Understanding the cryobiological responses of endothelial cells is important to ensure the successful cryopreservation of vascularized tissues and organs. The endothelium, a monolayer of endothelial cells comprising the inner lining of blood vessels and other tissues such as the cornea, is sensitive to cryoinjury. In this chapter, we update our published cryopreservation protocol, which we have since validated for several more cell types. Our optimized protocol involves culturing cells on Rinzl plastic coverslips, using a combination of a permeating cryoprotectant (5% dimethyl sulfoxide) and a non-permeating cryoprotectant (6% hydroxyethyl starch), addition of 2% chondroitin sulfate, controlled-rate slow cooling, storage in liquid nitrogen, and removal of cryoprotectants immediately after thaw. Our post-thaw assessments include membrane integrity and a functional assay immediately after thaw and after extended incubation in culture conditions.

Repeated intramuscular administration of progesterone (PRG) oily injections often causes macrophage-mediated injection-site reactions and pain, limiting patient compliance during long-term therapy. Herein, a PRG-loaded hydroxyethyl starch-coated semi-solid lipid microparticle system (HES-SLM) was developed for low-irritant sustained intramuscular delivery. Oleic acid-grafted HES was synthesized as an emulsifier, and HES-SLM was prepared by melt emulsification using glyceryl monostearate/medium-chain triglycerides as the lipid core, with PEG-SLM as a control. HES-SLM exhibited a uniform spherical morphology, a D50 of 34.36 ± 0.42 μm, low burst release within 2 h (4.17 ± 0.98%), and sustained release for 13 days. Compared with PEG-SLM, HES-SLM significantly reduced macrophage infiltration, inflammatory cytokine expression, collagen deposition, and profibrotic responses at the injection site, thereby improving the local release microenvironment and facilitating PRG transport into systemic circulation. Consequently, HES-SLM markedly increased PRG exposure, with an AUC0-t of 3649.113 ± 1066.590 ng·mL[-1]·h versus 1751.285 ± 357.668 ng·mL[-1]·h for PEG-SLM. In immature female rats, HES-SLM also achieved efficacy comparable to marketed PRG oily injection and reduced inflammatory factors and Nav1.8 expression, indicating reduced injection-site pain. This study provides a surface-modified microparticle strategy for sustained, low-irritant intramuscular PRG delivery with reduced dosing frequency.

Natural polymer based-hydrogels have attracted increasing attention as flexible wearable sensors owing to their excellent biocompatibility. However, hydrogels derived from natural polymers with balanced mechanical and adhesive properties still remain a challenge. To address this issue, a hybrid hydrogel network based on two natural polymers and poly(lipoic acid) (PLA) was fabricated. Specifically, gelatin (Gel) and oxidized hydroxyethyl starch (OHES) were crosslinked via Schiff base linkages to provide mechanical support. The PLA network imparted adhesion through the ring-opening polymerization of lipoic acid (LA). Tannic acid (TA) suppressed PLA depolymerization through Michael addition-mediated interactions, and simultaneously boosted the hydrogel's adhesive performance. Moreover, zinc ions (Zn[2+]) endowed the hydrogel with ionic conductivity and served as dynamic coordination crosslinks within the polymer network, leading to enhanced mechanical robustness and stretchability. Then, the hydrogel was assembled into a strain sensor capable of directly adhering to human joints. It could accurately detect both large-scale motions (e.g., joint and neck bending) and subtle movements (e.g., swallowing and smiling). In addition, the hydrogel functioned as a skin-conformal electrode for high-quality acquisition of physiological bioelectrical signals, including electrocardiogram (ECG), electromyogram (EMG), and electroencephalogram (EEG).

BACKGROUND Spontaneous intracranial hypotension is a challenging clinical entity, conventionally managed through epidural blood patching. In a subset of refractory patients who have exhausted all standard-of-care therapies, suboptimal response or recurrence are major clinical concerns and can lead to prolonged symptoms, repeated interventions, and a heavier healthcare burden. Therefore, we describe an innovative intervention-continuous epidural infusion of hydroxyethyl starch-for the treatment of spontaneous intracranial hypotension, which is intended to provide sustained modulation of epidural pressure and to potentially promote closure of cerebrospinal fluid leaks. CASE REPORT This retrospective case series included 4 individuals diagnosed with spontaneous intracranial hypotension, all with orthostatic headaches. Three had non-traumatic subdural hematomas, among whom 2 underwent hematoma evacuation along with intracranial pressure monitoring within the subdural space. In contrast to conventional management strategies, these patients were treated with continuous epidural infusion of hydroxyethyl starch via epidural catheterization, with infusion parameters titrated to clinical response and, when available, intracranial pressure measurements. All patients had favorable clinical outcomes, with marked symptom relief, functional recovery, and no recurrence during follow-up. No major neurological complications or rebound intracranial hypertension occurred. CONCLUSIONS The patients' responses to continuous epidural infusion of hydroxyethyl starch were uniformly positive, offering a promising outlook for the treatment of spontaneous intracranial hypotension. Larger prospective studies are needed to confirm its safety and efficacy.

