Antibody nanoparticle conjugate-based targeted immunotherapy for non-small cell lung cancer.

The use of immune checkpoint inhibitors, which activate T cells, is a paradigm shift in the treatment of non-small cell lung cancer. However, the overall response remains low. To address this limitation, here we describe a novel platform, termed antibody-conjugated drug-loaded nanotherapeutics (ADN), which combines immunotherapy and molecularly targeted therapy. An ADN was designed with an anti-CD47 and anti-programmed death ligand 1 (PDL1) antibody pair on the surface of the nanoparticle and a molecularly targeted inhibitor of the PI3K (phosphatidylinositol 3-kinase)/AKT/mTOR (mammalian target of rapamycin) pathway, PI103, entrapped in the nanoparticle. The anti-CD47-PDL1-ADN exhibited greater antitumor efficacy than current treatment options with a PDL1 inhibitor in vivo in an aggressive lung cancer immunocompetent mouse model. Dual antibody-drug-loaded nanotherapeutics can emerge as an attractive platform to improve outcomes with cancer immunotherapy.

Engine shutdown: migrastatic strategies and prevention of metastases.

Most cancer-related deaths among patients with solid tumors are caused by metastases. Migrastatic strategies represent a unique therapeutic approach to prevent all forms of cancer cell migration and invasion. Because the migration machinery has been shown to promote metastatic dissemination, successful migrastatic therapy may reduce the need for high-dose cytotoxic therapies that are currently used to prevent the risk of metastatic dissemination. In this review we focus on anti-invasive and antimetastatic strategies that hold promise for the treatment of solid tumors. The best targets for migrastatic therapy would be those that are required by all forms of motility, such as ATP availability, mitochondrial metabolism, and cytoskeletal dynamics and cell contractility.

Silicane Derivative Increases Doxorubicin Efficacy in an Ovarian Carcinoma Mouse Model: Fighting Drug Resistance.

The development of cancer resistance continues to represent a bottleneck of cancer therapy. It is one of the leading factors preventing drugs to exhibit their full therapeutic potential. Consequently, it reduces the efficacy of anticancer therapy and causes the survival rate of therapy-resistant patients to be far from satisfactory. Here, an emerging strategy for overcoming drug resistance is proposed employing a novel two-dimensional (2D) nanomaterial polysiloxane (PSX). We have reported on the synthesis of PSX nanosheets (PSX NSs) and proved that they have favorable properties for biomedical applications. PSX NSs evinced unprecedented cytocompatibility up to the concentration of 300 µg/mL, while inducing very low level of red blood cell hemolysis and were found to be highly effective for anticancer drug binding. PSX NSs enhanced the efficacy of the anticancer drug doxorubicin (DOX) by around 27.8-43.4% on average and, interestingly, were found to be especially effective in the therapy of drug-resistant tumors, improving the effectiveness of up to 52%. Fluorescence microscopy revealed improved retention of DOX within the drug-resistant cells when bound on PSX NSs. DOX bound on the surface of PSX NSs, i.e., PSX@DOX, improved, in general, the DOX cytotoxicity in vitro. More importantly, PSX@DOX reduced the growth of DOX-resistant tumors in vivo with 3.5 times better average efficiency than the free drug. Altogether, this paper represents an introduction of a new 2D nanomaterial derived from silicane and pioneers its biomedical application. As advances in the field of material synthesis are rapidly progressing, novel 2D nanomaterials with improved properties are being synthesized and await thorough exploration. Our findings further provide a better understanding of the mechanisms involved in the cancer resistance and can promote the development of a precise cancer therapy.

Oxidized polysaccharides for anticancer-drug delivery: What is the role of structure?

