A helical oncolytic polypeptide with potent membranolytic activity for cancer therapy.

Oncolytic peptides (OLPs) with membranolytic activity show great potential to combat multidrug-resistant cancer cells. Herein, we report a cationic helical oncolytic polypeptide (OLPP) with potent membranolytic activity for cancer therapy. The OLPP was synthesized by ring-opening polymerization of N-carboxyanhydrides (NCAs) and thiol-ene reaction. The OLPP was resistant to protease, showed high cytotoxicity to a series of cancer cells and caused cancer cell necrosis by quickly lysing cancer cell membrane independent of classic death-related intracellular pathways. Intra-tumoral injection of the OLPP effectively suppressed tumor growth in mice through the direct oncolytic effect. The OLPP represents a potential oncolytic chemotherapeutics for cancer therapy.

Immunomodulating nano-adaptors potentiate antibody-based cancer immunotherapy.

Modulating effector immune cells via monoclonal antibodies (mAbs) and facilitating the co-engagement of T cells and tumor cells via chimeric antigen receptor- T cells or bispecific T cell-engaging antibodies are two typical cancer immunotherapy approaches. We speculated that immobilizing two types of mAbs against effector cells and tumor cells on a single nanoparticle could integrate the functions of these two approaches, as the engineered formulation (immunomodulating nano-adaptor, imNA) could potentially associate with both cells and bridge them together like an 'adaptor' while maintaining the immunomodulatory properties of the parental mAbs. However, existing mAbs-immobilization strategies mainly rely on a chemical reaction, a process that is rough and difficult to control. Here, we build up a versatile antibody immobilization platform by conjugating anti-IgG (Fc specific) antibody (αFc) onto the nanoparticle surface (αFc-NP), and confirm that αFc-NP could conveniently and efficiently immobilize two types of mAbs through Fc-specific noncovalent interactions to form imNAs. Finally, we validate the superiority of imNAs over the mixture of parental mAbs in T cell-, natural killer cell- and macrophage-mediated antitumor immune responses in multiple murine tumor models.

Investigation of the in vivo integrity of polymeric micelles via large Stokes shift fluorophore-based FRET.

Polymeric micelles hold great potential for anticancer drug delivery. Sufficient integrity of polymeric micelles after intravenous injection is critical for successful drug delivery to solid tumors, but accurate measurement of the in vivo micellar integrity remains challenging. Methods based on Förster resonance energy transfer (FRET) to monitor the in vivo micellar integrity are frequently used. However, the self-quenching effect of these FRET fluorophores used has been improperly ignored and has caused inaccurate measurements. Herein, we report a FRET-based approach using the large Stokes shift (LSS) fluorophores NBD-X and MS735 as the donor and acceptor, respectively, to investigate the integrity of polyethylene glycol-block-poly(ε-caprolactone) (PEG-PCL) micelles. We established a mathematical formula for the integrity calculation, and an in vitro verification experiment showed that the formula results exactly matched the simulated results. Our results demonstrated that PEG-PCL micelles gradually dissociated in blood circulation, but approximately 60% of the micelles in plasma remained intact 72 h after intravenous (i.v.) injection. This LSS fluorophore-based FRET approach can be used to accurately monitor the integrity of nanoparticles, and this study demonstrates that most of PEG-PCL micelles maintain their aggregation state during blood circulation for anticancer drug delivery.

Protein Binding Affinity of Polymeric Nanoparticles as a Direct Indicator of Their Pharmacokinetics.

