Nanobody-mediated targeting of zinc phthalocyanine with polymer micelles as nanocarriers.

Photodynamic therapy (PDT) is a suitable alternative to currently employed cancer treatments. However, the hydrophobicity of most photosensitizers (e.g., zinc phthalocyanine (ZnPC)) leads to their aggregation in blood. Moreover, non-specific accumulation in skin and low clearance rate of ZnPC leads to long-lasting skin photosensitization, forcing patients with a short life expectancy to remain indoors. Consequently, the clinical implementation of these photosensitizers is limited. Here, benzyl-poly(ε-caprolactone)-b-poly(ethylene glycol) micelles encapsulating ZnPC (ZnPC-M) were investigated to increase the solubility of ZnPC and its specificity towards cancers cells. Asymmetric flow field-flow fractionation was used to characterize micelles with different ZnPC-to-polymer ratios and their stability in human plasma. The ZnPC-M with the lowest payload (0.2 and 0.4% ZnPC w/w) were the most stable in plasma, exhibiting minimal ZnPC transfer to lipoproteins, and induced the highest phototoxicity in three cancer cell lines. Nanobodies (Nbs) with binding specificity towards hepatocyte growth factor receptor (MET) or epidermal growth factor receptor (EGFR) were conjugated to ZnPC-M to facilitate cell targeting and internalization. MET- and EGFR-targeting micelles enhanced the association and the phototoxicity in cells expressing the target receptor. Altogether, these results indicate that ZnPC-M decorated with Nbs targeting overexpressed proteins on cancer cells may provide a better alternative to currently approved formulations.

Versatile Click Linker Enabling Native Peptide Release from Nanocarriers upon Redox Trigger.

Nanocarriers have shown their ability to extend the circulation time of drugs, enhance tumor uptake, and tune drug release. Therapeutic peptides are a class of drug compounds in which nanocarrier-mediated delivery can potentially improve their therapeutic index. To this end, there is an urgent need for orthogonal covalent linker chemistry facilitating the straightforward on-the-resin peptide generation, nanocarrier conjugation, as well as the triggered release of the peptide in its native state. Here, we present a copper-free clickable ring-strained alkyne linker conjugated to the N-terminus of oncolytic peptide LTX-315 via standard solid-phase peptide synthesis (SPPS). The linker contains (1) a recently developed seven-membered ring-strained alkyne, 3,3,6,6-tetramethylthiacycloheptyne sulfoximine (TMTHSI), (2) a disulfide bond, which is sensitive to the reducing cytosolic and tumor environment, and (3) a thiobenzyl carbamate spacer enabling release of the native peptide upon cleavage of the disulfide via 1,6-elimination. We demonstrate convenient "clicking" of the hydrophilic linker-peptide conjugate to preformed pegylated core-cross-linked polymeric micelles (CCPMs) of 50 nm containing azides in the hydrophobic core under aqueous conditions at room temperature resulting in a loading capacity of 8 mass % of peptide to polymer (56% loading efficiency). This entrapment of hydrophilic cargo into/to a cross-linked hydrophobic core is a new and counterintuitive approach for this class of nanocarriers. The release of LTX-315 from the CCPMs was investigated in vitro and rapid release upon exposure to glutathione (within minutes) followed by slower 1,6-elimination (within an hour) resulted in the formation of the native peptide. Finally, cytotoxicity of LTX CCPMs as well as uptake of sulfocyanine 5-loaded CCPMs was investigated by cell culture, demonstrating successful tumor cell killing at concentrations similar to that of the free peptide treatment.

Mechanistic Study on the Degradation of Hydrolysable Core-Crosslinked Polymeric Micelles.

Core-crosslinked polymeric micelles (CCPMs) are an attractive class of nanocarriers for drug delivery. Two crosslinking approaches to form CCPMs exist: either via a low-molecular-weight crosslinking agent to connect homogeneous polymer chains with reactive handles or via cross-reactive handles on polymers to link them to each other (complementary polymers). Previously, CCPMs based on methoxy poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl) methacrylamide-lactate] (mPEG-b-PHPMAmLacn) modified with thioesters were crosslinked via native chemical ligation (NCL, a reaction between a cysteine residue and thioester resulting in an amide bond) using a bifunctional cysteine containing crosslinker. These CCPMs are degradable under physiological conditions due to hydrolysis of the ester groups present in the crosslinks. The rapid onset of degradation observed previously, as measured by the light scattering intensity, questions the effectiveness of crosslinking via a bifunctional agent. Particularly due to the possibility of intrachain crosslinks that can occur using such a small crosslinker, we investigated the degradation mechanism of CCPMs generated via both approaches using various analytical techniques. CCPMs based on complementary polymers degraded slower at pH 7.4 and 37 °C than CCPMs with a crosslinker (the half-life of the light scattering intensity was approximately 170 h versus 80 h, respectively). Through comparative analysis of the degradation profiles of the two different CCPMs, we conclude that partially ineffective intrachain crosslinks are likely formed using the small crosslinker, which contributed to more rapid CCPM degradation. Overall, this study shows that the type of crosslinking approach can significantly affect degradation kinetics, and this should be taken into consideration when developing new degradable CCPM platforms.

Orthogonal Covalent Entrapment of Cargo into Biodegradable Polymeric Micelles via Native Chemical Ligation.