Study provides an in-depth analysis of the structure-function relationship of polysaccharide anticancer drug carriers and points out benefits and potential drawbacks of differences in polysaccharide glycosidic bonding, branching and drug binding mode of the carriers. Cellulose, dextrin, dextran and hyaluronic acid have been regioselectively oxidized to respective dicarboxylated derivatives, allowing them to directly conjugate cisplatin, while preserving their major structural features intact. The structure of source polysaccharide has crucial impact on conjugation effectiveness, carrier capacity, drug release rates, in vitro cytotoxicity and cellular uptake. For example, while branched structure of dextrin-based carrier partially counter the undesirable initial burst release, it also attenuates the cellular uptake and the cytotoxicity of carried drug. Linear polysaccharides containing ß-(1→4) glycosidic bonds and oxidized at C2 and C3 (cellulose and hyaluronate) have the best overall combination of structural features for improved drug delivery applications including potentiation of the cisplatin efficacy towards malignances.

Two-dimensional materials in biomedical, biosensing and sensing applications.

Two-dimensional (2D) materials are at the forefront of materials research. Here we overview their applications beyond graphene, such as transition metal dichalcogenides, monoelemental Xenes (including phosphorene and bismuthene), carbon nitrides, boron nitrides along with transition metal carbides and nitrides (MXenes). We discuss their usage in various biomedical and environmental monitoring applications, from biosensors to therapeutic treatment agents, their toxicity and their utility in chemical sensing. We highlight how a specific chemical, physical and optical property of 2D materials can influence the performance of bio/sensing, improve drug delivery and photo/thermal therapy as well as affect their toxicity. Such properties are determined by crystal phases electrical conductivity, degree of exfoliation, surface functionalization, strong photoluminescence, strong optical absorption in the near-infrared range and high photothermal conversion efficiency. This review conveys the great future of all the families of 2D materials, especially with the expanding 2D materials' landscape as new materials emerge such as germanene and silicene.

Locked and Loaded: Ruthenium(II)-Capped Cucurbit[n]uril-Based Rotaxanes with Antimetastatic Properties.

We report here the first coupling of Ru(II) units with cucurbit[6/7]uril-based pseudorotaxane ligands meant for biological application. The resulting ruthenium-capped rotaxanes were fully characterized, and a structure of one supramolecular system was determined by X-ray diffraction. Because the biological properties of Ru-based metallodrugs are tightly linked to the ligand-exchange processes, the effect of salt concentration on the hydrolysis of chlorides from the Ru(II) center was monitored by using 1H NMR spectroscopy. The biological activity of Ru(II)-based rotaxanes was evaluated for three selected mammalian breast cell lines, HBL-100, MCF-7, and MDA-MB-231. The antimetastatic activity of the assembled cationic Ru(II)-rotaxane systems, evaluated in migration assays against MCF-7 and MDA-MB-231 cell lines, is notably enhanced compared to that of RAPTA-C, a reference that was used. The indicated synergistic effect of combining Ru(II) with a pseudorotaxane unit opens a new direction in searching for anticancer supramolecular metallodrugs.

Unexpected therapeutic effects of cisplatin.

Cisplatin is a widely used chemotherapeutic agent that is clinically approved to fight both carcinomas and sarcomas. It has relatively high efficiency in treating ovarian cancers and metastatic testicular cancers. It is generally accepted that the major mechanism of cisplatin anti-cancer action is DNA damage. However, cisplatin is also effective in metastatic cancers and should, therefore, affect slow-cycling cancer stem cells in some way. In this review, we focused on the alternative effects of cisplatin that can support a good therapeutic response. First, attention was paid to the effects of cisplatin at the cellular level such as changes in intracellular pH and cellular mechanical properties. Alternative cellular targets of cisplatin, and the effects of cisplatin on cancer cell metabolism and ER stress were also discussed. Furthermore, the impacts of cisplatin on the tumor microenvironment and in the whole organism context were reviewed. In this review, we try to reveal possible causes of the unexpected effectiveness of this anti-cancer drug.

Graphene Oxide Nanoplatelets Potentiate Anticancer Effect of Cisplatin in Human Lung Cancer Cells.