Polymeric nanoparticles (NPs) are an important category of drug delivery systems, and their in vivo fate is closely associated with delivery efficacy. Analysis of the protein corona on the surface of NPs to understand the in vivo fate of different NPs has been shown to be reliable but complicated and time-consuming. In this work, we establish a simple approach for predicting the in vivo fate of polymeric NPs. We prepared a series of poly(ethylene glycol)-block-poly(d,l-lactide) (PEG-b-PLA) NPs with different protein binding behaviors by adjusting their PEG densities, which were determined by analyzing the serum protein adsorption. We further determined the protein binding affinity, denoted as the equilibrium association constant (KA), to correlate with in vivo fate of NPs. The in vivo fate, including blood clearance and Kupffer cell uptake, was studied, and the maximum concentration (Cmax), the area under the plasma concentration-time curve (AUC), and the mean residence time (MRT) were negatively linearly dependent, while Kupffer cell uptake was positively linearly dependent on KA. Subsequently, we verified the reliability of the approach for in vivo fate prediction using poly(methoxyethyl ethylene phosphate)-block-poly(d,l-lactide) (PEEP-b-PLA) and poly(vinylpyrrolidone)-block-poly(d,l-lactide) (PVP-b-PLA) NPs, and the linear relationship between the KA value and their PK parameters further suggests that the protein binding affinity of polymeric NPs can be a direct indicator of their pharmacokinetics.

Nanoparticle-delivered siRNA targeting Bruton's tyrosine kinase for rheumatoid arthritis therapy.

Rheumatoid arthritis (RA) is a systemic autoimmune disease that can cause irreversible joint deformity. There is still no cure for RA, and current therapeutics, including methotrexate and adalimumab, cause serious off-target effects and systemic immunosuppression, which in turn increases the risk of infection. Bruton's tyrosine kinase (BTK) in macrophages and B cells has been demonstrated to be a promising therapeutic target for RA. However, high doses of BTK inhibitors are required for efficient BTK suppression, which limits their clinical use. Small interfering RNA (siRNA) is promising for the silencing of specific genes and has been used for the treatment of multiple diseases. To deliver siRNA into macrophages and B cells for BTK gene silencing, we employed cationic lipid-assisted PEG-b-PLGA nanoparticles (CLANs) to encapsulate siRNA. We demonstrated that macrophages and B cells were able to efficiently ingest the CLANs both in vitro and in vivo. Thereafter, we encapsulated siRNA targeting BTK (siBTK) into the CLANs, denoted as CLANsiBTK, and demonstrated that CLANsiBTK significantly inhibited BTK expression in macrophages and B cells. In a collagen-induced mouse arthritis model, CLANsiBTK treatment dramatically reduced joint inflammation and other RA symptoms but showed no toxicity, proving that using CLANsiBTK is a promising approach for RA therapy.

In situ repurposing of dendritic cells with CRISPR/Cas9-based nanomedicine to induce transplant tolerance.

Organ transplantation is the only effective method to treat end-stage organ failure. However, it is continuously plagued by immune rejection, which is mostly caused by T cell-mediated reactions. Dendritic cells (DCs) are professional antigen-presenting cells, and blocking the costimulatory signaling molecule CD40 in DCs inhibits T cell activation and induces transplant tolerance. In this study, to relieve graft rejection, Cas9 mRNA (mCas9) and a guide RNA targeting the costimulatory molecule CD40 (gCD40) were prepared and encapsulated into poly(ethylene glycol)-block-poly(lactide-co-glycolide) (PEG-b-PLGA)-based cationic lipid-assisted nanoparticles (CLAN), denoted CLANmCas9/gCD40. CLAN effectively delivered mCas9/gCD40 into DCs and disrupted CD40 in DCs at the genomic level both in vitro and in vivo. After intravenous injection into an acute mouse skin transplant model, CLANmCas9/gCD40-mediated CD40 disruption significantly inhibited T cell activation, which reduced graft damage and prolonged graft survival. This work provides a promising strategy for reprogramming DCs with nanoparticles carrying the CRISPR/Cas9 system to abate transplant rejection.

Incorporation of a rhodamine B conjugated polymer for nanoparticle trafficking both in vitro and in vivo.

