Biodistribution of Poly(alkyl cyanoacrylate) Nanoparticles in Mice and Effect on Tumor Infiltration of Macrophages into a Patient-Derived Breast Cancer Xenograft

We have investigated the biodistribution and tumor macrophage infiltration after intravenous injection of the poly(alkyl cyanoacrylate) nanoparticles (NPs): PEBCA (poly(2-ethyl-butyl cyanoacrylate), PBCA (poly(n-butyl cyanoacrylate), and POCA (poly(octyl cyanoacrylate), in mice. These NPs are structurally similar, have similar PEGylation, and have previously been shown to give large variations in cellular responses in vitro. The PEBCA NPs had the highest uptake both in the patient-derived breast cancer xenograft MAS98.12 and in lymph nodes, and therefore, they are the most promising of these NPs for delivery of cancer drugs. High-resolution magic angle spinning magnetic resonance (HR MAS MR) spectroscopy did not reveal any differences in the metabolic profiles of tumors following injection of the NPs, but the PEBCA NPs resulted in higher tumor infiltration of the anti-tumorigenic M1 macrophages than obtained with the two other NPs. The PEBCA NPs also increased the ratio of M1/M2 (anti-tumorigenic/pro-tumorigenic) macrophages in the tumors, suggesting that these NPs might be used both as a vehicle for drug delivery and to modulate the immune response in favor of enhanced therapeutic effects

Overall survival after resection for colon cancer in a national cohort study was adversely affected by TNM stage, lymph node ratio, gender, and old age

Background A national surveillance program of colon cancer treatment was introduced in 2007. We examined prognostic factors for colon cancer operated in 2000 with an aim of improving survival in the new program and a special focus on the merit of lymph node yield. Methods A cohort of 269 patients, 152 women (56.5%), with a mean age of 71 years, was operated for colon cancer in 2000 at three teaching hospitals and followed up for 7 years. Results Overall 5-year survival was 58.0%, and overall hospital mortality was 5.2%, with 4.5% in elective cases and 12.5% after urgent surgery. In only 41.1% of the specimens were 12 or more lymph nodes retrieved, but this did not affect survival in the combined cohort, although one of the hospitals achieved a significantly better result with a harvest of 12 or more lymph nodes. In a multivariate analysis, old age, gender, a high lymph node ratio (LNR) at stage III, and tumor–node–metastasis stage were adverse factors for survival. Conclusions The operative mortality was high and should be reassessed. The lymph node count did not have a significant impact on outcome overall, whereas the LNR proved significant for stage III. A prospective protocol using overall lymph node yield as a surrogate measure for more radical surgery, nevertheless, seems warranted to improve the lymph node harvest according to international recommendations

Cellular uptake of nanoparticles: Involvement of caveolae?

Here we discuss some pitfalls and challenges briefly when investigating which endocytic mechanisms are involved in the cellular uptake of nanoparticles. We specifically discuss some common misunderstandings regarding studies claimed to demonstrate uptake via caveolae. Scientists in the nanomedicine field should be aware that reducing the membrane content of cholesterol by adding methyl-β-cyclodextrin removes caveolae and inhibits other uptake mechanisms, such as macropinocytosis as well. Furthermore, the general tyrosine kinase inhibitor genistein is not a specific inhibitor of uptake from caveolae. Moreover, one can still see that scientists in the field write that they want to direct transport of their particles to caveolae and caveosomes to avoid lysosomal degradation. However, caveosomes are artifacts caused by overexpression of caveolin-1 constructs, and ligands or particles taken up by caveolae are transported to endosomes and lysosomes as reported for other types of endocytosis

Biodistribution, pharmacokinetics and excretion studies of intravenously injected nanoparticles and extracellular vesicles: Possibilities and challenges

There is a large interest in developing nanoparticles and extracellular vesicles for delivery of therapeutics or imaging agents. Regulatory approval of such products requires knowledge about their biodistribution, metabolism and excretion. We here discuss possibilities and challenges of methods used for such studies, which most often are performed after labelling with radioactive isotopes or fluorescent molecules. It is important to evaluate if the labelled and unlabeled products can be expected to behave similarly in the body. Furthermore, one needs to critically consider whether the labels are still associated with the product at the time of analyses. We discuss advantages and disadvantages of different imaging modalities such as PET, SPECT, MRI, CT, ultrasound and optical imaging for whole-body biodistribution, and describe how to estimate the amount of labelled product in harvested organs and tissue. Microscopy of cells and tissues and various mass spectrometry methods are also discussed in this review