Cancer remains a major global public health challenge, posing a substantial threat to human health and socioeconomic development. 10-hydroxycamptothecin (10-HCPT), a potent topoisomerase I inhibitor, exhibits broad antitumor activity; however, its clinical application is limited by poor aqueous solubility, rapid lactone-ring opening under physiological pH, and low bioavailability. To address these limitations, we developed an enzyme- and pH-dual-responsive polymer prodrug nanocarrier based on hydroxyethyl starch (HES). 10-HCPT was conjugated to the HES backbone through a hydrolysable succinate ester linkage, yielding an amphiphilic HES-10-HCPT prodrug capable of self-assembling into micelles. The resulting polymer prodrug micelles exhibited favorable storage and plasma stability. Importantly, drug release was accelerated under acidic and enzymatic conditions, suggesting tumor microenvironment-responsive release behavior. In vitro cytotoxicity assays against 4T1 breast cancer cells showed that the high-drug-loading micelles, HH-2, displayed markedly enhanced potency, with an IC50 value of 0.651 μg/mL, compared with free 10-HCPT, which showed an IC50 value of 27.7 μg/mL. In vivo studies in 4T1 tumor-bearing mice demonstrated that, at an equivalent 10-HCPT dose, HH-2 achieved a tumor inhibition rate of 46.31%, compared with 19.70% for free 10-HCPT. Histological analysis further suggested a favorable preliminary safety profile. Overall, this HES-based polymer prodrug micelle system provides a promising strategy for improving the stability, therapeutic efficacy, and safety of 10-HCPT and may serve as a potential platform for cancer chemotherapy.

The cryopreservation of primary human brain vascular pericytes (HBVPs) has facilitated their commercial availability as an essential component of the blood-brain barrier. Among the commercial suppliers of HBVPs in suspension, only one provides information regarding the cryoprotectant and vehicle used, i.e., 5% dimethyl sulfoxide (Me2SO) in complete medium, with no cryopreservation procedure given. Published protocols for the cryopreservation of brain vascular pericytes derived from human-induced pluripotent stem cells use traditional 10% Me2SO. In this study, we characterized the cryobiological response of HBVPs using graded freezing, both in the absence of cryoprotectants, which identifies their susceptibility to the main types of cryoinjury, and in the presence of several different cryoprotectants with the aim of quantifying post-thaw outcome. We first validated our graded freezing technique using human cerebral microvascular endothelial cell/D3 clone (hCMEC/D3) by comparing the membrane integrity results in this study in the absence and presence of 5% Me2SO plus 6% hydroxyethyl starch (HES) in hCMEC/D3 complete medium, with those previously published by our group. We then assessed the cryobiological response of HBVPs to controlled slow cooling at 1 °C/min in the absence and presence of various cryoprotectants, namely 5% Me2SO, 10% Me2SO, 5% Me2SO plus 6% HES, or 10% glycerol, all in HBVP complete medium. We found that all cryoprotectants tested yielded immediate post-thaw membrane integrity >93%. Cryopreservation with 10% Me2SO resulted in 95.3 ± 0.7% membrane integrity; using a lower concentration of Me2SO (5%) alone, or in the presence of 6% HES, resulted in membrane integrities of 93.3 ± 0.8% and 94.2 ± 0.8%, respectively. For applications where avoiding Me2SO is preferred, we showed that HBVPs can be cryopreserved in 10% glycerol (post-thaw membrane integrity of 95.7 ± 0.5%). The AlamarBlue reduction assay with 3 h incubation time indicated that HBVPs cryopreserved with either 5% Me2SO, 10% Me2SO, 5% Me2SO plus 6% HES, or 10% glycerol exhibited post-thaw metabolic activities which were not statistically different from those of unfrozen controls. Our study presents various application-specific options for cryopreservation of primary HBVPs with validated post-thaw membrane integrity and metabolic activity.

Iron oxide nanoparticles (IONPs) offer promise for drug delivery and imaging, but their vascular safety requires thorough evaluation. Hydroxyethyl starch (HES) is a clinically used, biocompatible polysaccharide with potential as a nanoparticle coating to improve vascular safety. We synthesized novel hydroxyethyl starch-coated IONPs (IONPs@HES) and assessed their properties and effects on human microvascular endothelial cells (HMEC-1) under basal and inflammatory conditions. IONPs@HES showed magnetite cores, near-neutral charge, and reduced magnetic saturation, supporting biocompatibility. They were efficiently internalized without affecting viability or proliferation (20-500 μg/mL) and did not increase LPS-induced ICAM-1 expression. Autophagic activity, assessed by LC3 immunofluorescence and Cyto-ID flow cytometry, remained unchanged, suggesting preserved autophagic homeostasis. A modest increase in phosphorylated caveolin-1 (p-CAV1) was observed, with no enhancement under LPS stimulation. Co-treatment with indomethacin showed no additive toxicity. These findings support IONPs@HES as a biocompatible nanoplatform suitable for vascular-targeted cancer therapy, meriting further in vivo validation.

Polysaccharides and starch derivatives can play a role in the development of buccal films and patches. Buccal films are a valid approach to the transmucosal administration of drugs. These drug-delivery systems are adaptable to the mucosal surface, while being particularly suitable for the pediatric population. This study explores the combination of polysaccharide-based films for the buccal administration of the poorly soluble drug, ibuprofen. The films were obtained via the freeze-thaw technique using an innovative combination of two film-forming polymers of natural origin, i.e. hydroxyethylcellulose and a pea starch derivate (Linecaps®). Ibuprofen was selected as model drug to be loaded into the buccal formulation as a β-cyclodextrin complex. The produced films showed good physico-chemical and functional properties including homogeneous drug content, thickness, mechanical strength, mucoadhesion, thus guaranteeing suitable drug loading and applicability. In vitro, permeation studies of Ibuprofen buccal films were carried out using a synthetic biomimetic membrane, and rabbit mucosae tissue exhibited values of permeability fluxes in the same order of magnitude for the two tested membranes (1.51 × 10[-2] ± 0.33 × 10[-2] μg/cm[2]*s vs 1.21 × 10[-2] ± 0.18 × 10[-2] μg/cm[2]*s). The release of the drug from the buccal film, the taste-masking and permeation properties suggested that the proposed combination of polysaccharides represent a potential formulation of mucoadhesive buccal films.