Breast Cancer Immune Landscape: Interplay Between Systemic and Local Immunity.

Breast cancer (BC) is one of the most common malignancies in women worldwide. Numerous studies in immuno-oncology and successful trials of immunotherapy have demonstrated the causal role of the immune system in cancer pathogenesis. The interaction between the tumor and the immune system is known to have a dual nature. Despite cytotoxic lymphocyte activity against transformed cells, a tumor can escape immune surveillance and leverage chronic inflammation to maintain its own development. Research on antitumor immunity primarily focuses on the role of the tumor microenvironment, whereas the systemic immune response beyond the tumor site is described less thoroughly. Here, a comprehensive review of the formation of the immune profile in breast cancer patients is offered. The interplay between systemic and local immune reactions as self-sustaining mechanism of tumor progression is described and the functional activity of the main cell populations related to innate and adaptive immunity is discussed. Additionally, the interaction between different functional levels of the immune system and their contribution to the development of the pro- or anti-tumor immune response in BC is highlighted. The presented data can potentially inform the development of new immunotherapy strategies in the treatment of patients with BC.

Swelling, Rupture and Endosomal Escape of Biological Nanoparticles Per Se and Those Fused with Liposomes in Acidic Environment.

Biological nanoparticles (NPs), such as extracellular vesicles (EVs), exosome-mimetic nanovesicles (EMNVs) and nanoghosts (NGs), are perspective non-viral delivery vehicles for all types of therapeutic cargo. Biological NPs are renowned for their exceptional biocompatibility and safety, alongside their ease of functionalization, but a significant challenge arises when attempting to load therapeutic payloads, such as nucleic acids (NAs). One effective strategy involves fusing biological NPs with liposomes loaded with NAs, resulting in hybrid carriers that offer the benefits of both biological NPs and the capacity for high cargo loads. Despite their unique parameters, one of the major issues of virtually any nanoformulation is the ability to escape degradation in the compartment of endosomes and lysosomes which determines the overall efficiency of nanotherapeutics. In this study, we fabricated all major types of biological and hybrid NPs and studied their response to the acidic environment observed in the endolysosomal compartment. In this study, we show that EMNVs display increased protonation and swelling relative to EVs and NGs in an acidic environment. Furthermore, the hybrid NPs exhibit an even greater response compared to EMNVs. Short-term incubation of EMNVs in acidic pH corresponding to late endosomes and lysosomes again induces protonation and swelling, whereas hybrid NPs are ruptured, resulting in the decline in their quantities. Our findings demonstrate that in an acidic environment, there is enhanced rupture and release of vesicular cargo observed in hybrid EMNVs that are fused with liposomes compared to EMNVs alone. This was confirmed through PAGE electrophoresis analysis of mCherry protein loaded into nanoparticles. In vitro analysis of NPs colocalization with lysosomes in HepG2 cells demonstrated that EMNVs mostly avoid the endolysosomal compartment, whereas hybrid NPs escape it over time. To conclude, (1) hybrid biological NPs fused with liposomes appear more efficient in the endolysosomal escape via the mechanism of proton sponge-associated scavenging of protons by NPs, influx of counterions and water, and rupture of endo/lysosomes, but (2) EMNVs are much more efficient than hybrid NPs in actually avoiding the endolysosomal compartment in human cells. These results reveal biochemical differences across four major types of biological and hybrid NPs and indicate that EMNVs are more efficient in escaping or avoiding the endolysosomal compartment.

The Intricate Balance between Life and Death: ROS, Cathepsins, and Their Interplay in Cell Death and Autophagy.

Cellular survival hinges on a delicate balance between accumulating damages and repair mechanisms. In this intricate equilibrium, oxidants, currently considered physiological molecules, can compromise vital cellular components, ultimately triggering cell death. On the other hand, cells possess countermeasures, such as autophagy, which degrades and recycles damaged molecules and organelles, restoring homeostasis. Lysosomes and their enzymatic arsenal, including cathepsins, play critical roles in this balance, influencing the cell's fate toward either apoptosis and other mechanisms of regulated cell death or autophagy. However, the interplay between reactive oxygen species (ROS) and cathepsins in these life-or-death pathways transcends a simple cause-and-effect relationship. These elements directly and indirectly influence each other's activities, creating a complex web of interactions. This review delves into the inner workings of regulated cell death and autophagy, highlighting the pivotal role of ROS and cathepsins in these pathways and their intricate interplay.

