MMPs-responsive silk spheres for controlled drug release within tumor microenvironment.

Silk is a biocompatible and biodegradable material that enables the formation of various morphological forms, including nanospheres. The functionalization of bioengineered silk makes it possible to produce particles with specific properties. In addition to tumor cells, the tumor microenvironment (TME) includes stromal, immune, endothelial cells, signaling molecules, and the extracellular matrix (ECM). Matrix metalloproteinases (MMPs) are overexpressed in TME. We investigated bioengineered spider silks functionalized with MMP-responsive peptides to obtain targeted drug release from spheres within TME. Soluble silks MS12.2MS1, MS12.9MS1, and MS22.9MS2 and the corresponding silk spheres carrying MMP-2 or MMP-2/9 responsive peptides were produced, loaded with doxorubicin (Dox), and analyzed for their susceptibility to MMP-2/9 digestion. Although all variants of functionalized silks and spheres were specifically degraded by MMP-2/9, the MS22.9MS2 nanospheres showed the highest levels of degradation and release of Dox after enzyme treatment. Moreover, functionalized spheres were degraded in the presence of cancer cells releasing MMP-2/9. In the 2D and 3D spheroid cancer models, the MMP-2/9-responsive substrate was degraded and released from spheres when loaded into MS22.9MS2 particles but not into the control MS2 spheres. The present study demonstrated that a silk-based MMP-responsive delivery system could be used for controlled drug release within the tumor microenvironment.

Antioxidant Enzymes in Cancer Cells: Their Role in Photodynamic Therapy Resistance and Potential as Targets for Improved Treatment Outcomes.

Photodynamic therapy (PDT) is a selective tumor treatment that consists of a photosensitive compound-a photosensitizer (PS), oxygen, and visible light. Although each component has no cytotoxic properties, their simultaneous use initiates photodynamic reactions (PDRs) and sequentially generates reactive oxygen species (ROS) and/or free radicals as cytotoxic mediators, leading to PDT-induced cell death. Nevertheless, tumor cells develop various cytoprotective mechanisms against PDT, particularly the adaptive mechanism of antioxidant status. This review integrates an in-depth analysis of the cytoprotective mechanism of detoxifying ROS enzymes that interfere with PDT-induced cell death, including superoxide dismutase (SOD), catalase, glutathione redox cycle, and heme oxygenase-1 (HO-1). Furthermore, this review includes the use of antioxidant enzymes inhibitors as a strategy in order to diminish the antioxidant activities of tumor cells and to improve the effectiveness of PDT. Conclusively, PDT is an effective tumor treatment of which its effectiveness can be improved when combined with a specific antioxidant inhibitor.

Endotoxin reduction from biotech silk material inhibits the production of anti-silk antibodies in mice.

Eliminating endotoxins is a common problem in the development of biotechnologically produced pharmaceuticals or biomaterials. Residual endotoxins in the final sample may hamper the properties of the product or induce severe adverse effects. Developing an effective downstream purification protocol that ensures a lack of minimal endotoxin content in the final product can be a challenging task. In our previous studies, we developed nanospheres produced from bioengineered silks. Despite their good overall biocompatibility, in vivo characterization of spheres showed mild activation of the immune system (mainly in terms of anti-silk antibody production). Herein, we examined, if the endotoxins delivered with the silk spheres might have contributed to activating the adaptive immune response. We investigated various commercially available methods for endotoxin removal that can be applied as an extra step in downstream endotoxin removal from MS1-type silk proteins. We selected a method that allowed for a 10-fold reduction of endotoxin content in soluble silk and 2-fold in the final product (silk spheres). The reduced level of endotoxins improved the biocompatibility of the silk spheres as these particles induced negligible titers of anti-silk antibodies in an in vivo immune study. Since endotoxins can enhance life-threatening immune responses, it is crucial to optimize the method of their removal before clinical use not only of silk-based products but also of other biomolecules produced biotechnologically.

Oligonucleotide-Based Therapeutics for STAT3 Targeting in Cancer-Drug Carriers Matter.