Biodistribution, pharmacokinetics and excretion studies of intravenously injected nanoparticles and extracellular vesicles: Possibilities and challenges

There is a large interest in developing nanoparticles and extracellular vesicles for delivery of therapeutics or imaging agents. Regulatory approval of such products requires knowledge about their biodistribution, metabolism and excretion. We here discuss possibilities and challenges of methods used for such studies, which most often are performed after labelling with radioactive isotopes or fluorescent molecules. It is important to evaluate if the labelled and unlabeled products can be expected to behave similarly in the body. Furthermore, one needs to critically consider whether the labels are still associated with the product at the time of analyses. We discuss advantages and disadvantages of different imaging modalities such as PET, SPECT, MRI, CT, ultrasound and optical imaging for whole-body biodistribution, and describe how to estimate the amount of labelled product in harvested organs and tissue. Microscopy of cells and tissues and various mass spectrometry methods are also discussed in this review

Cell-Penetrating Peptides: Possibilities and Challenges for Drug Delivery in Vitro and in Vivo

In this review, we discuss how cell-penetrating peptides (CPPs) might get access to their intracellular targets. We specifically focus on the challenge of deciding whether the positively-charged CPPs are just bound to the negatively-charged cell surface and subsequently endocytosed or actually transported into the cytosol, either by direct plasma membrane penetration or after endocytosis. This discussion includes comments about pitfalls when using pharmacological inhibitors in such studies. The possibility of exploiting CPPs as carriers for the delivery of drugs of different sizes in vitro is discussed, as is the use of CPPs as carriers for therapeutic drugs or contrast agents in vivo. We conclude that in many cases, more studies are needed to demonstrate conclusively whether increased delivery of a substance attached to CPPs is due to a membrane-penetrating property or whether the increase is a consequence of just changing the charge of the substance to be delivered. Finally, the expected dose needed for the use of such conjugates in vivo is discussed, including aspects to consider in order to bring potential products into clinical use

Modulation of Ricin Intoxication by the Autophagy Inhibitor EACC

The compound EACC (ethyl (2-(5-nitrothiophene-2-carboxamido) thiophene-3-carbonyl) carbamate) was recently reported to inhibit fusion of autophagosomes with lysosomes in a reversible manner by inhibiting recruitment of syntaxin 17 to autophagosomes. We report here that this compound also provides a strong protection against the protein toxin ricin as well as against other plant toxins such as abrin and modeccin. The protection did not seem to be caused by inhibition of endocytosis and retrograde transport, but rather by inhibited release of the enzymatically active A-moiety to the cytosol. The TANK-binding kinase 1 (TBK1) has been reported to phosphorylate syntaxin 17 and be required for initiation of autophagy. The inhibitor of TBK1, MRT68601, induced in itself a strong sensitization to ricin, apparently by increasing transport to the Golgi apparatus. Importantly, MRT68601 increased Golgi transport of ricin even in the presence of EACC, but EACC was still able to inhibit intoxication, supporting the idea that EACC protects at a late step along the retrograde pathway. These results also indicate that phosphorylation of syntaxin 17 is not required for the protection observed

Endosome to Golgi Transport of Ricin Is Regulated by Cholesterol

We have here studied the role of cholesterol in transport of ricin from endosomes to the Golgi apparatus. Ricin is endocytosed even when cells are depleted for cholesterol by using methyl-β-cyclodextrin (mβCD). However, as here shown, the intracellular transport of ricin from endosomes to the Golgi apparatus, measured by quantifying sulfation of a modified ricin molecule, is strongly inhibited when the cholesterol content of the cell is reduced. On the other hand, increasing the level of cholesterol by treating cells with mβCD saturated with cholesterol (mβCD/chol) reduced the intracellular transport of ricin to the Golgi apparatus even more strongly. The intracellular transport routes affected include both Rab9-independent and Rab9-dependent pathways to the Golgi apparatus, since both sulfation of ricin after induced expression of mutant Rab9 (mRab9) to inhibit late endosome to Golgi transport and sulfation of a modified mannose 6-phosphate receptor (M6PR) were inhibited after removal or addition of cholesterol. Furthermore, the structure of the Golgi apparatus was affected by increased levels of cholesterol, as visualized by pronounced vesiculation and formation of smaller stacks. Thus, our results indicate that transport of ricin from endosomes to the Golgi apparatus is influenced by the cholesterol content of the cell