Prolonged and Controllable Release of Doxorubicin Hydrochloride from the Composite Electrospun Poly(ε-Caprolactone)/Polyvinylpyrrolidone Scaffolds.

Burst release, typical for the drug-loaded electrospun poly(ε-caprolactone) (PCL) scaffolds is unfavorable in case of cytostatics due to the toxic levels reached during the initial implantation period. In the present short communication, we report an unexpected ability of the composite scaffolds made of PCL and water-soluble polyvinylpyrrolidone (PVP) to provide long-term release of widely used anti-cancer drug doxorubicin hydrochloride (DOX-HCl). That effect was observed for electrospun DOX-HCl-loaded composite scaffolds based on PCL and PVP with various mass ratios (100/0, 95/5, 90/10, 75/25 and 50/50). After the morphology and water contact angle studies, it was concluded that PVP content has no effect on the average fiber diameter, while PVP content higher 10 wt. % changes the hydrophobic character of the scaffolds surface (water contact angle of 123.9 ± 3.5°) to superhydrophilic (water contact angle of 0°). Despite the dramatic change in water wettability, by high performance liquid chromatography (HPLC), it was revealed that the PVP content in the scaffolds reduces the DOX-HCl release rate under short (first hours) and long-term (during 1 month) exposure to phosphate buffer saline (PBS). These results are in good agreement with in vitro studies, in which the viability of HeLa cervical cancer cells was higher after 24 h of culture with scaffolds with high PVP content.

Expression, Intracellular Localization, and Maturation of Cysteine Cathepsins in Renal Embryonic and Cancer Cell Lines.

Cysteine cathepsins play an important role in tumor development and metastasis. The expression of these enzymes is often increased in many types of tumor cells. Cysteine cathepsins contribute to carcinogenesis through a number of mechanisms, including proteolysis of extracellular matrix and signaling molecules on the cell surface, as well as degradation of transcription factors and disruption of signaling cascades in the cell nucleus. Distinct oncogenic functions have been reported for several members of the cysteine cathepsin family in various types of cancer, but a comparative study of all eleven cysteine cathepsins in one experimental model is still missing. In this work, we assessed and compared the expression, localization, and maturation of all eleven cysteine cathepsins in embryonic kidney cells HEK293 and kidney cancer cell lines 769-P and A-498. We found that the expression of cathepsins V, B, Z, L, and S was 3- to 9-fold higher in kidney tumor cells than in embryonic cells. We also showed that all cysteine cathepsins were present in varying amounts in the nucleus of both embryonic and tumor cells. Notably, more than half of the cathepsin Z or K and over 88% of cathepsin F were localized in tumor cell nuclei. Moreover, mature forms of cysteine cathepsins were more prevalent in tumor cells than in embryonic cells. These results can be further used to develop novel diagnostic tools and may assist in the investigation of cysteine cathepsins as potential therapeutic targets.

Phase transition and potential biomedical applications of thermoresponsive compositions based on polysaccharides, proteins and DNA: A review.

Smart thermoresponsive polymers have long attracted attention as materials of a great potential for biomedical applications, mainly for drug delivery, tissue engineering and wound dressing, with a special interest to injectable hydrogels. Poly-N-isopropylacrylamide (PNIPAM) is the most important synthetic thermoresponsive polymer due to its physiologically relevant transition temperature. However, the use of unmodified PNIPAM encounters such problems as low biodegradability, low drug loading capacity, slow response to thermal stimuli, and insufficient mechanical robustness. The use of natural polysaccharides and proteins in combinations with PNIPAM, in the form of grafted copolymers, IPNs, microgels and physical mixtures, is aimed at overcoming these drawbacks and creating dual-functional materials with both synthetic and natural polymers' properties. When developing such compositions, special attention should be paid to preserving their key property, thermoresponsiveness. Addition of hydrophobic and hydrophilic fragments to PNIPAM is known to affect its transition temperature. This review covers various classes of natural polymers - polysaccharides, fibrous and non-fibrous proteins, DNA - used in combination with PNIPAM for the prospective biomedical purposes, with a focus on their phase transition temperatures and its relation to the natural polymer's structure.

