CD44-targeted nanoparticles for co-delivery of docetaxel and an Akt inhibitor against colorectal cancer.

New strategies to develop drug-loaded nanocarriers with improved therapeutic efficacy are needed for cancer treatment. Herein we report a novel drug-delivery nanosystem comprising encapsulation of the chemotherapeutic drug docetaxel (DTX) and recombinant fusion of a small peptide inhibitor of Akt kinase within an elastin-like recombinamer (ELR) vehicle. This combined approach is also precisely targeted to colorectal cancer cells by means of a chemically conjugated DNA aptamer specific for the CD44 tumor marker. This 53 nm dual-approach nanosystem was found to selectively affect cell viability (2.5 % survival) and proliferation of colorectal cancer cells in vitro compared to endothelial cells (50 % survival), and to trigger both apoptosis- and necrosis-mediated cell death. Our findings also show that the nanohybrid particles remain stable under physiological conditions, trigger sustained drug release and possess an adequate pharmacokinetic profile after systemic intravenous administration. In vivo assays showed that these dual-approach nanohybrids significantly reduced the number of tumor polyps along the colorectal tract in a murine colorectal cancer model. Furthermore, systemic administration of advanced nanohybrids induced tissue recovery by improving the morphology of gastrointestinal crypts and the tissue architecture. Taken together, these findings indicate that our strategy of an advanced dual-approach nanosystem allows us to achieve successful controlled release of chemotherapeutics in cancer cells and may have a promising potential for colorectal cancer treatment.

Bioinspired mineralization of engineered living materials to promote osteogenic differentiation.

In this work, Engineered Living Materials (ELMs), based on the combination of genetically-modified bacteria and mineral-reinforced organic matrices, and endowed with self-healing or regenerative properties and adaptation to specific biological environments were developed. Concretely, we produced ELMs combining human mesenchymal stem cells (hMSCs) and Lactococcus lactis (L. lactis), which was specifically programmed to deliver bone morphogenetic protein (BMP-2) upon external stimulation using nisin, into mineralized alginate matrices. The hybrid organic/inorganic matrix was built through a protocol, inspired by bone mineralization, in which alginate (Alg) assembly and apatite (HA) mineralization occurred simultaneously driven by calcium ions. Chemical composition, structure and reologhical properties of the hybrid 3D matrices were dedicately optimized prior the incorportation of the living entities. Then, the same protocol was reproduced in the presence of hMSC and engineered L. lactis that secrete BMP-2 resulting in 3D hybrid living hydrogels. hMSC viability and osteogenic differentiation in the absence and presence of the bacteria were evaluated by live/dead and quantitative real-time polymerase chain reaction (qPCR) and immunofluorescence assays, respectively. Results demonstrate that these 3D engineered living material support osteogenic differentiation of hMSCs due to the synergistic effect between HA and the growth factors BMP-2 delivered by L. lactis.

Inducible expression of interleukin-12 augments the efficacy of affinity-tuned chimeric antigen receptors in murine solid tumor models.

The limited number of targetable tumor-specific antigens and the immunosuppressive nature of the microenvironment within solid malignancies represent major barriers to the success of chimeric antigen receptor (CAR)-T cell therapies. Here, using epithelial cell adhesion molecule (EpCAM) as a model antigen, we used alanine scanning of the complementarity-determining region to fine-tune CAR affinity. This allowed us to identify CARs that could spare primary epithelial cells while still effectively targeting EpCAMhigh tumors. Although affinity-tuned CARs showed suboptimal antitumor activity in vivo, we found that inducible secretion of interleukin-12 (IL-12), under the control of the NFAT promoter, can restore CAR activity to levels close to that of the parental CAR. This strategy was further validated with another affinity-tuned CAR specific for intercellular adhesion molecule-1 (ICAM-1). Only in affinity-tuned CAR-T cells was NFAT activity stringently controlled and restricted to tumors expressing the antigen of interest at high levels. Our study demonstrates the feasibility of specifically gearing CAR-T cells towards recognition of solid tumors by combining inducible IL-12 expression and affinity-tuned CAR.