Graphene oxide (GO) has been widely explored by many in drug delivery strategies and toxicity assays. The toxicity of graphene oxide depends on the size of the sheets. Smaller sheets show lower toxicity, a quality which is essential for utilization in biomedical applications. However, despite vast research on GO, anticancer properties and drug carrier capabilities of graphene oxide nanoplatelets have yet to be fully explored. Herein, we have uniquely prepared graphene oxide nanoplatelets (GONPs) from well-defined stacked graphite nanofibers (SGNF) with a base of 50 × 50 nm2 for toxicity and drug potentiation studies when coadministered with the chemotherapeutic drug cisplatin (CP) in human lung cancer cells, A549 cells. Results obtained from our studies have found that not only were GONPs able to act as drug carriers, but they can also significantly potentiate anticancer effect of CP in A549 cells.

Selectively Oxidized Cellulose with Adjustable Molecular Weight for Controlled Release of Platinum Anticancer Drugs.

The synthesis of selectively oxidized cellulose, 2,3-dicarboxycellulose (DCC), is optimized for preparation of highly oxidized material for biological applications, which includes control over the molecular weight of the product during its synthesis. Conjugates of DCC and cisplatin simultaneously offer a very high drug binding efficiency (>90%) and drug loading capacity (up to 50 wt %), while retaining good aqueous solubility. The adjustable molecular weight of the DCC together with variances in drug feeding ratio allows to optimize cisplatin release profiles from delayed (<2% of cisplatin released during 6 h) to classical burst release with more than 60% of cisplatin released after 24 h. The release rates are also pH-dependent (up to 2 times faster release at pH 5.5 than at pH 7.4), which allows to exploit the acidic nature of tumor microenvironment. Extensive in vitro studies were performed on eight different cell lines for two cisplatin-DCC conjugates with different release profiles. In comparison with free cisplatin, both cisplatin-DCC conjugates demonstrated considerably lower cytotoxicity toward healthy cells. Conjugates with burst release profiles were found more effective against prostate cell lines, while DCC conjugates with slower release were more cytotoxic against ovarian and lung carcinoma cell lines. In vivo studies indicated a significantly longer survival rate, a reduction in tumor volume, and a higher accumulation of platinum in tumors of mice treated with the cisplatin-DCC conjugate in comparison to those treated by free cisplatin.

Black Phosphorus Cytotoxicity Assessments Pitfalls: Advantages and Disadvantages of Metabolic and Morphological Assays.

Black phosphorus (BP) belongs to a group of 2D nanomaterials and nowadays attracts constantly increasing attention. Parallel to the growing utilization of BP nanomaterial increase also the requirements for the thorough comprehension of its potential impact on human and animal health. The aim of this study was to compare and discuss five assays commonly used for the cytotoxicity assessments of nanomaterials with a special focus on BP nanoparticles. A comprehensive survey of factors and pitfalls is provided that should be accounted for when assessing their toxicity and pointed to their inconsistency. BP might introduce various levels of interference during toxicity assessments depending on its concentration applied. More importantly, the BP toxicity evaluation was found to be influenced by the nature of assay chosen. These are based on different principles and do not have to assess all the cellular events equally. A commercial assay based on the measurement of protease activity was identified to be the most suitable for the BP toxicity assessment. Further, the benefit of time-lapse quantitative phase imaging for nanomaterial toxicity evaluation was highlighted. Unlike the conventional assessments it provides real-time analysis of the processes accompanying BP administration and enables to understand them deeper and in the context.

Establishment of oral squamous cell carcinoma cell line and magnetic bead-based isolation and characterization of its CD90/CD44 subpopulations.