Electrospun Poly-L-Lactic Acid Scaffolds Surface-Modified via Reactive Magnetron Sputtering Using Different Mixing Ratios of Nitrogen and Xenon.

Controlled regeneration processes involving tissue growth using the surface and structure of scaffolds, are actively used in tissue engineering. Reactive magnetron sputtering is a versatile surface modification method of both metal and polymer substrates, as the properties of the formed coatings can be modified in a wide range by changing the process parameters. In magnetron sputtering, the working gas and its composition have an influence on the chemical composition and physical characteristics of the obtained coatings. However, there are no studies addressing the influence of the nitrogen/xenon gas mixture ratio in direct current magnetron sputtering on the deposition rate, physicochemical and in vitro properties of surface-modified biocompatible poly-L-lactic acid scaffolds. In this study, the application of mixtures of nitrogen and xenon in various ratios is demonstrated to modify the surface of non-woven poly-L-lactic acid scaffolds by direct current magnetron sputtering of a titanium target. It has been found that the magnetron sputtering parameters chosen do not negatively influence the morphology of the prepared scaffolds, but increase the hydrophilicity. Moreover, quantitative spectroscopic analysis results indicate that the formed coatings are primarily composed of titanium oxide and titanium oxynitride compounds and is dependent on the gas mixture ratio only to a certain extent. Atomic force microscopy investigations of the roughness of the fibers of the electrospun scaffolds and the thickness of the coatings formed on them show that the considerable variations observed in the intrinsic fiber reliefs are due to the formation of a fine layer on the fiber surfaces. The observed decrease in roughness after plasma modification is due to temperature and radiation effects of the plasma. In vitro experiments with human osteosarcoma cells show that the scaffolds investigated here have no cytotoxic effect on these cells. The cells adhere and proliferate well on each of the surface-modified electrospun scaffolds, with stimulation of cell differentiation in the osteogenic direction.

New Perspectives on the Role of Nuclear Proteases in Cell Death Pathways.

Multiple factors can trigger cell death via various pathways, and nuclear proteases have emerged as essential regulators of these processes. While certain nuclear proteases have been extensively studied and their mechanisms of action are well understood, others remain poorly characterized. Regulation of nuclear protease activity is a promising therapeutic strategy that could selectively induce favorable cell death pathways in specific tissues or organs. Thus, by understanding the roles of newly discovered or predicted nuclear proteases in cell death processes, we can identify new pharmacological targets for improving therapeutic outcomes. In this article, we delved into the role of nuclear proteases in several types of cell death and explore potential avenues for future research and therapeutic development.

The cell softening as a universal indicator of cell damage during cytotoxic effects.

Cytotoxicity assays are essential tests in studies on the safety and biocompatibility of various substances and on the efficiency of anticancer drugs. The most frequently used assays commonly require application of externally added labels and read only collective response of cells. Recent studies show that the internal biophysical parameters of cells can be associated with the cellular damage. Therefore, using atomic force microscopy, we assessed the changes in the viscoelastic parameters of cells treated with eight different common cytotoxic agents to gain a more systematic view of the occurring mechanical changes. With the robust statistical analysis to account for both the cell-level variability and the experimental reproducibility, we have found that cell softening is a common response after each treatment. More precisely, the combined changes in the viscoelastic parameters of power-law rheology model led to a significant decrease of the apparent elastic modulus. The comparison with the morphological parameters (cytoskeleton and cell shape) demonstrated a higher sensitivity of the mechanical parameters versus the morphological ones. The obtained results support the idea of cell mechanics-based cytotoxicity tests and suggest a common way of a cell responding to damaging actions by softening.