Nanomedicine for autophagy modulation in cancer therapy: a clinical perspective.

In recent years, progress in nanotechnology provided new tools to treat cancer more effectively. Advances in biomaterials tailored for drug delivery have the potential to overcome the limited selectivity and side effects frequently associated with traditional therapeutic agents. While autophagy is pivotal in determining cell fate and adaptation to different challenges, and despite the fact that it is frequently dysregulated in cancer, antitumor therapeutic strategies leveraging on or targeting this process are scarce. This is due to many reasons, including the very contextual effects of autophagy in cancer, low bioavailability and non-targeted delivery of existing autophagy modulatory compounds. Conjugating the versatile characteristics of nanoparticles with autophagy modulators may render these drugs safer and more effective for cancer treatment. Here, we review current standing questions on the biology of autophagy in tumor progression, and precursory studies and the state-of-the-art in harnessing nanomaterials science to enhance the specificity and therapeutic potential of autophagy modulators.

Anti-neoplastic sulfonamides alter the metabolic homeostasis and disrupt the suppressor activity of regulatory T cells.

Regulatory T cells (Tregs) are essential to maintain self-tolerance and immune homeostasis but, as components of the tumor microenvironment (TME), are also a major barrier to effective cancer immunosurveillance and immunotherapy. FH535 and its derivative Y3 are two N-aryl-benzene-sulfonamides (NABs) that inhibit HCC cell proliferation and tumor progression. However, the impact of NABs on the immune cells in the TME is not yet known. Analyses of explanted livers from patients with hepatocellular carcinoma (HCC) showed that high levels of tumor-infiltrating Tregs were associated with poor tumor differentiation. These results lead us to investigate the immunomodulatory effects of NABs in regulatory and effector T cells. Exposure of primary human Tregs to NABs induced a rapid but temporary increase of cell expansion, a gradual disruption of suppressor activity, and concomitant bioenergetics and autophagic flux dysregulations. In contrast to Tregs, no gross effects were observed in effector T cells. Addition of Rapamycin prevented the functional decay of Tregs and restored their metabolic profile, suggesting that NAB effects require the integrity of the mTOR pathway. This study revealed the immunomodulatory properties of NABs with a preferential impact on Treg activity and provided novel insights into the anti-tumor potential of sulfonamides.

Elastin-like Polymers as Nanovaccines: Protein Engineering of Self-Assembled, Epitope-Exposing Nanoparticles.

In this chapter we describe two unconventional strategies for the formulation of new nanovaccines. Both strategies are based on obtaining chimeric genes that code for proteins in which the major antigens of the pathogens are fused to an elastin-like recombinamer (ELR) as carrier. ELRs are a family of synthetic protein biopolymers obtained using DNA recombinant techniques. The ELRs employed in the present chapter are block copolymers that are able to assemble, under controlled conditions, into nanoparticles similar to virus-like particles and to provoke an immune response. We describe the biosynthesis of ELRs genetically fused to an antigenic sequence from Mycobacterium tuberculosis and a simple procedure for obtaining stable nanoparticles displaying the antigen in the first strategy. The second approach describes the production of a DNA vaccine library consisting of plasmids codifying for major antigens from Rift Valley fever virus fused to different ELR-based block copolymer architectures.The procedures described can be adapted for the production of other chimeric DNA-protein vaccines based on protein polymer carriers.

Smart Nanoparticles as Advanced Anti-Akt Kinase Delivery Systems for Pancreatic Cancer Therapy.