High expression and phosphorylation of signal transducer and transcription activator 3 (STAT3) are correlated with progression and poor prognosis in various types of cancer. The constitutive activation of STAT3 in cancer affects processes such as cell proliferation, apoptosis, metastasis, angiogenesis, and drug resistance. The importance of STAT3 in cancer makes it a potential therapeutic target. Various methods of directly and indirectly blocking STAT3 activity at different steps of the STAT3 pathway have been investigated. However, the outcome has been limited, mainly by the number of upstream proteins that can reactivate STAT3 or the relatively low specificity of the inhibitors. A new branch of molecules with significant therapeutic potential has emerged thanks to recent developments in the regulatory function of non-coding nucleic acids. Oligonucleotide-based therapeutics can silence target transcripts or edit genes, leading to the modification of gene expression profiles, causing cell death or restoring cell function. Moreover, they can reach untreatable targets, such as transcription factors. This review briefly describes oligonucleotide-based therapeutics that found application to target STAT3 activity in cancer. Additionally, this review comprehensively summarizes how the inhibition of STAT3 activity by nucleic acid-based therapeutics such as siRNA, shRNA, ASO, and ODN-decoy affected the therapy of different types of cancer in preclinical and clinical studies. Moreover, due to some limitations of oligonucleotide-based therapeutics, the importance of carriers that can deliver nucleic acid molecules to affect the STAT3 in cancer cells and cells of the tumor microenvironment (TME) was pointed out. Combining a high specificity of oligonucleotide-based therapeutics toward their targets and functionalized nanoparticles toward cell type can generate very efficient formulations.

Unmasking the Deceptive Nature of Cancer Stem Cells: The Role of CD133 in Revealing Their Secrets.

Cancer remains a leading cause of death globally, and its complexity poses a significant challenge to effective treatment. Cancer stem cells and their markers have become key players in tumor growth and progression. CD133, a marker in various cancer types, is an active research area as a potential therapeutic target. This article explores the role of CD133 in cancer treatment, beginning with an overview of cancer statistics and an explanation of cancer stem cells and their markers. The rise of CD133 is discussed, including its structure, functions, and occurrence in different cancer types. Furthermore, the article covers CD133 as a therapeutic target, focusing on gene therapy, immunotherapy, and approaches to affect CD133 expression. Nanoparticles such as gold nanoparticles and nanoliposomes are also discussed in the context of CD133-targeted therapy. In conclusion, CD133 is a promising therapeutic target for cancer treatment. As research in this area progresses, it is hoped that CD133-targeted therapies will offer new and effective treatment options for cancer patients in the future.

In vivo study of the immune response to bioengineered spider silk spheres.

Bioengineered MS1 silk is derived from major ampullate spidroin 1 (MaSp1) from the spider Nephila clavipes. The MS1 silk was functionalized with the H2.1 peptide to target Her2-overexpressing cancer cells. The immunogenic potential of drug carriers made from MS1-type silks was investigated. The silk spheres were administered to healthy mice, and then (i) the phenotypes of the immune cells that infiltrated the Matrigel plugs containing spheres (implanted subcutaneously), (ii) the presence of silk-specific antibodies (after two intravenous injections of the spheres), (iii) the splenocyte phenotypes and their activity after restimulation ex vivo in terms of proliferation and cytokine secretion (after single intravenous injection of the spheres) were analyzed. Although the immunogenicity of MS1 particles was minor, the H2.1MS1 spheres attracted higher levels of B lymphocytes, induced a higher anti-silk antibody titer, and, after ex vivo restimulation, caused the activation of splenocytes to proliferate and express more IFN-γ and IL-10 compared with the PBS and MS1 groups. Although the H2.1MS1 spheres triggered a certain degree of an immunological response, multiple injections (up to six times) neither hampered the carrier-dependent specific drug delivery nor induced toxicity, as previously indicated in a mouse breast cancer model. Both findings indicate that a drug delivery system based on MS1-type silk has great potential for the treatment of cancer and other conditions.

Systemic and Local Silk-Based Drug Delivery Systems for Cancer Therapy.

For years, surgery, radiotherapy, and chemotherapy have been the gold standards to treat cancer, although continuing research has sought a more effective approach. While advances can be seen in the development of anticancer drugs, the tools that can improve their delivery remain a challenge. As anticancer drugs can affect the entire body, the control of their distribution is desirable to prevent systemic toxicity. The application of a suitable drug delivery platform may resolve this problem. Among other materials, silks offer many advantageous properties, including biodegradability, biocompatibility, and the possibility of obtaining a variety of morphological structures. These characteristics allow the exploration of silk for biomedical applications and as a platform for drug delivery. We have reviewed silk structures that can be used for local and systemic drug delivery for use in cancer therapy. After a short description of the most studied silks, we discuss the advantages of using silk for drug delivery. The tables summarize the descriptions of silk structures for the local and systemic transport of anticancer drugs. The most popular techniques for silk particle preparation are presented. Further prospects for using silk as a drug carrier are considered. The application of various silk biomaterials can improve cancer treatment by the controllable delivery of chemotherapeutics, immunotherapeutics, photosensitizers, hormones, nucleotherapeutics, targeted therapeutics (e.g., kinase inhibitors), and inorganic nanoparticles, among others.