Hydroxychloroquine Enhances Cytotoxic Properties of Extracellular Vesicles and Extracellular Vesicle-Mimetic Nanovesicles Loaded with Chemotherapeutics.

Because of their high biocompatibility, biological barrier negotiation, and functionalization properties, biological nanoparticles have been actively investigated for many medical applications. Biological nanoparticles, including natural extracellular vesicles (EVs) and synthetic extracellular vesicle-mimetic nanovesicles (EMNVs), represent novel drug delivery vehicles that can accommodate different payloads. In this study, we investigated the physical, biological, and delivery properties of EVs and EMNVs and analyzed their ability to deliver the chemotherapeutic drug doxorubicin. EMNVs and EVs exhibit similar properties, but EMNVs are more effectively internalized, while EVs show higher intracellular doxorubicin release activity. In addition, these nanotherapeutics were investigated in combination with the FDA-approved drug hydroxychloroquine (HCQ). We demonstrate that HCQ-induced lysosome destabilization and could significantly increase nanoparticle internalization, doxorubicin release, and cytotoxicity. Altogether, these data demonstrate that, from the delivery standpoint in vitro, the internalization of EMNVs and EVs and their payload release were slightly different and both nanotherapeutics had comparable cytotoxic performance. However, the synthesis of EMNVs was significantly faster and cost-effective. In addition, we highlight the benefits of combining biological nanoparticles with the lysosome-destabilizing agent HCQ that increased both the internalization and the cytotoxic properties of the particles.

Anticancer Nanotherapeutics in Clinical Trials: The Work behind Clinical Translation of Nanomedicine.

The ultimate goal of nanomedicine has always been the generation of translational technologies that can ameliorate current therapies. Cancer disease represented the primary target of nanotechnology applied to medicine, since its clinical management is characterized by very toxic therapeutics. In this effort, nanomedicine showed the potential to improve the targeting of different drugs by improving their pharmacokinetics properties and to provide the means to generate new concept of treatments based on physical treatments and biologics. In this review, we considered different platforms that reached the clinical trial investigation, providing an objective analysis about their physical and chemical properties and the working mechanism at the basis of their tumoritr opic properties. With this review, we aim to help other scientists in the field in conceiving their delivering platforms for clinical translation by providing solid examples of technologies that eventually were tested and sometimes approved for human therapy.

Monocyte programming by cancer therapy.

Monocytes in peripheral blood circulation are the precursor of essential cells that control tumor progression, that include tumor-associated macrophages (TAMs), dendritic cells (DCs) and myeloid-derive suppressor cells (MDSC). Monocytes-derived cells orchestrate immune reactions in tumor microenvironment that control disease outcome and efficiency of cancer therapy. Four major types of anti-cancer therapy, surgery, radiotherapy, chemotherapy, and most recent immunotherapy, affect tumor-associated macrophage (TAM) polarization and functions. TAMs can also decrease the efficiency of therapy in a tumor-specific way. Monocytes is a major source of TAMs, and are recruited to tumor mass from the blood circulation. However, the mechanisms of monocyte programming in circulation by different therapeutic onsets are only emerging. In our review, we present the state-of-the art about the effects of anti-cancer therapy on monocyte progenitors and their dedifferentiation, on the content of monocyte subpopulations and their transcriptional programs in the circulation, on their recruitment into tumor mass and their potential to give origin for TAMs in tumor-specific microenvironment. We have also summarized very limited available knowledge about genetics that can affect monocyte interaction with cancer therapy, and highlighted the perspectives for the therapeutic targeting of circulating monocytes in cancer patients. We summarized the knowledge about the mediators that affect monocytes fate in all four types of therapies, and we highlighted the perspectives for targeting monocytes to develop combined and minimally invasive anti-cancer therapeutic approaches.

Monitoring of hydrolysis products of organophosphorus nerve agents in plant material and soil by liquid chromatography-tandem mass spectrometry.

