This study is about fine tuning the targeting capacity of peptide-decorated nanoparticles to discriminate between cells that express different integrin make-ups. Using microfluidic-assisted ...nanoprecipitation, we have prepared poly(lactic acid-co-glycolic acid) (PLGA) nanoparticles with a PEGylated surface decorated with two different arginine-glycine-aspartic acid (RGD) peptides: one is cyclic (RGDFC) and has specific affinity towards α
β
integrin heterodimers; the other is linear (RGDSP) and is reported to bind equally α
β
and α
β
. We have then evaluated the nanoparticle internalization in two cell lines with a markedly different integrin fingerprint: ovarian carcinoma A2780 (almost no α
β
, moderate in α
β
) and glioma U87MG (very high in α
β
, moderate/high in α
β
). As expected, particles with cyclic RGD were heavily internalized by U87MG (proportional to the peptide content and abrogated by anti-α
β
) but not by A2780 (same as PEGylated particles). The linear peptide, on the other hand, did not differentiate between the cell lines, and the uptake increase vs. control particles was never higher than 50%, indicating a possible low and unselective affinity for various integrins. The strong preference of U87MG for cyclic (vs. linear) peptide-decorated nanoparticles was shown in 2D culture and further demonstrated in spheroids. Our results demonstrate that targeting specific integrin make-ups is possible and may open the way to more precise treatment, but more efforts need to be devoted to a better understanding of the relation between RGD structure and their integrin-binding capacity.
This paper reports the first use of a novel completely optically based photothermal method (O-PTIR) for obtaining infrared spectra of both fixed and living cells using a quantum cascade laser (QCL) ...and optical parametric oscillator (OPO) laser as excitation sources, thus enabling all biologically relevant vibrations to be analyzed at submicron spatial resolution. In addition, infrared data acquisition is combined with concomitant Raman spectra from exactly the same excitation location, meaning the full vibrational profile of the cell can be obtained. The pancreatic cancer cell line MIA PaCa-2 and the breast cancer cell line MDA-MB-231 are used as model cells to demonstrate the capabilities of the new instrumentation. These combined modalities can be used to analyze subcellular structures in both fixed and, more importantly, live cells under aqueous conditions. We show that the protein secondary structure and lipid-rich bodies can be identified on the submicron scale.
Lipid nanoparticles (LNPs) are important delivery systems for RNA-based therapeutics, yet the mechanism of their interaction with endosomal membranes remains unclear. Here, the interactions of ...nucleic acid-loaded LNPs that contain an ionizable lipid with models of the early and late endosomal membranes are studied, for the first time, using different reflectometry techniques. Novel insight is provided with respect to the subphase pH, the stage of the endosome, and the nature of the nucleic acid cargo. It is found that the insertion of lipids from the LNPs into the model membrane is greatest at pH 6.5 and 5.5, whereas at higher pH, lipid insertion is suppressed with evidence instead for the binding of intact LNPs, demonstrating the importance of the pH in the fusion of LNPs undergoing the endosomal pathway. Furthermore, and independently of the pH, the effect of the early- versus late-stage endosomal models is minimal, suggesting that the increased fluidity and anionic nature of the late endosome has little effect on the extent of LNP interaction. Last, there is greater nucleic acid delivery from LNPs containing mRNA than Poly(A), indicating that the extent of interaction can be tuned according to the nature of the nucleic acid cargo. Such new information on the relative impact of factors influencing nucleic acid delivery by LNP interactions with endosomal membranes is important in the design and tuning of vehicles with improved nucleic acid delivery capacities.
