The RNA world: from experimental laboratory to "in silico" approach. Part 1: User friendly RNA expression databases portals.

Cellular information about "life instruction" is stored, transferred, and modified using different types of RNA molecules. During the last decades, a growing number of RNA data has been generated thanks to the development of microarray chips and next-generation sequencing (NGS) methods. Improvement of bioinformatics contributed to the discovery of many types of new non-coding RNAs (ncRNAs), mostly with regulatory functions that supplemented the knowledge about the world of RNA. All of it, as well as the Human Genome Project (HGP) and the Cancer Genome Atlas (TCGA) project, has resulted in the formation of data storage and analysis portals which are widely used in cancer research and moved science from in vitro to in silico research. In this review we presented and discussed the data storage and analysis portals used by us, such as cBioPortal, UALCAN, ENCORI, and others. During the revision of these sites, we paid attention to data integration, simplicity of analysis, and results visualization, which are important for users without bioinformatic or statistical skills. In our opinion, the RNA analysis online tools will rapidly develop during the next decade and it seems to be a way for personalization of cancer treatment.

SRY-Related Transcription Factors in Head and Neck Squamous Cell Carcinomas: In Silico Based Analysis.

Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cancer and the fifth cause of cancer-related deaths worldwide with a poor 5-year survival. SOX family genes play a role in the processes involved in cancer development such as epithelial-mesenchymal transition (EMT), the maintenance of cancer stem cells (CSCs) and the regulation of drug resistance. We analyzed the expression of SOX2-OT, SOX6, SOX8, SOX21, SOX30 and SRY genes in HNSCC patients using the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets, to assess their biological role and their potential utility as biomarkers. We demonstrated statistically significant differences in expression between normal and primary tumor tissues for SOX6, SOX8, SOX21 and SOX30 genes and pointed to SOX6 as the one that met the independent diagnostic markers criteria. SOX21 or SRY alone, or the panel of six SRY-related genes, could be used to estimate patient survival. SRY-related genes are positively correlated with immunological processes, as well as with keratinization and formation of the cornified envelope, and negatively correlated with DNA repair and response to stress. Moreover, except SRY, all analyzed genes were associated with a different tumor composition and immunological profiles. Based on validation results, the expression of SOX30 is higher in HPV(+) patients and is associated with patients' survival. SRY-related transcription factors have vast importance in HNSCC biology. SOX30 seems to be a potential biomarker of HPV infection and could be used as a prognostic marker, but further research is required to fully understand the role of SOX family genes in HNSCC.

ZNF714 Supports Pro-Oncogenic Features in Lung Cancer Cells.

Despite the ongoing progress in diagnosis and treatments, cancer remains a threat to more than one-third of the human population. The emerging data indicate that many Krüppel-associated box zinc finger proteins (KRAB-ZNF) belonging to a large gene family may be involved in carcinogenesis. Our previous study identified Zinc Finger Protein 714 (ZNF714), a KRAB-ZNF gene of unknown function, as being commonly overexpressed in many tumors, pointing to its hypothetical oncogenic role. Here, we harnessed The Cancer Genome Atlas (TCGA)-centered databases and performed functional studies with transcriptomic and methylomic profiling to explore ZNF714 function in cancer. Our pan-cancer analyses confirmed frequent ZNF714 overexpression in multiple tumors, possibly due to regional amplification, promoter hypomethylation, and Nuclear Transcription Factor Y Subunit Beta (NFYB) signaling. We also showed that ZNF714 expression correlates with tumor immunosuppressive features. The in vitro studies indicated that ZNF714 expression positively associates with proliferation, migration, and invasion. The transcriptomic analysis of ZNF714 knocked-down cells demonstrated deregulation of cell adhesion, migration, proliferation, apoptosis, and differentiation. Importantly, we provided evidence that ZNF714 negatively regulates the expression of several known TSGs indirectly via promoter methylation. However, as ZNF714 did not show nuclear localization in our research model, the regulatory mechanisms exerted by ZNF714 require further investigation. In conclusion, our results reveal, for the first time, that ZNF714 may support pro-oncogenic features in lung cancer cells.

ZNF643/ZFP69B Exerts Oncogenic Properties and Associates with Cell Adhesion and Immune Processes.

The global cancer burden remains high; thus, a better understanding of the molecular mechanisms driving carcinogenesis is needed to improve current prevention and treatment options. We previously detected the ZNF643/ZFP69B gene upregulated in multiple tumors, and we speculated it may play a role in tumor biology. To test this hypothesis, we employed TCGA-centered databases to correlate ZNF643 status with various clinicopathological parameters. We also performed RNA-seq analysis and in vitro studies assessing cancer cell phenotypes, and we searched for ZNF643-bound genomic loci. Our data indicated higher levels of ZNF643 in most analyzed tumors compared to normal samples, possibly due to copy number variations. ZNF643 mRNA correlated with diverse molecular and immune subtypes and clinicopathological features (tumor stage, grade, patient survival). RNA-seq analysis revealed that ZNF643 silencing triggers the deregulation of the genes implicated in various cancer-related processes, such as growth, adhesion, and immune system. Moreover, we observed that ZNF643 positively influences cell cycle, migration, and invasion. Finally, our ChIP-seq analysis indicated that the genes associated with ZNF643 binding are linked to adhesion and immune signaling. In conclusion, our data confirm the oncogenic properties of ZNF643 and pinpoint its impact on cell adhesion and immune processes.

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.

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 as an innovative biomaterial for cancer therapy.