Pancreatic cancer is one of the deadliest cancers partly due to late diagnosis, poor drug delivery to the target site, and acquired resistance to therapy. Therefore, more effective therapies are urgently needed to improve the outcome of patients. In this work, we have tested self-assembling genetically engineered polymeric nanoparticles formed by elastin-like recombinamers (ELRs), carrying a small peptide inhibitor of the protein kinase Akt, in both PANC-1 and patient-derived pancreatic cancer cells (PDX models). Nanoparticle cell uptake was measured by flow cytometry, and subcellular localization was determined by confocal microscopy, which showed a lysosomal localization of these nanoparticles. Furthermore, metabolic activity and cell viability were significantly reduced after incubation with nanoparticles carrying the Akt inhibitor in a time- and dose-dependent fashion. Self-assembling 73 ± 3.2 nm size nanoparticles inhibited phosphorylation and consequent activation of Akt protein, blocked the NF-κB signaling pathway, and triggered caspase 3-mediated apoptosis. Furthermore, in vivo assays showed that ELR-based nanoparticles were suitable devices for drug delivery purposes with long circulating time and minimum toxicity. Hence, the use of these smart nanoparticles could lead to the development of more effective treatment options for pancreatic cancer based on the inhibition of Akt.

Advanced nanomedicine and cancer: Challenges and opportunities in clinical translation.

Cancer has reached pandemic dimensions in the whole world. Although current medicine offers multiple treatment options against cancer, novel therapeutic strategies are needed due to the low specificity of chemotherapeutic drugs, undesired side effects and the presence of different incurable types of cancer. Among these new strategies, nanomedicine arises as an encouraging approach towards personalized medicine with high potential for present and future cancer patients. Therefore, nanomedicine aims to develop novel tools with wide potential in cancer treatment, imaging or even theranostic purposes. Even though numerous preclinical studies have been published with successful preliminary results, promising nanosystems have to face multiple obstacles before adoption in clinical practice as safe options for patients with cancer. In this MiniReview, we provide a short overview on the latest advances in current nanomedicine approaches, challenges and promising strategies towards more accurate cancer treatment.

Cohort Multicenter Study on the Role of Medications in Parenteral Nutrition-Related Alteration of Liver Function Tests in Adults.

BACKGROUND: Our objective was to assess in non-critically-ill adult inpatients receiving parenteral nutrition (PN) the risk of developing liver function test (LFT) alterations when receiving concomitant possibly hepatotoxic medications or others reported to improve LFTs during PN. METHODS: A multicenter retrospective analysis of prospectively collected data was performed on patients receiving PN. Two groups were recruited: group LALT (patients with any LFT alterations during PN), and group NOLALT (patients without such alterations). Exclusion criteria were previous sepsis, shock, renal failure, hyperglycemia, LFT alteration, or biliopancreatic surgical procedures. Medications were classified into 2 categories: medications reported to improve LFTs during PN (n = 8) and possibly hepatotoxic medications (n = 54), including a subgroup of possibly highly hepatotoxic medications (n = 30). RESULTS: The study included 200 patients, 136 (68.0%) in the LALT group. The groups differed in the number of patients requiring surgical intervention ≤7 days before PN (LALT, 94 [69.1%]; NOLALT, 29 [45.3%]; P < .002) and those receiving possibly hepatotoxic medications (LALT, 126 [92.6%]; NOLALT, 45 [70.3%]; P < .001). Variables in the final Cox regression model were possibly hepatotoxic medications, odds ratio (OR) 3.310 (1.678-6.530); surgical intervention prior to PN, OR 1.861 (1.277-2.711); baseline triglyceridemia, OR 1.005 (1.001-1.009); and creatinine, OR 1.861 (1.043-3.323). CONCLUSIONS: Patients who received PN and concomitantly possibly hepatotoxic medications had a 3-fold risk of developing LFT alterations. Medications reported to improve LFTs had no effect. The use of possibly hepatotoxic medications during PN was associated with LFT alterations.

Aptamer-Functionalized Natural Protein-Based Polymers as Innovative Biomaterials.

Biomaterials science is one of the most rapidly evolving fields in biomedicine. However, although novel biomaterials have achieved well-defined goals, such as the production of devices with improved biocompatibility and mechanical properties, their development could be more ambitious. Indeed, the integration of active targeting strategies has been shown to allow spatiotemporal control of cell-material interactions, thus leading to more specific and better-performing devices. This manuscript reviews recent advances that have led to enhanced biomaterials resulting from the use of natural structural macromolecules. In this regard, several structural macromolecules have been adapted or modified using biohybrid approaches for use in both regenerative medicine and therapeutic delivery. The integration of structural and functional features and aptamer targeting, although still incipient, has already shown its ability and wide-reaching potential. In this review, we discuss aptamer-functionalized hybrid protein-based or polymeric biomaterials derived from structural macromolecules, with a focus on bioresponsive/bioactive systems.