The development of delivery systems capable of tumor targeting represents a promising strategy to overcome issues related to nonspecific effects of conventional anticancer therapies. Currently, one ...of the most investigated agents for cancer targeting is hyaluronic acid (HA), since its receptor, CD44, is overexpressed in many cancers. However, most of the studies on CD44/HA interaction have been so far performed in cell-free or genetically modified systems, thus leaving some uncertainty regarding which cell-related factors influence HA binding and internalization (collectively called “uptake”) into CD44-expressing cells. To address this, the expression of CD44 (both standard and variants, designated CD44s and CD44v, respectively) was evaluated in human dermal fibroblasts (HDFs) and a large panel of cancer cell lines, including breast, prostate, head and neck, pancreatic, ovarian, colorectal, thyroid, and endometrial cancers. Results showed that CD44 isoform profiles and expression levels vary across the cancer cell lines and HDF and are not consistent within the cell origin. Using composite information of CD44 expression, HA binding, and internalization, we found that the expression of CD44v can negatively influence the uptake of HA, and, instead, when cells primarily expressed CD44s, a positive correlation was observed between expression and uptake. In other words, CD44shigh cells bound and internalized more HA compared to CD44slow cells. Moreover, CD44shigh HDFs were less efficient in uptaking HA compared to CD44shigh cancer cells. The experiments described here are the first step toward understanding the interplay between CD44 expression, its functionality, and the underlying mechanism(s) for HA uptake. The results show that factors other than the amount of CD44 receptor can play a role in the interaction with HA, and this represents an important advance with respect to the design of HA-based carriers and the selection of tumors to treat according to their CD44 expression profile.
CD44 is an endocytic hyaluronic acid (HA) receptor, and is overexpressed in many carcinomas. This has encouraged the use of HA to design CD44‐targeting carriers. This paper is about dissecting the ...mechanistic role of CD44. Here, HA‐decorated nanoparticles are used to deliver siRNA to both tumoral (AsPC‐1, PANC‐1, HT‐29, HCT‐116) and non‐tumoral (fibroblasts, differently polarized THP‐1 macrophages, HUVEC) human cell lines, evaluating the initial binding of the nanoparticles, their internalization rate, and the silencing efficiency (cyclophilin B (PPIB) gene). Tumoral cells internalize faster and experience higher silencing than non‐tumoral cells. This is promising as it suggests that, in a tumor, HA nanocarriers may have limited off‐target effects. More far‐reaching is the inter‐relation between the four parameters of the study: CD44 expression, HA binding on cell surfaces, internalization rate, and silencing efficiency. No correlation is found between binding (an early event) and any of the other parameters, whereas silencing correlates both with speed of the internalization process and CD44 expression. This study confirms on one hand that HA‐based carriers can perform a targeted action, but on the other it suggests that this may not be due to a selective binding event, but rather to a later recognition leading to selective internalization.
Hyaluronic acid (HA) is used for CD44‐targeted therapies. The evidence points to CD44 possibly being a selective internalizer rather than a primary binder for HA, which implies that a first (unselective?) receptor may be involved in HA capture before trafficking to CD44.
Two of the main limitations of conventional cancer drugs are their lack of ability to discriminate between cancer and normal cells, producing the well-known side effects, and the resistance to radio- ...and chemotherapy. The resistance is mainly due to the presence of hypoxic (low oxygen) regions in tumours where the Hypoxia Inducible Factor (HIF) transcriptional system regulates the expression of hypoxia-dependent prosurvival and drug resistance genes. The development of a delivery system capable of specifically targeting and penetrating tumours is a promising strategy to overcome these issues. Currently, one of the most investigated agents for cancer targeting is hyaluronic acid (HA), since its main receptor, CD44, is overexpressed in many cancers. However, it is still unclear which cell-related factors influence HA binding and internalisation (collectively called “uptake”) into CD44 expressing cells. To address this, the expression of CD44 (standard and variants isoforms, CD44s and CD44v respectively) was evaluated in human dermal fibroblasts (HDF) as healthy control and in a large panel of cancer cell lines. It was found that the expression of CD44v can negatively influence the uptake of HA but, interestingly, the healthy control HDF that expressed high levels of CD44s were less efficient in taking up HA when compared to high expressing CD44s cancer cells. Starting from this knowledge, HA-coated chitosan (CS)- based nanoparticles (NPs), engineered to contain siRNAs to knockdown HIF-1, were used to target CD44s expressing pancreatic cancer cell lines (MIA PaCa-2 and PANC1). Two different NPs were formulated using low or high molecular weight (LMW or HMW) CS evaluating differences in their internalisation by cells and correlating that with gene silencing studies. Cells showed a slower internalisation of HMW CS/HA NPs compared to the LMW counterpart. However, the latter were slightly more efficient in HIF-1α and its downstream target genes knockdown. LMW CS/HA NPs were able to penetrate deeply into multicellular spheroids, but when injected intravenously in tumour bearing mice they resulted not completely stable (with potential decomplexation) as 48 hours post-injection most of HA was detected in the liver and the siRNA in the kidneys. In conclusion, these results provide some understanding on the interplay between CD44 expression, its functionality and the underlying mechanism(s) for HA uptake and importantly, demonstrate that factors other than the amount of CD44 receptor can play a role in the interaction with HA. However, other methods for preparation or in vivo administration of HA/CS NPs need to be evaluated to assess the system stability in a route different from intravenous.