Nerve agents are organophosphorus compounds of the highest toxicity and danger. The production, transportation and use of these substances are prohibited by the Organization for the Prohibition of Chemical Weapons. Any fact of alleged use of nerve agents is a crime against humanity and must be investigated in detail by the world community. For this purpose, such analysis objects as biological fluids (urine, blood) and environmental objects (water, soil) are well studied. The current study demonstrates the possibility of using plants as a convenient material for retrospective analysis. Methyl phosphonic acid and some of its alkyl esters (ethyl, isopropyl, isobutyl, cyclohexyl, pinacolyl) were chosen as nerve agent metabolites. Hedera Helix growing in soil was used as a carrier of the markers. The selected markers were injected once in the soil and their content in the plant and soil was monitored for 4 weeks. A fast and simple way of sample homogenization with liquid nitrogen followed by ultrasonic liquid extraction was applied. The developed HPLC-MS/MS approach with the use of deuterated internal standards for quantitative analysis was validated. The research discovered that all the studied nerve agent markers could be detected and determined both in the soil and the plant for at least one month. The results indicate the promising use of plants as additional objects of analysis in investigation of incidents involving the use of chemical warfare agents.

Redox Regulation of Signaling Complex between Caveolin-1 and Neuronal Calcium Sensor Recoverin.

Caveolin-1 is a cholesterol-binding scaffold protein, which is localized in detergent-resistant membrane (DRM) rafts and interacts with components of signal transduction systems, including visual cascade. Among these components are neuronal calcium sensors (NCSs), some of which are redox-sensitive proteins that respond to calcium signals by modulating the activity of multiple intracellular targets. Here, we report that the formation of the caveolin-1 complex with recoverin, a photoreceptor NCS serving as the membrane-binding regulator of rhodopsin kinase (GRK1), is a redox-dependent process. Biochemical and biophysical in vitro experiments revealed a two-fold decreased affinity of recoverin to caveolin-1 mutant Y14E mimicking its oxidative stress-induced phosphorylation of the scaffold protein. At the same time, wild-type caveolin-1 demonstrated a 5-10-fold increased affinity to disulfide dimer of recoverin (dRec) or its thiol oxidation mimicking the C39D mutant. The formation of dRec in vitro was not affected by caveolin-1 but was significantly potentiated by zinc, the well-known mediator of redox homeostasis. In the MDCK cell model, oxidative stress indeed triggered Y14 phosphorylation of caveolin-1 and disulfide dimerization of recoverin. Notably, oxidative conditions promoted the accumulation of phosphorylated caveolin-1 in the plasma membrane and the recruitment of recoverin to the same sites. Co-localization of these proteins was preserved upon depletion of intracellular calcium, i.e., under conditions reducing membrane affinity of recoverin but favoring its interaction with caveolin-1. Taken together, these data suggest redox regulation of the signaling complex between recoverin and caveolin-1. During oxidative stress, the high-affinity interaction of thiol-oxidized recoverin with caveolin-1/DRMs may disturb the light-induced translocation of the former within photoreceptors and affect rhodopsin desensitization.

DNA Copy Number Aberrations and Expression of ABC Transporter Genes in Breast Tumour: Correlation with the Effect of Neoadjuvant Chemotherapy and Prognosis of the Disease.

One of the important reasons for the ineffectiveness of chemotherapy in breast cancer (BC) is considered to be the formation of a multidrug resistance phenotype in tumour cells, which is caused by the expression of energy-dependent ABC transporters. The aim of this work was to assess chromosomal aberrations and the level of transcripts of all 49 known ABC transporter genes in breast tumours. MATERIALS AND METHODS: The study included 129 patients with breast cancer. A microarray study of all tumour samples was carried out on microchips. RESULTS: This study established that the presence of a deletion in genes ABCB1, ABCB4, ABCB8, ABCC7, ABCC11, ABCC12, ABCF2, and ABCG4 is associated with an objective response to treatment (p ≤ 0.05). A decrease in the expression of genes was associated with a good response to chemotherapy, whereas an increase in expression caused the progression and stabilization of the tumour. Analysis of metastatic-free survival rates showed that the presence of ABCB1/4 and ABCC1/6 deletions was associated with 100% survival (log-rank test p = 0.01 and p = 0.03). CONCLUSIONS: The study showed that the aberrant state of ABC transporter genes, as well as a decrease in the expression of these genes, is a predictor of the effectiveness of therapeutic treatment and a potential prognostic marker of metastatic survival.