Silk has been used for centuries in the textile industry and as surgical sutures. In addition to its unique mechanical properties, silk possesses other properties, such as biocompatibility, biodegradability and ability to self-assemble, which make it an interesting material for biomedical applications. Although silk forms only fibers in nature, synthetic techniques can be used to control the processing of silk into different morphologies, such as scaffolds, films, hydrogels, microcapsules, and micro- and nanospheres. Moreover, the biotechnological production of silk proteins broadens the potential applications of silk. Synthetic silk genes have been designed. Genetic engineering enables modification of silk properties or the construction of a hybrid silk. Bioengineered hybrid silks consist of a silk sequence that self-assembles into the desired morphological structure and the sequence of a polypeptide that confers a function to the silk biomaterial. The functional domains can comprise binding sites for receptors, enzymes, drugs, metals or sugars, among others. Here, we review the current status of potential applications of silk biomaterials in the field of oncology with a focus on the generation of implantable, injectable and targeted drug delivery systems and the three-dimensional cancer models based on silk scaffolds for cancer research. However, the systems described could be applied in many biomedical fields.

Functionalized spider silk spheres as drug carriers for targeted cancer therapy.

Bioengineered spider silk is a biomaterial that combines the properties of self-assembly, biocompatibility and biodegradability with reasonable accessibility and a simple purification procedure. Moreover, genetic engineering enables the functionalization of silk by adding the peptide coding sequences of the desired attribute. Hybrids composed of Her2 binding peptides (H2.1 or H2.2) and bioengineered silk MS1 (based on the MaSp1 sequence from N. clavipes) were designed. Bioengineered silks were expressed in a bacterial system and purified using a tag-free thermal method. The hybrid silks with N-terminal functionalization were bound more efficiently to cells that were overexpressing Her2 than those with the C-terminal fusion. Moreover, the functionalization did not impede the self-assembly property of bioengineered silk, enabling the processing of silk proteins into spheres. The binding domains were exposed on the surface of the spheres, because the functionalized particles specifically bound and internalized into Her2-overexpressing cells. The binding of the functionalized spheres to Her2-positive cells was significantly higher compared with the control sphere and Her2-negative cell binding. Silk spheres were loaded with doxorubicin and showed pH-dependent drug release. The silk spheres were not cytotoxic, unless they were loaded with the drug doxorubicin. This study indicates the ability of drug-loaded functionalized spider silk spheres to serve as a carrier for targeted cancer therapy.

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.

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.

Physiological and tumor-associated angiogenesis: Key factors and therapy targeting VEGF/VEGFR pathway.

Cancer remains one of the leading causes of death worldwide and poses a significant challenge to effective treatment due to its complexity. Angiogenesis, the formation of new blood vessels, is one of the cancer hallmarks and is a critical process in tumor growth and metastasis. The pivotal role of angiogenesis in cancer development has made antiangiogenic treatment a promising strategy for cancer therapy. To develop an effective therapy, it is essential to understand the basics of the physiological and tumor angiogenesis process. This review presents the primary factors related to physiological and tumor angiogenesis and the mechanisms of angiogenesis in tumors. We summarize potential molecular targets for cancer treatment by focusing on the vasculature, with the VEGF/VEGFR pathway being one of the most important and well-studied. Additionally, we present the advantages and limitations of currently used clinical protocols for cancer treatment targeting the VEGF/VEGFR pathway.

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.

[Engineered spider silk: the intelligent biomaterial of the future. Part I]. / Inzynierowany jedwab pajeczy: inteligentny biomaterial przyszlosci. Czesc I.

The unique properties of spider silk such as strength, extensibility, toughness, biocompatibility and biodegradability are the reasons for the recent development in silk biomaterial technology. For a long time scientific progress was impeded by limited access to spider silk. However, the development of the molecular biology strategy was a breaking point in synthetic spider silk protein design. The sequences of engineered spider silk are based on the consensus motives of the corresponding natural equivalents. Moreover, the engineered silk proteins may be modified in order to gain a new function. The strategy of the hybrid proteins constructed on the DNA level combines the sequence of engineered silk, which is responsible for the biomaterial structure, with the sequence of polypeptide which allows functionalization of the silk biomaterial. The functional domains may comprise receptor binding sites, enzymes, metal or sugar binding sites and others. Currently, advanced research is being conducted, which on the one hand focuses on establishing the particular silk structure and understanding the process of silk thread formation in nature. On the other hand, there are attempts to improve methods of engineered spider silk protein production. Due to acquired knowledge and recent progress in synthetic protein technology, the engineered silk will turn into intelligent biomaterial of the future, while its industrial production scale will trigger a biotechnological revolution.

[Engineered spider silk: the intelligent biomaterial of the future. Part II]. / Inzynierowany jedwab pajeczy: inteligentny biomaterial przyszlosci. Czesc II.

The development and progress in engineered spider silk manufacturing has enabled its practical application. Recombinant spider silk can assemble in several morphological forms such as films, hydrogels, fibers, scaffolds, microcapsules, and micro- and nanospheres. The in vitro fiber formation takes place by mimicking the natural spinning process in the spider spinning gland: in the presence of phosphate ions and dragging forces. Films are obtained by evaporation of solvent from the silk solution, while the result of evaporation of the solvent in the presence of porogens is a silk scaffold. Hydrogels are formed by spontaneous polymerization of silk particles in solutions at low pH. The silk film assembled at the interface of two immiscible phases forms microcapsules. The smallest of the described forms--silk spheres--are obtained by salting out the silk protein solution after addition of the phosphate ions. Common properties of the silk biomaterials are biocompatibility and biodegradability, which make them suitable for a number of applications in medicine and pharmacy. Moreover, the strategy of hybrid proteins which provides the desired function to biomaterial will further expand their potential use.

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.

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.

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.

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.