To evaluate the effectiveness of the measles-mumps-rubella (MMR) vaccine in reducing hospitalizations for infectious disease, targeted and not targeted, as well as from respiratory diseases in ...children in Rome.
The cohort was recomposed through record linkage of 2 archives (vaccination register and hospital discharge records.
The analysis included 11,004 children. 20.9% did not receive the MMR vaccination, 49% and 30.1% received one and 2 doses. There were no hospitalizations for rubella, 2 for mumps, and 12 for measles. The vaccine was highly protective against measles and mumps hospitalizations (HR = 0.10; 95% CI: 0.03.0.34). Regarding all infectious diseases there were 414 hospitalizations, and the vaccine was protective (HR = 0.29; 95% CI: 0.25 to 0.34). Concerning respiratory diseases, there were 809 admissions (7.4%), and the vaccine was highly protective (HR: 0.18; 95% CI: 0.07 to 0.48).
MMR vaccination is effective for the primary prevention of target and not targeted infectious diseases and may also limit hospitalizations for respiratory diseases.
The risk of getting influenza and pneumococcal disease is higher in cancer patients, and serum antibody levels tend to be lower in patients with hematological malignancy.
To assess flu and ...pneumococcal vaccinations efficacy, effectiveness, and safety in onco-hematological patients.
Two systematic reviews and possible meta-analysis were conducted to summarize the results of all primary study in the scientific literature about the flu and pneumococcal vaccine in onco-hematological patients. Literature searches were performed using Pub-Med and Scopus databases. StatsDirect 2.8.0 was used for the analysis.
22 and 26 studies were collected respectively for flu and pneumococcal vaccinations. Protection rate of booster dose was 30% (95% CI=6-62%) for H1N1. Pooled prevalence protection rate of H3N2 and B was available for meta-analysis only for first dose, 42.6% (95% CI=23.2 - 63.3 %) and 39.6 % (95% CI=26%-54.1%) for H3N2 and B, respectively. Response rate of booster dose resulted 35% (95% CI=19.7-51.2%) for H1N1, 23% (95% CI=16.6-31.5%) for H3N2, 29% (95% CI=21.3-37%) for B.
Despite the low rate of response, flu, and pneumococcal vaccines are worthwhile for patients with hematological malignancies. Patients undergoing chemotherapy in particular rituximab, splenectomy, transplant recipient had lower and impaired response. No serious adverse events were reported for both vaccines.
To describe the Italian Obstetric Surveillance System (ItOSS) investigating maternal death through incident case reporting and confidential enquiries.
All maternal deaths occurred in any public and ...private health facility in 8 Italian regions covering 73% of national births have been notified to the ItOSS. Every incident case is confidentially reviewed to assess quality of care and establish the cause and avoidability of the death.
A total of 106 maternal deaths among 1 455 545 live births have been notified to the surveillance system in 2013-17. Haemorrhage, sepsis and hypertensive disorders of pregnancy are the leading causes of direct maternal deaths due to obstetric causes.
A maternal mortality surveillance system, including incidence reporting and confidential enquiries along with a retrospective analysis of administrative data sources, emerged as the best option for case ascertainment and for preventing avoidable maternal deaths.
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