In Silico, In Vitro, and Clinical Investigations of Cathepsin B and Stefin A mRNA Expression and a Correlation Analysis in Kidney Cancer.

The cysteine protease Cathepsin B (CtsB) plays a critical role in multiple signaling pathways, intracellular protein degradation, and processing. Endogenous inhibitors regulate its enzymatic activity, including stefins and other cystatins. Recent data proved that CtsB is implicated in tumor extracellular matrix remodeling, cell invasion, and metastasis: a misbalance between cathepsins and their natural inhibitors is often considered a sign of disease progression. In the present study, we investigated CtsB and stefin A (StfA) expression in renal cell carcinoma (RCC). mRNA analysis unveiled a significant CTSB and STFA increase in RCC tissues compared to adjacent non-cancerogenic tissues and a higher CtsB expression in malignant tumors than in benign renal neoplasms. Further analysis highlighted a positive correlation between CtsB and StfA expression as a function of patient sex, age, tumor size, grade, lymph node invasion, metastasis occurrence, and survival. Alternative overexpression and silencing of CtsB and StfA confirmed the correlation expression between these proteins in human RCC-derived cells through protein analysis and fluorescent microscopy. Finally, the ectopic expression of CtsB and StfA increased RCC cell proliferation. Our data strongly indicated that CtsB and StfA expression play an important role in RCC development by mutually stimulating their expression in RCC progression.

Biomimetic approaches for targeting tumor-promoting inflammation.

With the ultimate goal of increasing tumor accumulation of therapeutics, various nanocarriers have been designed to overcome biological barriers encountered at each stage, from drug administration to the cancerous lesion. Stabilizing circulation and functionalization of the targeting surface impart high tumor accumulation properties to nanocarriers. However, various cells can recognize and infiltrate the tumor microenvironment more efficiently than synthetic carriers via overexpression of adhesive ligands, particularly in inflamed stroma of tumors. Thus, a new field of nanomedicine, called biomimicry, has evolved to generate nanoparticles with the same biological characteristics as cells that naturally infiltrate tumors. Revolutionary synthetic processes have been developed to transfer the cell membrane of leukocytes and mesenchymal cells to synthetic carriers. In addition, cells can generate their own "nanocarriers," known as exosomes, to transport molecular messages to distant sites, while biomimicry of viral and bacterial agents allows high targeting efficiency towards inflammatory immune cells. Alterations in the protein expression in cancer cells caused by inflammation can also be exploited for drug delivery. Finally, new developments in biomimetic drug delivery focus on turning the infiltrating cells into microcarriers that can actively perfuse the tumor and eventually release their therapeutic payload. In this review, we summarize recent developments in biomimetic drug delivery with a particular focus on targeting the tumor inflammatory microenvironment.

Predictive and Prognostic Significance of mRNA Expression and DNA Copies Aberrations of ERCC1, RRM1, TOP1, TOP2A, TUBB3, TYMS, and GSTP1 Genes in Patients with Breast Cancer.