Functional characterization of an enzymatically degradable multi-bioactive elastin-like recombinamer.

One of the main goals in both tissue engineering and regenerative medicine is to design innovative synthetic scaffolds that can simulate and control the communication pathways between cells and the extracellular matrix (ECM). In this context, we describe herein the characterization of protein polymer, a recombinant elastin-like recombinamer (ELR) designed for developing tissue-engineered devices for use in vascular regeneration. This ELR is composed of an elastin-like backbone that contains a fibronectin domain, which provides specific, endothelial cell adhesion, and a protease target domain directed towards specific proteases involved in ECM remodeling. We also compare the specific response of endothelial and fibroblast cells to ELR scaffolds and show that cell adhesion and spreading on this ELR is significantly higher for endothelial cells than for fibroblasts. The reactivity of this polymer and its hydrogels to specific enzymatic degradation is demonstrated in vitro. As with natural elastin, enzymatic hydrolysis of the ELR produces elastin-derived peptides, or "matrikines", which, in turn, are potentially able to regulate important cell activities.

A DNA Vaccine Delivery Platform Based on Elastin-Like Recombinamer Nanosystems for Rift Valley Fever Virus.

This work analyzes the immunogenicity of six genetically engineered constructs based on elastin-like recombinamers (ELRs) fused to the Gn glycoprotein from Rift Valley fever virus (RVFV). Upon transfection, all constructs showed no effect on cell viability. While fusion constructs including ELR blocks containing hydrophobic amino acids (alanine or isoleucine) did not increase the expression of viral Gn in eukaryotic cells, glutamic acid- or valine-rich fusion proteins showed enhanced expression levels compared with the constructs encoding the viral antigen alone. However, in vivo DNA plasmid immunization assays determined that the more hydrophobic constructs reduced viremia levels after RVFV challenge to a higher extent than glutamic- or valine-rich encoding plasmids and were better inducers of cellular immunity as judged by in vitro restimulation experiments. Although the Gn-ELR fusion constructs did not surpass the protective efficacy of a plasmid vaccine expressing nonfused Gn, our results warrant further experiments directed to take advantage of the immunomodulatory potential of ELR biomaterials for improving vaccines against infectious diseases.

Self-Assembling ELR-Based Nanoparticles as Smart Drug-Delivery Systems Modulating Cellular Growth via Akt.

This work investigates the physicochemical properties and in vitro accuracy of a genetically engineered drug-delivery system based on elastin-like block recombinamers. The DNA recombinant techniques allowed us to create this smart complex polymer containing bioactive sequences for internalization, lysosome activation under acidic pH, and blockage of cellular growth by a small peptide inhibitor. The recombinant polymer reversibly self-assembled when the temperature was increased above 15 °C into nanoparticles with a diameter of 72 nm and negative surface charge. Furthermore, smart nanoparticles were shown to enter in the cells via clathrin-dependent endocytosis and properly blocked phosphorylation and consequent activation of Akt kinase. This system provoked apoptosis-mediated cell death in breast and colorectal cancer cells, which possess higher expression levels of Akt, whereas noncancerous cells, such as endothelial cells, fibroblasts, and mesenchymal stem cells, were not affected. Hence, we conclude that the conformational complexity of this smart elastin-like recombinamer leads to achieving successful drug delivery in targeted cells and could be a promising approach as nanocarriers with bioactive peptides to modulate multiple cellular processes involved in different diseases.

Genetically Engineered Elastin-based Biomaterials for Biomedical Applications.