In this study, we describe the establishment of the human papillomavirus 18-positive, stage II, grade 1, T2N0M0 head and neck tumor primary cell line derived from oral squamous cell carcinoma of a non-smoking patient by using two different protocols. Furthermore, a preparation of subpopulations derived from this primary cell line according to the cluster of differentiation molecules CD44/CD90 status using magnetic bead-based separation and their characterization was performed. Impedance-based real-time cell analysis, enzyme-linked immunsorbant assay (ELISA), wound-healing assay, flow-cytometry, gene expression analysis, and MTT assay were used to characterize these four subpopulations (CD44+/CD90-, CD44-/CD90-, CD44+/CD90+, CD44-/CD90-). We optimised methodics for establishement of primary cell lines derived from oral squamous cell carcinoma tissue samples and subsequent separation of mesenchymal (CD90+) and epithelial (CD90-) types of tumorous cells. Primary cell line prepared by using trypsin proteolysis was more viable than the one prepared by using collagenase. According to our results, CD90 separation is a necessary step in preparation of permanent tumor-tissue derived cell lines. Based on the wound-healing assay, CD44+ cells exhibited stronger migratory capacity than CD44- subpopulations. CD44+ subpopulations had also significantly higher expression of BIRC5 and SOX2, lower expression of FLT1 and IL6, and higher levels of basal autophagy compared to CD44- subpopulations. Furthermore, co-cultivation experiments revealed that CD44-/CD90+ cells supported growth of epithelial tumor cells (CD44+/CD90-). On the contrary, factors released by CD44+/CD90+ type of cells seem to have rather inhibiting effect. The most cisplatin-resistant subpopulation with the shortest doubling time was CD44-/CD90+, but this subpopulation had a low migratory capacity.

Reduction of Doxorubicin-Induced Cardiotoxicity Using Nanocarriers: A Review.

BACKGROUND: Anthracycline antibiotic doxorubicin (DOX) is a very potent and extensively prescribed chemotherapeutic drug. It is widely utilized in the therapy of variety of haematological and solid tumours, although its administration is commonly accompanied with several severe side effects. The most serious one is a development of dose-dependent and cumulative cardiotoxicity. In the course of time, many strategies have been investigated in order to avoid or at least to diminish DOX-induced cardiac dysfunction; these include reduction of toxic effect by coadministration with iron chelators (dexrazoxane), trastuzumab, taxanes, statins, and ACE-inhibitors. However, the attenuation of cardiotoxic effect is still not satisfactory yet. OBJECTIVE: This review represents an overall appraisal of studies concerning with the utilization of various doxorubicinloaded nanoparticles in the cancer treatment with specific emphasis on those studies evaluating their influence on the reduction of heart tissue damage. CONCLUSION: Introduction of nanoscale drug delivery systems undoubtedly represents nowadays one of the most promising tools for lowering systemic toxicity. Nanoparticles enable to target the therapeutic payload directly towards the tumor tissue, thus leading to the increased accumulation of the drug in the desired tissue and simultaneously protecting surrounding healthy tissues.

Evaluation of EGFR as a prognostic and diagnostic marker for head and neck squamous cell carcinoma patients.

Approximately 90% of all head and neck tumors are squamous cell carcinomas. The overall survival of patients with head and neck squamous cell carcinoma (HNSCC) is low (≤50%). A non-invasive marker of disease progression is sorely required. The present study focused on the plasmatic levels of epidermal growth factor receptor (EGFR) in HNSCC patients (N=92) compared with healthy (N=29) and diabetic [type 2 diabetes mellitus (T2DM); N=26] controls. Enzyme-linked immunosorbent assay using antibodies against the extracellular region of EGFR (L25-S645) was performed. No significant changes were observed between diabetic and healthy controls. However, there were significantly higher EGFR plasma levels in HNSCC patients compared with both control groups (P=0.001 and 0.005, respectively). Receiver operating characteristic curve analysis identified a sensitivity of 76.09%, a specificity of 67.27% and an area under curve of 0.727 for this comparison. No significant association was observed between EGFR plasma levels and tumor stage, tumor grade, lymph node or distant metastasis occurrence, smoking habit or hypertension. However, the presence of human papillomavirus infection and T2DM in HNSCC patients had borderline effect on the plasma EGFR levels. Survival analysis revealed no significant influence of plasmatic EGFR levels on the overall and disease-specific survival of HNSCC patients. In conclusion, EGFR plasma levels appear to be a relatively promising diagnostic, but poor prognostic, HNSCC marker.

