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Cell-penetrating peptides (CPPs), such as penetratin, are often investigated as drug delivery vectors and incorporating d-amino acids, rather than the natural l-forms, to enhance proteolytic stability could improve their delivery efficiency. The present study aimed to compare membrane association, cellular uptake, and delivery capacity for all-l and all-d enantiomers of penetratin (PEN) by using different cell models and cargos. The enantiomers displayed widely different distribution patterns in the examined cell models, and in Caco-2 cells, quenchable membrane binding was evident for d-PEN in addition to vesicular intracellular localization for both enantiomers. The uptake of insulin in Caco-2 cells was equally mediated by the two enantiomers, and while l-PEN did not increase the transepithelial permeation of any of the investigated cargo peptides, d-PEN increased the transepithelial delivery of vancomycin five-fold and approximately four-fold for insulin at an extracellular apical pH of 6.5. Overall, while d-PEN was associated with the plasma membrane to a larger extent and was superior in mediating the transepithelial delivery of hydrophilic peptide cargoes compared to l-PEN across Caco-2 epithelium, no enhanced delivery of the hydrophobic cyclosporin was observed, and intracellular insulin uptake was induced to a similar degree by the two enantiomers.

Chemical modification of peptides and proteins, such as PEGylation and lipidation, creates conjugates with new properties. However, they are typically not dynamic or stimuli-responsive. Self-assembly controlled by a stimulus will allow adjusting properties directly. Here, we report that conjugates of oligogalacturonic acids (OGAs), isolated from plant-derived pectin, are Ca2+-responsive. We report the conjugation of OGA to human insulin (HI) to create new glyco-insulins. In addition, we coupled OGA to model peptides. We studied their self-assembly by dynamic light scattering, small-angle X-ray scattering, and circular dichroism, which showed that the self-assembly to form nanostructures depended on the length of the OGA sequence and Zn2+ and Ca2+ concentrations. Subcutaneous administration of OGA12-HI with Zn2+ showed a stable decrease in blood glucose over a longer period of time compared to HI, despite the lower receptor binding affinity.

The inherent low oral bioavailability of therapeutic peptides can be enhanced by the cell-penetrating peptide penetratin and its analogues shuffle and penetramax applied as carriers for delivery of insulin. In this study, the objective was to gain mechanistic insights on the effect of the carrier peptide stereochemistry on their interactions with insulin and on insulin delivery. Insulin-carrier peptide interactions were investigated using small-angle X-ray scattering and cryogenic transmission electron microscopy, while the insulin and peptide stability and transepithelial insulin permeation were evaluated in the Caco-2 cell culture model along with the carrier peptide-induced effects on epithelial integrity and cellular metabolic activity. Interestingly, the insulin transepithelial permeation was influenced by the degree of insulin-carrier peptide complexation and depended on the stereochemistry of penetramax but not of penetratin and shuffle. The l-form of the peptides initially decreased the epithelial integrity comparable to that induced by the d-peptides, suggesting a comparable mechanism of action. The immediate decrease was reversible during exposure of the Caco-2 epithelium to the l-peptides but not during exposure to the d-peptides, likely a result of their higher stability. Overall, exploration of the stereochemistry showed to be an interesting strategy for carrier peptide-mediated insulin delivery.

PURPOSE: On the basis of low risk of local recurrence in elderly patients with breast cancer after conservative surgery followed by whole breast irradiation (WBI), the Danish Breast Cancer Group initiated the noninferiority external-beam partial breast irradiation (PBI) trial (ClinicalTrials.gov identifier: NCT00892814). We hypothesized that PBI was noninferior to WBI regarding breast induration. METHODS: Patients operated with breast conservation for relatively low-risk breast cancer were randomly assigned to WBI versus PBI, and all had 40 Gy/15 fractions. The primary end point was 3-year grade 2-3 breast induration. RESULTS: In total, 865 evaluable patients (434 WBI and 431 PBI) were enrolled between 2009 and 2016. Median follow-up was 5.0 years (morbidity) and 7.6 years (locoregional recurrence). The 3-year rate of induration was 9.7% for WBI and 5.1% for PBI (P = .014). Large breast size was significantly associated with induration with a 3-year incidence of 13% (WBI) and 6% (PBI) for large-breasted patients versus 6% (WBI) and 5% (PBI) for small-breasted patients. PBI showed no increased risk of dyspigmentation, telangiectasia, edema, or pain, and patient satisfaction was high. Letrozole and smoking did not increase the risk of radiation-associated morbidity. Sixteen patients had a locoregional recurrence (six WBI and 10 PBI; P = .28), 20 patients had a contralateral breast cancer, and eight patients had distant failure (five WBI and three PBI). A nonbreast second cancer was detected in 73 patients (8.4%), and there was no difference between groups. CONCLUSION: External-beam PBI for patients with low-risk breast cancer was noninferior to WBI in terms of breast induration. Large breast size was a risk factor for radiation-associated induration. Few recurrences were detected and unrelated to PBI.

Purpose: The relation between breast induration grade 2–3 at 3 years after radiation therapy and irradiated breast volume was investigated for patients in the Danish Breast Cancer Group (DBCG) Partial Breast Irradiation (PBI) trial. METHODS Treatment plan data was obtained from the Danish radiotherapy plan database. Dosimetric parameters for breast and organs at risk were determined. Breast induration data was obtained from the DBCG database. The volume of the whole breast (CTVp_breast) treated to various dose levels was determined for treatment plans in both arms. Logistic regression was used to assess the frequency of induration on breast volume irradiated to ≥40 Gy. RESULTS PBI and WBI was given to 433 and 432 patients, respectively. Median and interquartile ranges (IQR) for CTVp_breast were 710 mL (467–963 mL; PBI) and 666 mL (443–1012 mL; WBI) (p = 0.98). Median and IQR for CTVp_breast treated to ≥40 Gy was 24.9% (18.6–32.6%; PBI) and 59.8% (53.6–68.5%; WBI). Grade 2–3 induration was observed in 5% (PBI) and 10% (WBI) of the patients. A dose–response relationship was established between irradiated breast volume and frequency of breast induration. From the model, 5% and 10% risks of breast induration were observed for ≥40 Gy delivered to CTVp_breast volumes of 177 mL (95%CI, 94–260 mL) and 426 mL (95%CI, 286–567 mL), respectively. CONCLUSION The frequency of breast induration increased significantly with increasing irradiated breast volume, strongly favouring small volumes and PBI. Thus, treated breast volume – not the breast size itself - is the risk factor for induration. This is the first report directly linking the 40 Gy irradiated breast volume to breast induration.