Modular Synthesis of Semiconducting Graft Copolymers to Achieve "Clickable" Fluorescent Nanoparticles with Long Circulation and Specific Cancer Targeting.

Semiconducting polymer nanoparticles (SPNs) are explored for applications in cancer theranostics because of their high absorption coefficients, photostability, and biocompatibility. However, SPNs are susceptible to aggregation and protein fouling in physiological conditions, which can be detrimental for in vivo applications. Here, a method for achieving colloidally stable and low-fouling SPNs is described by grafting poly(ethylene glycol) (PEG) onto the backbone of the fluorescent semiconducting polymer, poly(9,9'-dioctylfluorene-5-fluoro-2,1,3-benzothiadiazole), in a simple one-step substitution reaction, postpolymerization. Further, by utilizing azide-functionalized PEG, anti-human epidermal growth factor receptor 2 (HER2) antibodies, antibody fragments, or affibodies are site-specifically "clicked" onto the SPN surface, which allows the functionalized SPNs to specifically target HER2-positive cancer cells. In vivo, the PEGylated SPNs are found to have excellent circulation efficiencies in zebrafish embryos for up to seven days postinjection. SPNs functionalized with affibodies are then shown to be able to target HER2 expressing cancer cells in a zebrafish xenograft model. The covalent PEGylated SPN system described herein shows great potential for cancer theranostics.

Differences in Human Plasma Protein Interactions between Various Polymersomes and Stealth Liposomes as Observed by Fluorescence Correlation Spectroscopy.

A significant factor hindering the clinical translation of polymersomes as vesicular nanocarriers is the limited availability of comparative studies detailing their interaction with blood plasma proteins compared to liposomes. Here, polymersomes are self-assembled via film rehydration, solvent exchange, and polymerization-induced self-assembly using five different block copolymers. The hydrophilic blocks are composed of anti-fouling polymers, poly(ethylene glycol) (PEG) or poly(2-methyl-2-oxazoline) (PMOXA), and all the data is benchmarked to PEGylated "stealth" liposomes. High colloidal stability in human plasma (HP) is confirmed for all but two tested nanovesicles. In situ fluorescence correlation spectroscopy measurements are then performed after incubating unlabeled nanovesicles with fluorescently labeled HP or the specific labeled plasma proteins, human serum albumin, and clusterin (apolipoprotein J). The binding of HP to PMOXA-polymersomes could explain their relatively short circulation times found previously. In contrast, PEGylated liposomes also interact with HP but accumulate high levels of clusterin, providing them with their known prolonged circulation time. The absence of significant protein binding for most PEG-polymersomes indicates mechanistic differences in protein interactions and associated downstream effects, such as cell uptake and circulation time, compared to PEGylated liposomes. These are key observations for bringing polymersomes closer to clinical translation and highlighting the importance of such comparative studies.

Block Length-Dependent Protein Fouling on Poly(2-oxazoline)-Based Polymersomes: Influence on Macrophage Association and Circulation Behavior.

Polymersomes are vesicular structures self-assembled from amphiphilic block copolymers and are considered an alternative to liposomes for applications in drug delivery, immunotherapy, biosensing, and as nanoreactors and artificial organelles. However, the limited availability of systematic stability, protein fouling (protein corona formation), and blood circulation studies hampers their clinical translation. Poly(2-oxazoline)s (POx) are valuable antifouling hydrophilic polymers that can replace the current gold-standard, poly(ethylene glycol) (PEG), yet investigations of POx functionality on nanoparticles are relatively sparse. Herein, a systematic study is reported of the structural, dynamic and antifouling properties of polymersomes made of poly(2-methyl-2-oxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) (PMOXA-b-PDMS-b-PMOXA). The study relates in vitro antifouling performance of the polymersomes to atomistic molecular dynamics simulations of polymersome membrane hydration behavior. These observations support the experimentally demonstrated benefit of maximizing the length of PMOXA (degree of polymerization (DP) > 6) while keeping PDMS at a minimal length that still provides sufficient membrane stability (DP > 19). In vitro macrophage association and in vivo blood circulation evaluation of polymersomes in zebrafish embryos corroborate these findings. They further suggest that single copolymer presentation on polymersomes is outperformed by blends of varied copolymer lengths. This study helps to rationalize design rules for stable and low-fouling polymersomes for future medical applications.

