An injectable degradable porous polymer scaffold for tissue engineering and drug delivery

Cell transplantation on biodegradable scaffolds is an established approach in tissue engineering to the problem of the regeneration of diseased or damaged tissues. As cells grow and organise themselves, they secrete their own extracellular matrix, while the polymer degrades into natural metabolites resulting in eventual natural tissue replacement. Polymeric materials used for these scaffolds must satisfy a number of requirements. These include defined cell-interactive properties, porosity, biodegradability, mechanical and controlled release properties. To date, scaffolds have been designed to conform to these requirements. However, the need to perform defined three-dimensional structures requires prior knowledge of the dimensions of the defect or cavity to be filled. Furthermore the general use of toxic solvents in the processing of these scaffolds prevents the incorporation of biological agents and cells during fabrication. Therefore, poor transportation of cells through the scaffolds can result in low cell seeding efficiencies. Finally such scaffolds require an invasive operation for transplantation of the material. In contrast a number of injectable materials have been proposed and investigated. The transformation from liquid pre-cursor to gel in such systems can, however, require cell harmful trigger signals such as UV exposure or pH changes. Furthermore, these injectable gels lack a porous structure preventing effective cell migration and restricting tissue formation and vascularisation tothe barrier of diffusion for signalling and nutrient molecules. The work in this thesis presents a scaffold that is both injectable and conforms to the requirements of water-insoluble porous scaffolds. This starts with the synthesis of a biotinylated poly (lactic acid)-poly (ethylene glycol) (PLA-PEG) copolymer. The polymer is degradable, protein resistant and cell interactive when used in conjunction with biotinylated cell adhesive peptides. The biotin unit tethered to the PEG-PLA also provides the polymer with self-assembling properties when used in conjunction with avidin. In contrast to alternative injectable materials, the scaffold presented in this thesis is porous. This porosity is necessary for tissue ingrowth and vascularization. Therefore, before progressing on to the manufacture of the scaffold, a systematic study of two cell types involved in vascularisation was carried out over defined pore features. These studies revealed that cell behaviour over pore features was related to cell type, cell density and pore size. This had significant implications for the injectable scaffold in development because proposed advantages were delivery of a variety of cell types, controlled porous structure, and efficient cell seeding. Microparticles were then manufactured from the PLA-PEG-biotin using a single emulsion manufacturing process. Surface Plasmon Resonance (SPR) confirmed that these microparticles would bind efficiently to avidin. The condition for optimum self-assembling of particles was then determined using aggregation studies. These studies showed that a critical quantity of avidin was required for microparticles to aggregate together. The ability to aggregate particles of different sizes leads to the potential for controlling scaffold porosity. Rheological testing showed that the scaffold's mechanical properties could be tailored to that of the tissue in which regeneration is required. The self-assembly of microparticles was also demonstrated to form complex three-dimensional scaffolds without the use of toxic solvents. Scaffolds prepared in simulated tissues maintained shape upon injection. Scaffolds were then self-assembled with cells entrapped within them. Cell viability within the self-assembling scaffolds was confirmed by Alamar Blue assays. In vivo studies have demonstrated that cell-scaffold composites permit tissue ingrowth and thus readily undergo vascularisation. The novel molecular-interaction mechanism of self-assembly of these scaffolds differentiates this material from other injectable systems. The formation of porous scaffolds within a cavity or a soft-tissue could be a pre-requisite for tissue remodelling using new cell sources that are dependent on vascularisation and tissue ingrowth. The basic component of the scaffold is a biodegradable microparticle that presents a protein resistant surface with biotinylated moieties. Therefore, standard controlled release technologies and biotin-avidin mediated surface engineering can be combined with the self-assembly to form biomimetic scaffolds that stimulate integrin-mediated cell adhesion and then release growth factors

Mapping the immune environment in clear cell renal carcinoma by single-cell genomics

Clear cell renal cell carcinoma (ccRCC) is one of the most immunologically distinct tumor types due to high response rate to immunotherapies, despite low tumor mutational burden. To characterize the tumor immune microenvironment of ccRCC, we applied single-cell-RNA sequencing (SCRS) along with T-cell-receptor (TCR) sequencing to map the transcriptomic heterogeneity of 25,688 individual CD4

Topically Applied Resiquimod versus Imiquimod as a Potential Adjuvant in Melanoma Treatment

Melanoma is the most lethal form of skin cancer and surgery remains the preferred and most effective treatment. Nevertheless, there are cases where surgery is not a viable method and alternative treatments are therefore adopted. One such treatment that has been tested is topical 5% imiquimod (IMQ) cream, which, although showing promise as a treatment for melanoma, has been found to have undesirable off-target effects. Resiquimod (RSQ) is an immunomodulatory molecule that can activate immune responses by binding to Toll-like receptors (TLR) 7 and 8 and may be more effective than IMQ in the context of melanoma treatment. RSQ can cross the stratum corneum (SC) easily without requiring pretreatment of the skin. In a gel formulation, RSQ has been studied as a monotherapy and adjuvant for melanoma treatment in pre-clinical studies and as an adjuvant in clinical settings. Although side effects of RSQ in gel formulation were also reported, they were never severe enough for the treatment to be suspended. In this review, we discuss the potential use of RSQ as an adjuvant for melanoma treatment

The combination of a low-dose chemotherapeutic agent, 5-fluorouracil, and an adenoviral tumor vaccine has a synergistic benefit on survival in a tumor model system.

Standard cancer therapies, particularly those involving chemotherapy, are in need of modifications that both reduce short-term and long-term side effects as well as improve the overall survival of cancer patients. Here we show that combining low-dose chemotherapy with a therapeutic vaccination using an adenovirus encoding a model tumor-associated antigen, ovalbumin (Ad5-OVA), had a synergistic impact on survival in tumor-challenged mice. Mice that received the combinatorial treatment of Ad5-OVA plus low-dose 5-fluorouracil (5-FU) had a 95% survival rate compared to 7% and 30% survival rates for Ad5-OVA alone and 5-FU alone respectively. The presence of 5-FU enhanced the levels of OVA-specific CD8(+) T lymphocytes in the spleens and draining lymph nodes of Ad5-OVA-treated mice, a phenomenon that was dependent on the mice having been tumor-challenged. Thus 5-FU may have enhanced survival of Ad5-OVA-treated mice by enhancing the tumor-specific immune response combined with eliminating tumor bulk. We also investigated the possibility that the observed therapeutic benefit may have been derived from the capacity of 5-FU to deplete MDSC populations. The findings presented here promote the concept of combining adenoviral cancer vaccines with low-dose chemotherapy

Potential Role of Selenium in the Treatment of Cancer and Viral Infections

Selenium has been extensively evaluated clinically as a chemopreventive agent with variable results depending on the type and dose of selenium used. Selenium species are now being therapeutically evaluated as modulators of drug responses rather than as directly cytotoxic agents. In addition, recent data suggest an association between selenium base-line levels in blood and survival of patients with COVID-19. The major focus of this mini review was to summarize: the pathways of selenium metabolism; the results of selenium-based chemopreventive clinical trials; the potential for using selenium metabolites as therapeutic modulators of drug responses in cancer (clear-cell renal-cell carcinoma (ccRCC) in particular); and selenium usage alone or in combination with vaccines in the treatment of patients with COVID-19. Critical therapeutic targets and the potential role of different selenium species, doses, and schedules are discussed