Intracellular protein delivery is critical to the development of protein-based biopharmaceuticals and therapies. However, current delivery vectors often suffer from complicated syntheses, low generality among various proteins, and insufficient serum stability. Herein, we developed an enlightened cytosolic protein delivery strategy by dynamically crosslinking epigallocatechin gallate (EGCG), low-molecular-weight polyethylenimine (PEI 1.8k), and 2-acetylphenylboric acid (2-APBA) on the protein surface, hence forming the EPP-protein nanocapsules (NCs). EGCG enhanced protein encapsulation via hydrogen bonding, and reduced the positive charge density of PEI to endow the NCs with high serum tolerance, thereby enabling effective cellular internalization in serum. The formation of reversible imine and boronate ester among 2-APBA, EGCG, and PEI 1.8k allowed acid-triggered dissociation of EPP-protein NCs in the endolysosomes, which triggered efficient intracellular release of the native proteins. Such strategy therefore showed high efficiency and universality for diversities of proteins with different molecular weights and isoelectric points, including enzyme, toxin, antibody, and CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 ribonucleoprotein (RNP), outperforming the commercial protein transduction reagent PULSin and RNP transfection reagent lipofectamine CMAX. Moreover, intravenously (i.v.) injected EPP-saporin NCs efficiently delivered saporin into 4T1 tumor cells to provoke robust antitumor effect. This simple, versatile, and robust cytosolic protein delivery system holds translational potentials for the development of protein-based therapeutics.

MicroRNA-208a (miR-208a) plays critical roles in the severe fibrosis and heart failure post myocardial ischemia/reperfusion (IR) injury. MiR-208a inhibitor (mI) with complementary RNA sequence can silence the expression of miR-208a, while it is challenging to achieve efficient and myocardium-targeted delivery. Herein, biomimetic nanocomplexes (NCs) reversibly coated with red blood cell membrane (RM) were developed for the myocardial delivery of mI. To construct the NCs, membrane-penetrating helical polypeptide (PG) was first adopted to condense mI and form the cationic inner core, which subsequently adsorbed catalase (CAT) via electrostatic interaction followed by surface coating with RM. The membrane-coated NCs enabled prolonged blood circulation after systemic administration, and could accumulate in the injured myocardium via passive targeting. In the oxidative microenvironment of injured myocardium, CAT decomposed H₂O₂ to produce O₂ bubbles, which drove the shedding of the outer RM to expose the positively charged inner core, thus facilitated effective internalization by cardiac cells. Based on the combined contribution of mI-mediated miR-208a silencing and CAT-mediated alleviation of oxidative stress, NCs effectively ameliorated the myocardial microenvironment, hence reducing the infarct size as well as fibrosis and promoting recovery of cardiac functions. This study provides an effective strategy for the cytosolic delivery of nucleic acid cargoes in the myocardium, and it renders an enlightened approach to resolve the blood circulation/cell internalization dilemma of cell membrane-coated delivery systems.

Myocardial ischemia reperfusion (IR) injury is closely related to the overwhelming inflammation in the myocardium. Herein, cardiomyocyte-targeted nanotherapeutics were developed for the reactive oxygen species (ROS)-ultrasensitive co-delivery of dexamethasone (Dex) and RAGE small interfering RNA (siRAGE) to attenuate myocardial inflammation. PPTP, a ROS-degradable polycation based on PGE₂-modified, PEGylated, ditellurium-crosslinked polyethylenimine (PEI) was developed to surface-decorate the Dex-encapsulated mesoporous silica nanoparticles (MSNs), which simultaneously condensed siRAGE and gated the MSNs to prevent the Dex pre-leakage. Upon intravenous injection to IR-injured rats, the nanotherapeutics could be efficiently transported into the inflamed cardiomyocytes via PGE₂-assisted recognition of over-expressed E-series of prostaglandin (EP) receptors on the cell membranes. Intracellularly, the over-produced ROS degraded PPTP into small segments, promoting the release of siRAGE and Dex to mediate effective RAGE silencing (72%) and cooperative anti-inflammatory effect. As a consequence, the nanotherapeutics notably suppressed the myocardial fibrosis and apoptosis, ultimately recovering the systolic function. Therefore, the current nanotherapeutics represent an effective example for the co-delivery and on-demand release of nucleic acid and chemodrug payloads, and might find promising utilities toward the synergistic management of myocardial inflammation.