MS1-type bioengineered spider silk nanoparticles do not exhibit toxicity in an in vivo mouse model.

Background: Due to factors such as silk sequence, purification, degradation, morphology and functionalization, each silk variant should be individually tested for potential toxicity. Aim:  In vivo toxicological evaluation of the previously characterized bioengineered H2.1MS1 spider silk particles that can deliver chemotherapeutics to human epidermal growth factor receptor 2-positive breast cancer. Materials & methods: Silk nanoparticles (H2.1MS1 and control MS1) were administered intravenously to mice, and then the organismal response was assessed. Several parameters of acute and subchronic toxicity were analyzed, including animal mortality and behavior, nanosphere biodistribution, and histopathological analysis of internal organs. Also, the complete blood count, as well as the concentration of biochemical parameters and cytokines in the serum, were examined. Results & conclusion: No toxicity of the systemically administrated silk nanosphere was observed, indicating their potential application in biomedicine.

Application of a three-dimensional (3D) breast cancer model to study macrophage polarization.

Knowledge of the tumor microenvironment is crucial for developing an effective strategy to treat cancer. Recently, anticancer therapies targeting macrophages have been intensively investigated. Increased understanding of the importance of the tumor microenvironment has led to the development of three-dimensional (3D) in vitro tumor models. However, established techniques for studying tumor-associated macrophages in vitro are limited. We have previously characterized a 3D breast cancer model consisting of breast cancer cells and fibroblasts cocultured on a silk scaffold. In the present study, the influence of this model on macrophage polarization was investigated. The expression of macrophage markers was studied using reverse transcription-quantitative PCR and flow cytometry. The activity of nitric oxide synthase and arginase in macrophages was also measured. The presented model appeared to induce the polarization of macrophages towards an M2 phenotype. In this 3D tumor model, the in vivo behavior of macrophages could be reproduced. This model may be beneficial for the study of tumor biology and for screening drugs.

Hyperthermia treatment of cancer cells by the application of targeted silk/iron oxide composite spheres.

Magnetic iron oxide nanoparticles (IONPs) are one of the most extensively studied materials for theranostic applications. IONPs can be used for magnetic resonance imaging (MRI), delivery of therapeutics, and hyperthermia treatment. Silk is a biocompatible material and can be used for biomedical applications. Previously, we produced spheres made of H2.1MS1 bioengineered silk that specifically carried a drug to the Her2-overexpressing cancer cells. To confer biocompatibility and targeting properties to IONPs, we blended these particles with bioengineered spider silks. Three bioengineered silks (MS1Fe1, MS1Fe2, and MS1Fe1Fe2) functionalized with the adhesion peptides F1 and F2, were constructed and investigated to form the composite spheres with IONPs carrying a positive or negative charge. Due to its highest IONP content, MS1Fe1 silk was used to produce spheres from the H2.1MS1:MS1Fe silk blend to obtain a carrier with cell-targeting properties. Composite H2.1MS1:MS1Fe1/IONP spheres made of silks blended at different ratios were obtained. Although the increased content of MS1Fe1 silk in particles resulted in an increased affinity of the spheres to IONPs, it decreased the binding of the composite particles to cancer cells. The H2.1MS1:MS1Fe1 particles prepared at a ratio of 8:2 and loaded with IONPs exhibited the ability to bind to the targeted cancer cells similar to the control spheres without IONPs. Moreover, when exposed to the alternating magnetic field, these particles generated 2.5 times higher heat. They caused an almost three times higher percentage of apoptosis in cancer cells than the control particles. The blending of silks enabled the generation of cancer-targeting spheres with a high affinity for iron oxide nanoparticles, which can be used for anti-cancer hyperthermia therapy.

Functionalized silk spheres selectively and effectively deliver a cytotoxic drug to targeted cancer cells in vivo.