Protein-based polymers are some of the most promising candidates for a new generation of innovative biomaterials as recent advances in genetic-engineering and biotechnological techniques mean that protein-based biomaterials can be designed and constructed with a higher degree of complexity and accuracy. Moreover, their sequences, which are derived from structural protein-based modules, can easily be modified to include bioactive motifs that improve their functions and material-host interactions, thereby satisfying fundamental biological requirements. The accuracy with which these advanced polypeptides can be produced, and their versatility, self-assembly behavior, stimuli-responsiveness and biocompatibility, means that they have attracted increasing attention for use in biomedical applications such as cell culture, tissue engineering, protein purification, surface engineering and controlled drug delivery. The biopolymers discussed in this review are elastin-derived protein-based polymers which are biologically inspired and biomimetic materials. This review will also focus on the design, synthesis and characterization of these genetically encoded polymers and their potential utility for controlled drug and gene delivery, as well as in tissue engineering and regenerative medicine.

Multicenter study of parenteral nutrition in non-critically ill elderly patients compared to younger patients - Study IAIOS-PN (Incidence and Assessment of Iatrogenesis in Older Subjects receiving Parenteral Nutrition).

OBJECTIVE: this study assessed the incidence of hyperglycemia, hypertriglyceridemia, and liver function test (LFT) alterations among patients older and younger than 65 years receiving parenteral nutrition (PN). A secondary objective was to compare the incidence of any of these three events. MATERIAL AND METHODS: inclusion criteria were non-critically ill adult inpatients receiving PN for ≥ 7 days in 15 hospitals in Spain. Exclusion criteria were hyperglycemia, hypertriglyceridemia, LFT alterations, sepsis, shock, pancreatic/hepatobiliary surgery, renal failure, diabetes mellitus (DM) type 1, insulin-treated DM type 2, acute DM complications, or obesity prior to PN. Patients were classified into groups YOUNG (aged 35-64) and OLD (aged 65-95). RESULTS: this study recruited 200 patients. Group YOUNG included 63 (31.5%) patients and OLD, 137 (68.5%). Hyperglycemia appeared in 37 (18.5%) patients, eight (12.7%) in group YOUNG and 29 (21.2%) in group OLD (p = 0.174). Hypertriglyceridemia appeared in only one (0.7%) patient. LFT alterations appeared in 141 (70.5%) patients, 44 (69.8%) in group YOUNG and 97 (70.8%) in group OLD (p = 1.000). The model for hyperglycemia included DM type 2, previous surgical procedure, and use of hyperglycemia-inducing medications. The model for LFT alteration included previous surgical procedure, amount of lipids and amino acids, medications causing LFT alterations and a trend for age group. The model for any event included surgical procedure, DM type 2, and medications causing alterations. CONCLUSION: patients of ≥ 65 years receiving PN had similar incidences of hyperglycemia, hypertriglyceridemia, and LFT alterations as younger patients. Additionally, older patients had trends toward lower LFT alterations.

[Effectiveness of a perioperative chronic medication reconciliation program in patients scheduled for elective surgery]. / Efectividad de un programa de conciliación perioperatoria de la medicación crónica en pacientes de cirugía programada.

BACKGROUND AND OBJECTIVE: Evaluation of the effectiveness of a perioperative medication reconciliation program in patients scheduled for elective surgery on perioperative medication management protocol adherence. MATERIAL AND METHODS: Ambispective cross-cohort study: prospective cohort (Group A), and retrospective control cohort (Group B) without intervention. Forty eight hours before admission a pharmacist generates a proposal on medication active prior to admission to be maintained/discontinued during the perioperative period according to the hospital protocols. The Pharmacy proposal is validated/modified by an anesthesiologist 24h before admission. At the time of admission, after checking with the patient the medication list, the nurse initiates scheduled medication administration pre and postoperatively. The indicator for program effectiveness assessment is the degree of compliance with perioperative medication management protocols at 24, 48 and 72h of admission for intervention and control cohorts. RESULTS: Five hundred and three patients were included. One thousand two hundred and eleven pharmacy medication management recommendations were performed in Group A (intervention), of which 94.9% agreed with protocol. In Group B (control before program implementation) action taken by physicians agreed with protocol only in 32.4% of all cases. CONCLUSIONS: The medication reconciliation program significantly increased compliance with the recommendations on chronic medication perioperative management.