Tumor-Targeting Cholesterol-Decorated DNA Nanoflowers for Intracellular Ratiometric Aptasensing.

Probing endogenous molecular profiles is of fundamental importance to understand cellular function and processes. Despite the promise of programmable nucleic-acid-based aptasensors across the breadth of biomolecular detection, target-responsive aptasensors enabling intracellular detection are as of yet infrequently realized. Several challenges remain, including the difficulties in quantification/normalization of quencher-based intensiometric signals, stability issues of the probe architecture, and complex sensor operations often necessitating extensive structural modeling. Here, the biomimetic crystallization-empowered self-assembly of a tumor-targetable DNA-inorganic hybrid nanocomposite aptasensor is presented, which enables Förster resonance energy transfer (FRET)-based quantitative interpretation of changes in the cellular target abundance. Leveraging the design programmability and high-throughput fabrication of rolling circle amplification-driven DNA nanoarchitecture, this designer platform offers a method to self-assemble a robust nanosensor from a multifunctionality-encoded template that includes a cell-targeting aptamer, a ratiometric aptasensor, and a cholesterol-decorating element. Taking prostate cancer cells and intracellular adenosine triphosphate molecules as a model system, a synergistic effect in the targeted delivery by cholesterol and aptamers, and the feasibility of quantitative intracellular aptasensing are demonstrated. It is envisioned that this approach provides a highly generalizable strategy across wide-ranging target systems toward a biologically deliverable nanosensor that enables quantitative monitoring of the abundance of endogenous biomolecules.

Tuneable peptide cross-linked nanogels for enzyme-triggered protein delivery.

Many diseases are associated with the dysregulated activity of enzymes, such as matrix metalloproteinases (MMPs). This dysregulation can be leveraged in drug delivery to achieve disease- or site-specific cargo release. Self-assembled polymeric nanoparticles are versatile drug carrier materials due to the accessible diversity of polymer chemistry. However, efficient loading of sensitive cargo, such as proteins, and introducing functional enzyme-responsive behaviour remain challenging. Herein, peptide-crosslinked, temperature-sensitive nanogels for protein delivery were designed to respond to MMP-7, which is overexpressed in many pathologies including cancer and inflammatory diseases. The incorporation of N-cyclopropylacrylamide (NCPAM) into N-isopropylacrylamide (NIPAM)-based copolymers enabled us to tune the polymer lower critical solution temperature from 33 to 44 °C, allowing the encapsulation of protein cargo and nanogel-crosslinking at slightly elevated temperatures. This approach resulted in nanogels that were held together by MMP-sensitive peptides for enzyme-specific protein delivery. We employed a combination of cryogenic transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS), small angle neutron scattering (SANS), and fluorescence correlation spectroscopy (FCS) to precisely decipher the morphology, self-assembly mechanism, enzyme-responsiveness, and model protein loading/release properties of our nanogel platform. Simple variation of the peptide linker sequence and combining multiple different crosslinkers will enable us to adjust our platform to target specific diseases in the future.

Neutrophils Enable Local and Non-Invasive Liposome Delivery to Inflamed Skeletal Muscle and Ischemic Heart.

Uncontrolled inflammation is a major pathological factor underlying a range of diseases including autoimmune conditions, cardiovascular disease, and cancer. Improving localized delivery of immunosuppressive drugs to inflamed tissue in a non-invasive manner offers significant promise to reduce severe side effects caused by systemic administration. Here, a neutrophil-mediated delivery system able to transport drug-loaded nanocarriers to inflamed tissue by exploiting the inherent ability of neutrophils to migrate to inflammatory tissue is reported. This hybrid system (neutrophils loaded with liposomes ex vivo) efficiently migrates in vitro following an inflammatory chemokine gradient. Furthermore, the triggered release of loaded liposomes and reuptake by target macrophages is studied. The migratory behavior of liposome-loaded neutrophils is confirmed in vivo by demonstrating the delivery of drug-loaded liposomes to an inflamed skeletal muscle in mice. A single low-dose injection of the hybrid system locally reduces inflammatory cytokine levels. Biodistribution of liposome-loaded neutrophils in a human-disease-relevant myocardial ischemia reperfusion injury mouse model after i.v. injection confirms the ability of injected neutrophils to carry loaded liposomes to inflammation sites. This strategy shows the potential of nanocarrier-loaded neutrophils as a universal platform to deliver anti-inflammatory drugs to promote tissue regeneration in inflammatory diseases.

