, Ki-67 is continuously regulated and degraded via proteasome system in both cell types, suggesting an active control of the protein. However we also observed a putative extranuclear elimination pathway of Ki-67 where it is transported to the Golgi apparatus. Our evidence in the different expression of the splice variants may represent a milestone for the development of new targets for cancer diagnostic and prognostic. Additionally, the unexpected extranuclear elimination of Ki-67 strongly suggests that this protein must be looked at also outside of the “nuclear box”, as thought to date.</p></div

3D Surface Functionalization of Emulsion-Templated Polymeric Foams

We describe the preparation of porous polymeric scaffolds via polymerization of the oil phase in high internal phase water-in-oil-emulsions using amphiphilic block copolymers polystyrene-<i>b</i>-poly­(ethylene oxide), polystyrene-<i>b</i>-poly­(acrylic acid), poly­(1,4-butadiene)-<i>b</i>-poly­(ethylene oxide), and poly­(1,4-butadiene)-<i>b</i>-poly­(acrylic acid) as surfactants. We show that the block copolymers anchor to the polymerized oil phase via the lipophilic block, which can occur by chemical and/or physical entanglement and consequent presentation of the hydrophilic block on the pore surfaces. The <i>in situ</i> polymerization enables the full surface functionalization of the porous materials with the final surface chemistry dictated by the hydrophilic block. Furthermore, the foam physical architecture may be tailored through controlling emulsion parameters such as the initiator, shear rate, and aqueous phase volume fraction

Proposed molecular regulation and extranuclear pathway of Ki-67.

<p>Scheme representing the proposed Ki-67 regulation in non-cancerous (HDF) and cancerous cells (MDA-MB-231) before and after serum deprivation. In non-cancerous cells (left column), prolonged serum deprivation produces the down-expression of both the α and β splice variants of Ki-67. Furthermore, the already expressed Ki-67 is degraded <i>via</i> the proteasome. On the other hand, cancerous cells (right column), subjected at the same starving conditions, show the continuous expression of the α splice variant of the protein. Moreover, the level of the detected Ki-67 in cancerous cells is not affected overtime by the degradative action of the proteasome. The “feedback elimination mechanism” of Ki-67 degradation, which is related to the ER-Golgi secretory machinery, is schematised in the box at the bottom of the figure. In this proposed mechanism, Ki-67 is initially transferred to the ER, where it colocalises with BiP <b>(1)</b>. Subsequently, Ki-67 buds from the ER into specific COPII coated vesicles <b>(2)</b>, which transport their cargo to the Golgi apparatus <b>(3)</b> where Ki-67 may be further recycled and/or degraded. Non-cancer and cancer cells translocate Ki-67 with the same mechanism. However the obtained data indicate that cancerous cells could be characterised by a defective ER-Golgi secretory machinery. <b>(4)</b> Moreover, the Ki-67 translocation in cancerous cells could be unbalanced by the non down-regulation of the α variant of the protein.</p

Ki-67 and proteasome interaction.

<p>Confocal assays and quantification graphs (on the bottom of the picture) of the distribution and interaction between Ki-67 and the proteasome system, in both HDF (left column) and MDA-MB-231 cells (right column). The analyses were carried out with and without the presence of FBS. (t-test, p-value * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001).</p

Ki-67 mRNA quantification.

<p>RT-PCR (left column), and RT-qPCR (right column) quantification of the α and β splice variants of Ki-67, as a function of nutrient starvation in HDF and MDA-MB-231 cells. Bottom figure: (left) RT-PCR analysis of β-actin (A) and GAPDH (B) as internal controls, before and after nutrient deprivation. (Right) RT-qPCR quantification of the internal control GAPDH after serum starvation. (t-test, p-value * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001).</p

Ki-67 gene knock-down experiment.

<p><b>(A)</b> Confocal analysis of Ki-67 expression in HDF and MDA-MB-231 cells after shRNA knock-down. Bottom part: graph showing the Ki-67 protein quantification. <b>(B)</b> RT-qPCR quantification of the α and β splice variants of Ki-67, and GAPDH (G), after virus knock-down. The green graph represents the internal control GAPDH. For both cell models the relative quantification of GAPDH internal control (Ctrl) during Ki-67 gene knockdown experiments is also reported. (t-test, p-value * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001). Scale bar: 15 μm.</p

Ki-67 protein quantification.

<p>Simple Western^™, Standard Western, and quantification profile of Ki-67 in <b>(A)</b> HDF and <b>(B)</b> MDA-MB-231 breast cancer cells, growing in both media supplemented (+FBS) and lacking of FBS (-FBS). In the figure it is also reported the Simple Western^™ quantification profile of the internal control ERK 1/2 in HDF and MDA-MB-231 breast cancer cells, before and after FBS deprivation. (t-test, p-value * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001).</p