Sulforaphane-induced apoptosis involves the type 1 IP3 receptor.

In this study we show that anti-tumor effect of sulforaphane (SFN) is partially realized through the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1). This effect was verified in vitro on three different stable cell lines and also in vivo on the model of nude mice with developed tumors. Early response (6 hours) of A2780 ovarian carcinoma cells to SFN treatment involves generation of mitochondrial ROS and increased transcription of NRF2 and its downstream regulated genes including heme oxygenase 1, NAD(P)H:quinine oxidoreductase 1, and KLF9. Prolonged SFN treatment (24 hours) upregulated expression of NRF2 and IP3R1. SFN induces a time-dependent phosphorylation wave of HSP27. Use of IP3R inhibitor Xestospongin C (Xest) attenuates both SFN-induced apoptosis and the level of NRF2 protein expression. In addition, Xest partially attenuates anti-tumor effect of SFN in vivo. SFN-induced apoptosis is completely inhibited by silencing of IP3R1 gene but only partially blocked by silencing of NRF2; silencing of IP3R2 and IP3R3 had no effect on these cells. Xest inhibitor does not significantly modify SFN-induced increase in the rapid activity of ARE and AP1 responsive elements. We found that Xest effectively reverses the SFN-dependent increase of nuclear content and decrease of reticular calcium content. In addition, immunofluorescent staining with IP3R1 antibody revealed that SFN treatment induces translocation of IP3R1 to the nucleus. Our results clearly show that IP3R1 is involved in SFN-induced apoptosis through the depletion of reticular calcium and modulation of transcription factors through nuclear calcium up-regulation.

Effect of HPV on tumor expression levels of the most commonly used markers in HNSCC.

Approximately 90 % of head and neck cancers are squamous cell carcinomas (HNSCC), and the overall 5-year survival rate is not higher than 50 %. There is much evidence that human papillomavirus (HPV) infection may influence the expression of commonly studied HNSCC markers. Our study was focused on the possible HPV-specificity of molecular markers that could be key players in important steps of cancerogenesis (MKI67, EGF, EGFR, BCL-2, BAX, FOS, JUN, TP53, MT1A, MT2A, VEGFA, FLT1, MMP2, MMP9, and POU5F). qRT-PCR analysis of these selected genes was performed on 74 biopsy samples of tumors from patients with histologically verified HNSCC (22 HPV-, 52 HPV+). Kaplan-Meier analysis was done to determine the relevance of these selected markers for HNSCC prognosis. In conclusion, our study confirms the impact of HPV infection on commonly studied HNSCC markers MT2A, MMP9, FLT1, VEGFA, and POU5F that were more highly expressed in HPV-negative HNSCC patients and also shows the relevance of studied markers in HPV-positive and HPV-negative HNSCC patients.

Prognostic significance of the tumour-adjacent tissue in head and neck cancers.