BACKGROUND: Chemotherapy is often a first-line therapeutic approach for the treatment of a wide variety of cancers. Targeted drug delivery systems (DDSs) can potentially resolve the problem of chemotherapeutic drug off-targeting effects. Herein, we examined in vivo models to determine the efficacy of Her2-targeting silk spheres (H2.1MS1) as DDSs for delivering doxorubicin (Dox) to Her2-positive and Her2-negative primary and metastatic mouse breast cancers. RESULTS: The specific accumulation of H2.1MS1 spheres was demonstrated at the site of Her2-positive cancer. Dox delivered only by functionalized H2.1MS1 particles selectively inhibited Her2-positive cancer growth in primary and metastatic models. Moreover, the significant effect of the Dox dose and the frequency of treatment administration on the therapeutic efficacy was indicated. Although the control MS1 spheres accumulated in the lungs in Her2-positive metastatic breast cancer, the Dox-loaded MS1 particles did not treat cancer. Histopathological examination revealed no systemic toxicity after multiple administrations and at increased doses of Dox-loaded silk spheres. Although the studies were performed in immunocompetent mice, the H2.1MS1 silk spheres efficiently delivered the drug, which exerted a therapeutic effect. CONCLUSION: Our results indicated that functionalized silk spheres that enable cell-specific recognition, cellular internalization, and drug release represent an efficient strategy for cancer treatment in vivo.

Silk Particles as Carriers of Therapeutic Molecules for Cancer Treatment.

Although progress is observed in cancer treatment, this disease continues to be the second leading cause of death worldwide. The current understanding of cancer indicates that treating cancer should not be limited to killing cancer cells alone, but that the target is the complex tumor microenvironment (TME). The application of nanoparticle-based drug delivery systems (DDS) can not only target cancer cells and TME, but also simultaneously resolve the severe side effects of various cancer treatment approaches, leading to more effective, precise, and less invasive therapy. Nanoparticles based on proteins derived from silkworms' cocoons (like silk fibroin and sericins) and silk proteins from spiders (spidroins) are intensively explored not only in the oncology field. This natural-derived material offer biocompatibility, biodegradability, and simplicity of preparation methods. The protein-based material can be tailored for size, stability, drug loading/release kinetics, and functionalized with targeting ligands. This review summarizes the current status of drug delivery systems' development based on proteins derived from silk fibroin, sericins, and spidroins, which application is focused on systemic cancer treatment. The nanoparticles that deliver chemotherapeutics, nucleic acid-based therapeutics, natural-derived agents, therapeutic proteins or peptides, inorganic compounds, as well as photosensitive molecules, are introduced.

Drug affinity and targeted delivery: double functionalization of silk spheres for controlled doxorubicin delivery into Her2-positive cancer cells.

BACKGROUND: The optimal drug delivery system should be biocompatible, biodegradable, and allow the sustained release of the drug only after it reaches the target cells. Silk, as a natural polymer, is a great candidate for building drug carriers. Genetically engineered silks offer the possibility of functionalization. Previously, we characterized bioengineered silk spheres that were functionalized with H2.1 peptide that selectively delivered a drug to Her2-positive cancer cells. However, drug leakage from the silk spheres showed the need for improved control. RESULTS: To control the drug loading and release, we designed and produced functional silk (DOXMS2) that contains a DOX peptide with an affinity for doxorubicin. The DOXMS2 spheres showed the decreased release of doxorubicin compared with MS2 particles. Next, the DOXMS2 silk was blended with the H2.1MS1 polymer to improve the control of doxorubicin binding and release into Her2-positive cancer cells. The H2.1MS1:DOXMS2 particles showed the highest doxorubicin-loading capacity and binding per cell, which resulted in the highest cytotoxic effect compared with that of other sphere variants. Since drug release at a pH of 7.4 from the blended H2.1MS1:DOXMS2 particles was significantly lower than from blended spheres without DOXMS2 silk, this indicated that such particles could control the release of the drug into the circulatory system before the carrier reached the tumor site. CONCLUSIONS: This strategy, which is based on the blending of silks, allows for the generation of particles that deliver drugs in a controlled manner.

Implementation of a dynamic culture condition to the heterotypic 3D breast cancer model.

Cell culture system is used for a wide range of research and biotechnology production. Majority of in vitro cell studies are conducted as static, two dimensional (2D) dish culture system where cells grow in a monolayer. However, to better reflect the in vivo condition, three dimensional (3D) culture systems were introduced that allow investigating the cell-cell and cell-microenvironment interactions. In this work, the 3D breast cancer model was investigated. Previously, we developed a 3D breast cancer model that constituted of fibroblasts and breast cancer cells seeded on the silkworm silk scaffold. The dynamic culture condition that provides the medium flow and shear forces was implemented to the model. The dynamic conditions were compared to the static cultivation regarding its influence on the number of cells, their viability, scaffold penetration, and cells co-localization. The implication of the dynamic condition to the 3D cultures resulted in a higher number and viability of the cells compared with the static 3D cultures. In contrast to the static culture condition, during the dynamic cultivation cells penetrated entirely and evenly the inner parts of the scaffold. Moreover, in coculture, the transitions like a ratio of fibroblast to the cancer cells, fibroblast morphology, and their localization were similar in both types of culture conditions, but they proceeded much faster during the dynamic cultivation. The implementation of dynamic culture condition shortened the time needed to establish the settle 3D breast cancer model. The established dynamic cancer model can be used to study tumor biology and drug screening.