Increasingly, many researchers are focusing on the sensitivity in breast tumors (BC) to certain chemotherapy drugs and have personalized their research based on the assessment of this sensitivity. One such personalized approach is to assess the chemotherapy's gene expression, as well as aberrations in the number of DNA copies-deletions and amplifications with the ability to have a significant effect on the gene's activity. Thus, the aim of this work was to study the predictive and prognostic significance of the expression and chromosomal aberrations of eight chemosensitivity genes in breast cancer patients. MATERIAL AND METHODS: The study involved 97 patients with luminal B breast cancer IIB-IIIB stages. DNA and RNA were isolated from samples of tumor tissue before and after treatment. Microarray analysis was performed for all samples on high-density microarrays (DNA chips) of Affymetrix (USA) CytoScanTM HD Array and Clariom™ S Assay, human. Detection of expression level of seven chemosensitivity genes-RRM1, ERCC1, TOP1, TOP2a, TUBB3, TYMS, and GSTP1-was performed using PCR real-time (RT-qPCR). RESULTS: The expression of the RRM1 (AC scheme), TOP2α, TYMS, and TUBB3 genes in patients with an objective response to treatment (complete and partial regression) is higher than in patients with stabilization and progression (p < 0.05). According to our results, the presence of a high level of GSTP1 in a tumor biopsy is associated with the low efficiency of the NAC CP scheme (p = 0.05). The presence of RRM1 deletion is associated with complete and partial regression, as for the TOP1 and TUBB3 genes (p < 0.05). Higher rates of metastatic survival are associated with a high level of expression and amplification of the GSTP1 gene (log-rank test p = 0.02 and p = 0.05). CONCLUSION: Thus, a complex assessment of the chemotherapy's gene expression is important not only for understanding the heterogeneity and molecular biology of breast cancer but also to obtain a more accurate disease prognosis.

A Collagen Basketweave from the Giant Squid Mantle as a Robust Scaffold for Tissue Engineering.

The growing applications of tissue engineering technologies warrant the search and development of biocompatible materials with an appropriate strength and elastic moduli. Here, we have extensively studied a collagenous membrane (GSCM) separated from the mantle of the Giant squid Dosidicus Gigas in order to test its potential applicability in regenerative medicine. To establish the composition and structure of the studied material, we analyzed the GSCM by a variety of techniques, including amino acid analysis, SDS-PAGE, and FTIR. It has been shown that collagen is a main component of the GSCM. The morphology study by different microscopic techniques from nano- to microscale revealed a peculiar packing of collagen fibers forming laminae oriented at 60-90 degrees in respect to each other, which, in turn, formed layers with the thickness of several microns (a basketweave motif). The macro- and micromechanical studies showed high values of the Young's modulus and tensile strength. No significant cytotoxicity of the studied material was found by the cytotoxicity assay. Thus, the GSCM consists of a reinforced collagen network, has high mechanical characteristics, and is non-toxic, which makes it a good candidate for the creation of a scaffold material for tissue engineering.

Thin Thermoresponsive Polymer Films for Cell Culture: Elucidating an Unexpected Thermal Phase Behavior by Atomic Force Microscopy.

Application of poly-N-isopropylacrylamide (PNIPAM) and its more hydrophobic copolymers with N-tert-butylacrylamide (NtBA) as supports for cell sheets has been validated in numerous studies. The binary systems of these polymers with water are characterized by a lower critical solution temperature (LCST) in a physiologically favorable region. Upon lowering the temperature below the LCST, PNIPAM chains undergo a globule-to-coil transition, causing the film dissolution and cell sheet detachment. The character of the PNIPAM-water miscibility behavior is rather complex and not completely understood. Here, we applied atomic force microscopy to track the phase transition in thin films of linear thermoresponsive (co)polymers (PNIPAM and PNIPAM-co-NtBA) prepared by spin-coating. We studied the films' Young's modulus, roughness, and thickness in air and in distilled water in a full thermal cycle. In dry films, in the absence of water, all the measured parameters remained invariant. The swollen films in water above the LCST were softer by 2-3 orders of magnitude and about 10 times rougher than the corresponding dry films. Upon lowering the temperature to the LCST, the films passed through the phase transition observed as a drastic drop of Young's modulus (about an order of magnitude) and decrease in roughness in both polymers in a narrow temperature range. However, the films did not lose their integrity and demonstrated almost fully reversible changes in the mechanical properties and roughness. The thermal dependence of the films' thickness confirmed that they dissolved only partially and required an external force to induce the complete destruction. The reversible thermal behavior which is generally not expected from non-cross-linked polymers is a key finding, especially with respect to their practical application in cell culture. Both the thermodynamic and kinetic factors, as well as the confinement effect, may be responsible for this peculiar film robustness, which requires overcooling and the aid of an external force to destroy the film.