Polymeric Nanoparticles with Neglectable Protein Corona.

The current understanding of nanoparticle-protein interactions indicates that they rapidly adsorb proteins upon introduction into a living organism. The formed protein corona determines thereafter identity and fate of nanoparticles in the body. The present study evaluates the protein affinity of three core-crosslinked polymeric nanoparticles with long circulation times, differing in the hydrophilic polymer material forming the particle surface, namely poly(N-2-hydroxypropylmethacrylamide) (pHPMA), polysarcosine (pSar), and poly(ethylene glycol) (PEG). This includes the nanotherapeutic CPC634, which is currently in clinical phase II evaluation. To investigate possible protein corona formation, the nanoparticles are incubated in human blood plasma and separated by asymmetrical flow field-flow fractionation (AF4). Notably, light scattering shows no detectable differences in particle size or polydispersity upon incubation with plasma for all nanoparticles, while in gel electrophoresis, minor amounts of proteins can be detected in the particle fraction. Label-free quantitative proteomics is additionally applied to analyze and quantify the composition of the proteins. It proves that some proteins are enriched, but their concentration is significantly less than one protein per particle. Thus, most of the nanoparticles are not associated with any proteins. Therefore, this work underlines that polymeric nanoparticles can be synthesized, for which a protein corona formation does not take place.

Effect of Core-Crosslinking on Protein Corona Formation on Polymeric Micelles.

Most nanomaterials acquire a protein corona upon contact with biological fluids. The magnitude of this effect is strongly dependent both on surface and structure of the nanoparticle. To define the contribution of the internal nanoparticle structure, protein corona formation of block copolymer micelles with poly(N-2-hydroxypropylmethacrylamide) (pHPMA) as hydrophilic shell, which are crosslinked-or not-in the hydrophobic core is comparatively analyzed. Both types of micelles are incubated with human blood plasma and separated by asymmetrical flow field-flow fractionation (AF4). Their size is determined by dynamic light scattering and proteins within the micellar fraction are characterized by gel electrophoresis and quantified by liquid chromatography-high-resolution mass spectrometry-based label-free quantitative proteomics. The analyses reveal only very low amounts of plasma proteins associated with the micelles. Notably, no significant enrichment of plasma proteins is detectable for core-crosslinked micelles, while noncrosslinked micelles show a significant enrichment of plasma proteins, indicative of protein corona formation. The results indicate that preventing the reorganization of micelles (equilibrium with unimers) by core-crosslinking is crucial to reduce the interaction with plasma proteins.

HPMA-Based Nanocarriers for Effective Immune System Stimulation.

The selective activation of the immune system using nanoparticles as a drug delivery system is a promising field in cancer therapy. Block copolymers from HPMA and laurylmethacrylate-co-hymecromone-methacrylate allow the preparation of multifunctionalized core-crosslinked micelles of variable size. To activate dendritic cells (DCs) as antigen presenting cells, the carbohydrates mannose and trimannose are introduced into the hydrophilic corona as DC targeting units. To activate DCs, a lipophilic adjuvant (L18-MDP) is incorporated into the core of the micelles. To elicit an immune response, a model antigen peptide (SIINFEKL) is attached to the polymeric nanoparticle-in addition-via a click reaction with the terminal azide. Thereafter, the differently functionalized micelles are chemically and biologically characterized. While the core-crosslinked micelles without carbohydrate units are hardly bound by DCs, mannose and trimannose functionalization lead to a strong binding. Flow cytometric analysis and blocking studies employing mannan suggest the requirement of the mannose receptor and DC-SIGN for effective micelle binding. It could be suppressed by blocking with mannan. Adjuvant-loaded micelles functionalized with mannose and trimannose activate DCs, and DCs preincubated with antigen-conjugated micelles induce proliferation of antigen-specific CD8+ T cells.

Long-term biodistribution study of HPMA-ran-LMA copolymers in vivo by means of 131I-labeling.