Composite spheres made of bioengineered spider silk and iron oxide nanoparticles for theranostics applications.

Bioengineered spider silk is a biomaterial that has exquisite mechanical properties, biocompatibility, and biodegradability. Iron oxide nanoparticles can be applied for the detection and analysis of biomolecules, target drug delivery, as MRI contrast agents and as therapeutic agents for hyperthermia-based cancer treatments. In this study, we investigated three bioengineered silks, MS1, MS2 and EMS2, and their potential to form a composite material with magnetic iron oxide nanoparticles (IONPs). The presence of IONPs did not impede the self-assembly properties of MS1, MS2, and EMS2 silks, and spheres formed. The EMS2 spheres had the highest content of IONPs, and the presence of magnetite IONPs in these carriers was confirmed by several methods such as SEM, EDXS, SQUID, MIP-OES and zeta potential measurement. The interaction of EMS2 and IONPs did not modify the superparamagnetic properties of the IONPs, but it influenced the secondary structure of the spheres. The composite particles exhibited a more than two-fold higher loading efficiency for doxorubicin than the plain EMS2 spheres. For both the EMS2 and EMS2/IONP spheres, the drug revealed a pH-dependent release profile with advantageous kinetics for carriers made of the composite material. The composite spheres can be potentially applied for a combined cancer treatment via hyperthermia and drug delivery.

Cellular uptake, intracellular distribution and degradation of Her2-targeting silk nanospheres.

BACKGROUND: The development of nanocarrier technology has attracted great interest in the last decade. Biodegradable spheres made of functionalized silk have considerable potential to be used as drug delivery systems for cancer treatment. A targeting ligand displayed at the surface of a carrier, with a specific affinity towards a particular receptor, can further enhance the accumulation and uptake of nanoparticles at the site of a tumor. MATERIALS AND METHODS: The hybrid constructs were obtained by adding a Her2-binding peptide (H2.1) to MS1 and MS2 bioengineered silks based on the MaSp1 and MaSp2 proteins from N. clavipes, respectively. The H2.1MS1 and H2.1MS2 proteins were blended at a weight ratio of 8:2. Stable silk particles were formed by mixing a soluble protein with potassium phosphate using a micromixing technique. We used specific inhibitors of endocytosis to determine the cellular uptake pathway of the silk nanoparticles in human Her2-positive breast cancer cells. The subcellular distribution of silk particles was investigated by evaluating the signal colocalization with organelle-specific tracker. Moreover, lysosomal and exosomal inhibitors were implemented to evaluate their impact on the silk spheres behavior and degradation. RESULTS: The functionalized spheres were specifically taken up by Her2-positive cancer cells. Silk particles facilitated the entry into cells through both the clathrin- and caveola-dependent pathways of endocytosis. Upon entering the cells, the particles accumulated in the lysosomes, where intracellular degradation occurred. CONCLUSIONS: The present study demonstrated directly that the lysosomal function was essential for silk-based carrier elimination. The degradation of the carrier is of great importance to develop an optimal drug delivery system.

Optimization of spider silk sphere formation processing conditions to obtain carriers with controlled characteristics.

Bioengineered spider silk is a focus of research due to its biocompatibility, biodegradability, and excellent mechanical properties. Functionalized silk can be processed into spheres and employed as selective drug delivery vehicles in targeted cancer treatment. Efficient, repeatable and controllable processing conditions are essential to drug carrier development. This study aimed to optimize the processing conditions of silk spheres formation, scale-up, and automation of the silk spheres production process. The automated micromixing system provided substantial amounts of silk spheres under repetitive production conditions. Micromixing resulted in smaller sphere sizes and narrower sphere size distributions than mixing with a pipette. Furthermore, the particle size and size distribution of silk spheres could be tailored by varying mixing process parameters, such as protein concentration, silk and salting out buffer ratio, mixing speed, and the size of the tubes and mixing zone. In addition, the implementation of ultrafiltration techniques provided a fast and efficient concentration of spheres in water. Furthermore, the shear forces introduced by micromixing did not impede the properties of the Her2 binding peptide (H2.1) since the functionalized H2.1MS1:H2.1MS2 silk spheres selectively were internalized by Her2-positive cancer cells. This study indicated that micromixing in combination with ultrafiltration enabled scale-up of the sphere production process under controllable and repeatable conditions. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 3211-3221, 2018.