Polymeric nanoparticles as drug delivery systems have the potential to improve the therapeutic efficacy and reduce the toxicity of chemotherapeutic drugs by enhancing the drug selectivity in vivo. The efficacy is directly dependent on the polymeric nanoparticles' in vivo fate. Therefore, it is very important to develop a method to stably label the polymeric nanoparticles for detecting the in vivo fate. Here, we report a method to stably label self-assembled nanoparticles by the incorporation of rhodamine B-conjugated poly(ε-caprolactone) (PCL-RhoB). Only 1% of PCL-RhoB was released from the RhoB-labeled polymeric nanoparticles (RhoB-PNPs) in phosphate buffer within 12 hours, which suggested that the signal of PCL-RhoB can be used to represent the behaviors of polymeric nanoparticles both in vitro and in vivo. PCL-RhoB could be effectively extracted and quantitatively detected by ultra-high-performance liquid chromatography (UPLC) in various media, such as PBS, a cell culture medium containing 10% FBS (pH = 7.4 and pH = 6.8), mouse serum, simulated intestinal fluid and cell or tissue lysis. The intracellular contents of PCL-RhoB in MDA-MB-231 cells detected by UPLC were linearly correlated to the concentration of the RhoB-PNPs. In addition, the contents of PCL-RhoB in plasma and the spleen were proportional to the injected dose of RhoB-PNPs in vivo. As an application example, the pharmacokinetics and biodistribution of the nanoparticles over time in vivo were analyzed following intravenous injection to confirm the feasibility of this method.

Cationic lipid-assisted nanoparticles for delivery of mRNA cancer vaccine.

Message RNA-based vaccines with prominent advantages such as facile production, no requirement for nuclear entry and high safety without the need for integration into host genome have been shown to be potent activators of the cytotoxic immune system. However, wider applications of mRNA-based therapeutics have been hindered because of their intrinsically high vulnerability to expressed nucleases and difficulty while entering antigen-presenting cells (APCs) directly. Here, we investigated the potential of cationic lipid-assisted nanoparticles (CLAN), which form a clinically translatable nucleic acid delivery system working as a carrier of an mRNA vaccine. We found that CLAN encapsulating mRNA encoding antigen could effectively stimulate the maturation of dendritic cells (DCs) and promote the activation and proliferation of antigen-specific T cells both in vitro and in vivo. Intravenous immunization of mice with CLAN containing mRNA encoding ovalbumin (OVA) provoked a strong OVA-specific T-cell response and slowed tumor growth in an aggressive E·G7-OVA lymphoma model. Collectively, CLAN proved to be a promising platform for mRNA vaccine delivery.

Tumor acidity-sensitive linkage-bridged block copolymer for therapeutic siRNA delivery.

The design of ideal nanoparticle delivery systems should be capable of meeting the requirements of several stages of drug delivery, including prolonged circulation, enhanced accumulation and penetration in the tumor, facilitated cellular internalization and rapid release of the active drug in the tumor cells. However, among the current design strategies, meeting the requirements of one stage often conflicts with the other. Herein, a tumor pH-labile linkage-bridged block copolymer of poly(ethylene glycol) with poly(lacide-co-glycolide) (PEG-Dlinkm-PLGA) was used for siRNA delivery to fulfill all aforementioned requirements of these delivery stages. The obtained siRNA-encapsulating PEG-Dlinkm-PLGA nanoparticle gained efficiently prolonged circulation in the blood and preferential accumulation in tumor sites via the PEGylation. Furthermore, the PEG surface layer was detached in response to the tumor acidic microenvironment to facilitate cellular uptake, and the siRNA was rapidly released within tumor cells due to the hydrophobic PLGA layer. Hence, PEG-Dlinkm-PLGA nanoparticles met the requirements of several stages of drug delivery, and resulted in the enhanced therapeutic effect of the nanoparticular delivery systems.

Multivalent manganese complex decorated amphiphilic dextran micelles as sensitive MRI probes.

T1 contrast agents based on Mn(II) were conjugated on amphiphilic dextran micelles via click chemistry. The obtained paramagnetic nanomicelle contrast agent has a higher T1 relaxivity (13.3 Mn mmol-1 s-1) and better sensitivity than those of free Mn(II) complexes. Studies carried out in vivo suggest that this contrast agent has a better and long-acting vascular enhancement effect at a lower manganese dosage (0.1 Mn mmol kg-1 BW).