Even with significant advances in operative skills and adjuvant therapies, the overall survival of patients suffering with head and neck squamous cancers (HNSCC) is unsatisfactory. Accordingly, no clinically useful prognostic biomarkers have been found yet for HNSCC. Many studies analysed the expression of potential markers in tumour tissues compared to adjacent tissues. Nevertheless, due to the sharing of the same microenvironment, adjacent tissues show molecular similarity to tumour tissues. Thus, gene expression patterns of 94 HNSCC tumorous tissues were compared with 31 adjacent tissues and with 10 tonsillectomy specimens of non-cancer individuals. The genes analysed at RNA level using quantitative RT-PCR and correlated with clinico-pathological conditions were as follows: EGF, EGFR, MKI67, BCL2, BAX, FOS, JUN, TP53, VEGF, FLT1, MMP2, MMP9, MT1A and MT2A. The elevated MT2A, BAX, EGF and JUN expression was associated with the influence of tumour cells on the rearrangement of healthy tissues, as well as a significant shift in the BAX/BCL2 ratio. Our investigation also indicated that adjacent tissues play an important role in cancerogenesis by releasing several tumour-supporting factors such as EGF. A gradual increase in the metallothionein expression, from the lowest one in tonsillectomy samples to the highest ones in tumour samples, suggests that MT expression might be tissue reaction to the presence of tumour cells. The results of this study confirmed the significance of metallothionein in tumori-genesis and gave evidences for its use as a potential HNSCC biomarker. Furthermore, this study highlighted the importance of histologically normal tumour-adjacent tissue in prediction of HNSCC progress.

Modulation of induced cytotoxicity of doxorubicin by using apoferritin and liposomal cages.

Doxorubicin is an effective chemotherapeutic drug, however, its toxicity is a significant limitation in therapy. Encapsulation of doxorubicin inside liposomes or ferritin cages decreases cardiotoxicity while maintaining anticancer potency. We synthesized novel apoferritin- and liposome-encapsulated forms of doxorubicin ("Apodox" and "lip-8-dox") and compared its toxicity with doxorubicin and Myocet on prostate cell lines. Three different prostatic cell lines PNT1A, 22Rv1, and LNCaP were chosen. The toxicity of the modified doxorubicin forms was compared to conventional doxorubicin using the MTT assay, real-time cell impedance-based cell growth method (RTCA), and flow cytometry. The efficiency of doxorubicin entrapment was 56% in apoferritin cages and 42% in the liposome carrier. The accuracy of the RTCA system was verified by flow-cytometric analysis of cell viability. The doxorubicin half maximal inhibition concentrations (IC50) were determined as 170.5, 234.0, and 169.0 nM for PNT1A, 22Rv1, and LNCaP, respectively by RTCA. Lip8-dox is less toxic on the non-tumor cell line PNT1A compared to doxorubicin, while still maintaining the toxicity to tumorous cell lines similar to doxorubicin or epirubicin (IC50 = 2076.7 nM for PNT1A vs. 935.3 and 729.0 nM for 22Rv1 and LNCaP). Apodox IC50 was determined as follows: 603.1, 1344.2, and 931.2 nM for PNT1A, 22Rv1, and LNCaP.

Utilization of paramagnetic microparticles for automated isolation of free circulating mRNA as a new tool in prostate cancer diagnostics.

Determination of serum mRNA gained a lot of attention in recent years, particularly from the perspective of disease markers. Streptavidin-modified paramagnetic particles (SMPs) seem an interesting technique, mainly due to possible automated isolation and high efficiency. The aim of this study was to optimize serum isolation protocol to reduce the consumption of chemicals and sample volume. The following factors were optimized: amounts of (i) paramagnetic particles, (ii) oligo(dT)20 probe, (iii) serum, and (iv) the binding sequence (SMPs, oligo(dT)20 , serum vs. oligo(dT)20 , serum and SMPs). RNA content was measured, and the expression of metallothionein-2A as possible prostate cancer marker was analyzed to demonstrate measurable RNA content with ability for RT-PCR detection. Isolation is possible on serum volume range (10-200 µL) without altering of efficiency or purity. Amount of SMPs can be reduced up to 5 µL, with optimal results within 10-30 µL SMPs. Volume of oligo(dT)20 does not affect efficiency, when used within 0.1-0.4 µL. This optimized protocol was also modified to fit needs of automated one-step single-tube analysis with identical efficiency compared to conventional setup. One-step analysis protocol is considered a promising simplification, making RNA isolation suitable for automatable process.