Bioengineering the spider silk sequence to modify its affinity for drugs.

BACKGROUND: Silk is a biocompatible and biodegradable material, able to self-assemble into different morphological structures. Silk structures may be used for many biomedical applications, including carriers for drug delivery. The authors designed a new bioengineered spider silk protein, EMS2, and examined its property as a carrier of chemotherapeutics. MATERIALS AND METHODS: To obtain EMS protein, the MS2 silk monomer (that was based on the MaSp2 spidroin of Nephila clavipes) was modified by the addition of a glutamic acid residue. Both bioengineered silks were produced in an Escherichia coli expression system and purified by thermal method. The silk spheres were produced by mixing with potassium phosphate buffer. The physical properties of the particles were characterized using scanning electron microscopy, atomic force microscopy, Fourier-transform infrared spectroscopy, and zeta potential measurements. The MTT assay was used to examine the cytotoxicity of spheres. The loading and release profiles of drugs were studied spectrophotometrically. RESULTS: The bioengineered silk variant, EMS2, was constructed, produced, and purified. The EMS2 silk retained the self-assembly property and formed spheres. The spheres made of EMS2 and MS2 silks were not cytotoxic and had a similar secondary structure content but differed in morphology and zeta potential values; EMS2 particles were more negatively charged than MS2 particles. Independently of the loading method (pre- or post-loading), the loading of drugs into EMS2 spheres was more efficient than the loading into MS2 spheres. The advantageous loading efficiency and release rate made EMS2 spheres a good choice to deliver neutral etoposide (ETP). Despite the high loading efficiency of positively charged mitoxantrone (MTX) into EMS2 particles, the fast release rate made EMS2 unsuitable for the delivery of this drug. A faster release rate from EMS2 particles compared to MS2 particles was observed for positively charged doxorubicin (DOX). CONCLUSION: By modifying its sequence, silk affinity for drugs can be controlled.

Heterotypic breast cancer model based on a silk fibroin scaffold to study the tumor microenvironment.

An intensive investigation of the development of in vitro models to study tumor biology has led to the generation of various three-dimensional (3D) culture methods that better mimic in vivo conditions. The tumor microenvironment (TME) is shaped by direct interactions among cancer cells, cancer-associated cells and the extracellular matrix (ECM). Recognizing the need to incorporate both tissue dimensionality and the heterogeneity of cells, we have developed a 3D breast cancer model. NIH3T3 fibroblasts and EMT6 breast cancer cell lines were seeded in various ratios onto a silk fibroin scaffold. The porosity of the silk scaffold was optimized to facilitate the growth of cancer cells. EMT6 and NIH3T3 cells were modified to express GFP and turboFP635, respectively, which enabled the direct analysis of the cell morphology and colonization of the scaffold and for the separation of the cells after their co-culture. Use of 3D mono-culture and 3D co-culture methods resulted in the modification of cell morphology and in a significant increase in ECM production. These culture methods also induced cellular changes related to EMT (epithelial-mesenchymal transition) and CAF (cancer-associated fibroblast) markers. The presented model is an easy to manufacture, well-characterized tool that can be used to study processes of the TME.

Delivery of chemotherapeutics using spheres made of bioengineered spider silks derived from MaSp1 and MaSp2 proteins.

AIM: Analysis of the properties and chemotherapeutics delivery potential of spheres made of bioengineered spider silks MS1 and MS2. MATERIALS & METHODS: MS1 and MS2 derived from Nephila clavipes dragline silks - MaSp1 and MaSp2, respectively - formed spheres that were compared in terms of physicochemical properties, cytotoxicity and loading/release of chemotherapeutics. RESULTS: MS2 spheres were more dispersed, smaller, of solid core, of higher beta-sheet structure content, and of opposite (negative) charge than MS1 spheres. Preloaded MS2 showed greater applicability for mitoxantrone, while postloaded for etoposide delivery compared with MS1 spheres. However, MS1 spheres were a better choice for doxorubicin delivery than MS2. CONCLUSION: Bioengineered silks can be tailored to develop a system with optimal drug loading and release properties.