Renal clearable catalytic gold nanoclusters for in vivo disease monitoring.

Ultrasmall gold nanoclusters (AuNCs) have emerged as agile probes for in vivo imaging, as they exhibit exceptional tumour accumulation and efficient renal clearance properties. However, their intrinsic catalytic activity, which can enable an increased detection sensitivity, has yet to be explored for in vivo sensing. By exploiting the peroxidase-mimicking activity of AuNCs and the precise nanometre-size filtration of the kidney, we designed multifunctional protease nanosensors that respond to disease microenvironments to produce a direct colorimetric urinary readout of the disease state in less than one hour. We monitored the catalytic activity of AuNCs in the collected urine of a mouse model of colorectal cancer in which tumour-bearing mice showed a 13-fold increase in colorimetric signal compared to healthy mice. The nanosensors were eliminated completely through hepatic and renal excretion within four weeks of injection with no evidence of toxicity. We envision that this modular approach will enable the rapid detection of a diverse range of diseases by exploiting their specific enzymatic signatures.

Nanoparticle-based highly sensitive MRI contrast agents with enhanced relaxivity in reductive milieu.

Current magnetic resonance imaging (MRI) contrast agents often produce insufficient contrast for diagnosis of early disease stages, and do not sense their biochemical environments. Herein, we report a highly sensitive nanoparticle-based MRI probe with r1 relaxivity up to 51.7 ± 1.2 mM(-1) s(-1) (3T). Nanoparticles were co-assembled from Gd(3+) complexed to heparin-poly(dimethylsiloxane) copolymer, and a reduction-sensitive amphiphilic peptide serving to induce responsiveness to environmental changes. The release of the peptide components leads to a r1 relaxivity increase under reducing conditions and increases the MRI contrast. In addition, this MRI probe has several advantages, such as a low cellular uptake, no apparent cellular toxicity (tested up to 1 mM Gd(3+)), absence of an anticoagulation property, and a high shelf stability (no increase in free Gd(3+) over 7 months). Thus, this highly sensitive T1 MRI contrast nanoparticle system represents a promising probe for early diagnosis through possible accumulation and contrast enhancement within reductive extracellular tumour tissue.

An amphiphilic graft copolymer-based nanoparticle platform for reduction-responsive anticancer and antimalarial drug delivery.

Medical applications of anticancer and antimalarial drugs often suffer from low aqueous solubility, high systemic toxicity, and metabolic instability. Smart nanocarrier-based drug delivery systems provide means of solving these problems at once. Herein, we present such a smart nanoparticle platform based on self-assembled, reduction-responsive amphiphilic graft copolymers, which were successfully synthesized through thiol-disulfide exchange reaction between thiolated hydrophilic block and pyridyl disulfide functionalized hydrophobic block. These amphiphilic graft copolymers self-assembled into nanoparticles with mean diameters of about 30-50 nm and readily incorporated hydrophobic guest molecules. Fluorescence correlation spectroscopy (FCS) was used to study nanoparticle stability and triggered release of a model compound in detail. Long-term colloidal stability and model compound retention within the nanoparticles was found when analyzed in cell media at body temperature. In contrast, rapid, complete reduction-triggered disassembly and model compound release was achieved within a physiological reducing environment. The synthesized copolymers revealed no intrinsic cellular toxicity up to 1 mg mL(-1). Drug-loaded reduction-sensitive nanoparticles delivered a hydrophobic model anticancer drug (doxorubicin, DOX) to cancer cells (HeLa cells) and an experimental, metabolically unstable antimalarial drug (the serine hydroxymethyltransferase (SHMT) inhibitor (±)-1) to Plasmodium falciparum-infected red blood cells (iRBCs), with higher efficacy compared to similar, non-sensitive drug-loaded nanoparticles. These responsive copolymer-based nanoparticles represent a promising candidate as smart nanocarrier platform for various drugs to be applied to different diseases, due to the biocompatibility and biodegradability of the hydrophobic block, and the protein-repellent hydrophilic block.