BACKGROUND: For the evaluation of macromolecular drug delivery systems suitable pre-clinical monitoring of potential nanocarrier systems is needed. In this regard, both short-term as well as long-term in vivo tracking is crucial to understand structure-property relationships of polymer carrier systems and their resulting pharmacokinetic profile. Based on former studies revealing favorable in vivo characteristics for 18F-labeled random (ran) copolymers consisting of N-(2-hydroxypropyl)methacrylamide (HPMA) and lauryl methacrylate (LMA) - including prolonged plasma half-life as well as enhanced tumor accumulation - the presented work focuses on their long-term investigation in the living organism. METHODS: In this respect, four different HPMA-based polymers (homopolymers as well as random copolymers with LMA as hydrophobic segment) were synthesized and subsequent radioactive labeling was accomplished via the longer-lived radioisotope 131I. In vivo results, concentrating on the pharmacokinetics of a high molecular weight HPMA-ran-LMA copolymer, were obtained by means of biodistribution and metabolism studies in the Walker 256 mammary carcinoma model over a time-span of up to three days. Besides, a direct comparison with the 18F-radiolabeled polymer was drawn. To consider physico-chemical differences between the differently labeled polymer (18F or 131I) on the critical micelle concentration (CMC) and the size of the polymeric micelles, those properties were determined using the 19F- or 127I-functionalized polymer. Special emphasis was laid on the time-dependent correlation between blood circulation properties and corresponding tumor accumulation, particularly regarding the enhanced permeability and retention (EPR) effect. RESULTS: Studies revealed, at first, differences in the short time (2h) body distribution, despite the very similar properties (molecular structure, CMC and size of the micellar aggregates) of the non-radioactive 19F- and 127I-functionalized polymers. Long-term investigations with the 131I-labeled polymer demonstrated that, despite a polymer clearance from the blood within 72h, there was still an increase in tumor uptake observed over time. Regarding the stability of the 131I-label, ex vivo biodistribution experiments, considering the uptake in the thyroid, indicated low metabolism rates. CONCLUSION: The observed in vivo characteristics strongly underline the EPR effect. The findings illustrate the need to combine information of different labeling approaches and in vivo evaluation techniques to generate an overall pharmacokinetic picture of potential nanocarriers in the pre-clinical setting. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENTS: The in vivo behavior of the investigated HPMA-ran-LMA copolymer demonstrates great potential in terms of an effective accumulation in the tumor.

Targeting cells of the immune system: mannosylated HPMA-LMA block-copolymer micelles for targeting of dendritic cells.

BACKGROUND: Successful tumor immunotherapy depends on the induction of strong and sustained tumor antigen-specific immune responses by activated antigen-presenting cells (APCs) such as dendritic cells (DCs). Since nanoparticles have the potential to codeliver tumor-specific antigen and DC-stimulating adjuvant in a DC-targeting manner, we wanted to assess the suitability of mannosylated HPMA-LMA block polymers for immunotherapy. MATERIALS & METHODS: Fluorescence-labeled block copolymer micelles derived from P(HPMA)-block-P(LMA) copolymers and according statistical copolymers were synthesized via RAFT polymerization, and loaded with the APC activator L18-MDP. Both types of copolymers were conjugated with D-mannose to target the mannose receptor as expressed by DCs and macrophages. The extent and specificity of micelle binding and activation of APCs was monitored using mouse spleen cells and bone marrow-derived DC (BMDC). RESULTS: Nontargeting HPMA-LMA statistical copolymers showed strong unspecific cell binding. HPMA-LMA block copolymers bound DC only when conjugated with mannose, and in a mannose receptor-specific manner. Mannosylated HPMA-LMA block copolymers were internalized by DC. DC-targeting HPMA-LMA block copolymers mediated DC activation when loaded with L18-MDP. CONCLUSION: Mannosylated HPMA-LMA block copolymers are a promising candidate for the delvopment of DC-targeting nanovaccines.

Endovascular management of tandem extracranial internal carotid artery aneurysms with a covered stent.

PURPOSE: To illustrate the endovascular technique for treating tandem arterial aneurysms in the distal carotid artery. CASE REPORT: A 49-year-old woman presented with progressive swelling of the neck caused by a pulsatile tumor. Angiography demonstrated 2 aneurysms of the extracranial internal carotid artery near the skull base, so surgery was not a therapeutic option. Both aneurysms were successfully excluded with deployment of a single Wallgraft across both lesions; no complications were encountered. At 2-year follow-up, the patient is doing well, without any sign of aneurysm reperfusion. CONCLUSIONS: The presented case demonstrates that stenting of tandem aneurysms may be performed even in the carotid artery. Nevertheless, this treatment should be considered as a therapeutic option only in selected cases.