Polymeric micelles (PMs) are promising platforms for enhanced tissue targeting of entrapped therapeutic agents. Strategies to circumvent premature release of entrapped drugs include cross-linking of the micellar core as well as covalent attachment of the drug cargo. The chemistry employed to obtain cross-linked micelles needs to be mild to also allow entrapment of fragile molecules, such as certain peptides, proteins, oligonucleotides, and fluorescent dyes. Native chemical ligation (NCL) is a mild bio-orthogonal reaction between a N-terminal cysteine residue and a thioester that proceeds under physiological conditions. Here, we designed a trifunctional cross-linker containing two cysteine residues for the micelle core-cross-linking reaction and an azide residue for ring-strained alkyne conjugation of fluorescent dyes. We applied this approach to thermosensitive methoxypolyethylene glycol-b-N-(2-hydroxypropyl)methacrylamide-lactate (mPEG-b-HPMAmLacn) based block copolymers of a core-cross-linked polymeric micelle (CCPM) system by attaching thioester residues (using ethyl thioglycolate-succinic anhydride, ETSA) for NCL cross-linking with the trifunctional cross-linker under physiological conditions. By use of mild copper-free click chemistry, we coupled fluorescent dyes, Sulfo.Cy5 and BODIPY, to the core via the azide residue present on the cross-linker by triazole ring formation. In addition, we employed a recently developed cycloheptyne strain promoted click reagent (TMTHSI, CliCr) in comparison to the frequently employed cyclooctyne derivative (DBCO), both achieving successful dye entrapment. The size of the resulting CCPMs could be tuned between 50 and 100 nm by varying the molecular weight of the thermosensitive block and ETSA content. In vitro cell experiments showed successful internalization of the dye entrapped CCPMs, which did not affect cell viability up to a polymer concentration of 2 mg/mL in PC3 cells. These fluorescent dye entrapped CCPMs can be applied in diagnostic imaging and the chemistry developed in this study serves as a steppingstone toward covalently entrapped fragile drug compounds with tunable release in CCPMs.

Gold Nanoclusters: Imaging, Therapy, and Theranostic Roles in Biomedical Applications.

For the past two decades, atomic gold nanoclusters (AuNCs, ultrasmall clusters of several to 100 gold atoms, having a total diameter of <2 nm) have emerged as promising agents in the diagnosis and treatment of cancer. Owing to their small size, significant quantization occurs to their conduction band, which leads to emergent photonic properties and the disappearance of the plasmonic responses observed in larger gold nanoparticles. For example, AuNCs exhibit native luminescent properties, which have been well-explored in the literature. Using proteins, peptides, or other biomolecules as structural scaffolds or capping ligands, required for the stabilization of AuNCs, improves their biocompatibility, while retaining their distinct optical properties. This paved the way for the use of AuNCs in fluorescent bioimaging, which later developed into multimodal imaging combined with computer tomography and magnetic resonance imaging as examples. The development of AuNC-based systems for diagnostic applications in cancer treatment was then made possible by employing active or passive tumor targeting strategies. Finally, the potential therapeutic applications of AuNCs are extensive, having been used as light-activated and radiotherapy agents, as well as nanocarriers for chemotherapeutic drugs, which can be bound to the capping ligand or directly to the AuNCs via different mechanisms. In this review, we present an overview of the diverse biomedical applications of AuNCs in terms of cancer imaging, therapy, and combinations thereof, as well as highlighting some additional applications relevant to biomedical research.

Engineered Polymersomes for the Treatment of Fish Odor Syndrome: A First Randomized Double Blind Olfactory Study.

Trimethylamine (TMA) is a metabolite overtly present in patients suffering from trimethylaminuria (TMAU), a rare genetic disorder characterized by a strong "fishy" body odor. To date, no approved pharmacological treatment to sequester excess TMA on the skin of patients exists. Here, transmembrane pH gradient poly(isoprene)-block-poly(ethylene glycol) (PI-b-PEG) polymersomes are investigated for the topical removal of TMA. PI-b-PEG amphiphiles of varying chain length are synthesized and evaluated for their ability to form vesicular structures in aqueous media. The optimization of the PI/PEG ratio of transmembrane pH gradient polymersomes allows for the rapid and efficient capture of TMA both in solution and after incorporation into a topical hydrogel matrix at the pH of the skin. A subsequent double blind olfactory study reveals a significant decrease in perceived odor intensity after application of the polymersome-based formulation on artificial skin substrates that has been incubated in TMA-containing medium. This simple and novel approach has the potential to ease the burden of people suffering from TMAU.

Ammonia uptake by transmembrane pH gradient poly(isoprene)-block-poly(ethylene glycol) polymersomes.

Transmembrane pH gradient poly(isoprene)-block-poly(ethylene glycol) (PI-b-PEG) polymersomes were investigated for their potential use in the detoxification of ammonia, a metabolite that is excessively present in patients suffering from urea cycle disorders and advanced liver diseases, and which causes neurotoxic effects (e.g., hepatic encephalopathy). Polymers varying in PI and PEG block length were synthesized via nitroxide-mediated polymerization and screened for their ability to self-assemble into polymersomes in aqueous media. Ammonia sequestration by the polymersomes was investigated in vitro. While most vesicular systems were able to capture ammonia in simulated intestinal fluids, uptake was lost in partially dehydrated medium mimicking conditions in the colon. Polymeric crosslinking of residual olefinic bonds in the PI block increased polymersome stability, partially preserving the ammonia capture capacity in the simulated colon environment. These more stable vesicular systems hold promise for the chronic oral treatment of hyperammonemia.