Insufficient intratumoral penetration greatly hurdles the anticancer performance of nanomedicine. To realize highly efficient tumor penetration in a precisely and spatiotemporally controlled manner, far-red light-responsive nanoclusters (NCs) capable of size shrinkage and charge conversion were developed and co-administered with iRGD to synergistically improve the intratumoral penetration and the anticancer efficacy. The NCs were constructed using the singlet oxygen-sensitive (SOS) polyethylene glycol- polyurethane-polyethylene glycol (PEG-(¹O₂)PU-PEG) triblock copolymer to encapsulate the doxorubicin (DOX)-loaded, chlorin e6 (Ce6)-conjugated polyamindoamine (PAMAM) dendrimer (DCD) via the double-emulsion method. Co-administration of iRGD notably increased the permeability of NCs within tumor vasculature and tumor tissues. In addition, upon far-red light irradiation (660 nm) of tumors at low optical density (10 mW/cm²), the generated ¹O₂ could disintegrate the NCs and release the DCD with positive surface charge and ultra-small size (~ 5 nm), which synergized with iRGD to enable deep intratumoral penetration. Consequently, the local ¹O₂ at lethal concentrations along with the released DOX efficiently and cooperatively eradicated tumor cells. This study provides a convenient approach to spatiotemporally promote the intratumoral penetration of nanomedicine and mediate programmed anticancer therapy.

Polymeric micelles have demonstrated wide utility for chemodrug delivery, which however, still suffer from shortcomings such as undesired drug loading, disassembly upon dilution, pre-leakage of drug cargoes during systemic circulation, and lack of cancer-selective drug release. Herein, a poly(ethylene glycol) (PEG)-polyphosphoester-based, reactive oxygen species (ROS)-responsive, core-cross-linked (CCL) micellar system was developed to encapsulate both chemodrug (doxorubicin, Dox) and photosensitizer (chlorin e6, Ce6). The hydrophobic core of the micelles was cross-linked via a thioketal (TK)-containing linker, which notably enhanced the drug loading and micelle stability. In tumor cells, far-red light irradiation of Ce6 generated ROS to cleave the TK linkers and disrupt the micelle cores. As such, micelles were destabilized and Dox release was promoted, which thereafter imparted synergistic anti-cancer effect with ROS-mediated photodynamic therapy. This study provides an effective approach to realize the precise control over drug loading, formulation stability, and cancer-selective drug release using polymeric micelles, and would render promising utilities for the programmed anti-cancer combination therapy.

Co-delivery of anti-inflammatory siRNA and hydrophilic drug provides a promising approach for the treatment of ulcerative colitis (UC). However, lack of a suitable and efficient co-delivery carrier poses critical challenge against their utilization. We herein developed macrophage-targeting, reversibly crosslinked polymersomes (TKPR-RCP) based on the TKPR-modified, poly(ethylene glycol)-b-poly(trimethylene carbonate-co-dithiolane trimethylene carbonate)-b-polyethylenimine (PEG-P(TMC-DTC)-PEI) triblock copolymer, which could efficiently encapsulate TNF-α siRNA and dexamethasone sodium phosphate (DSP) in their hydrophilic core. The cationic PEI segments provided additional electrostatic interactions with cargo molecules to promote the encapsulation, and disulfide crosslinking of the polymersome membrane endowed the TKPR-RCP with high colloidal stability. Because the cationic PEI was embedded in the hydrophilic core, the polymersomes displayed neutral surface charge and thus possessed high serum stability. The TKPR-RCP co-encapsulating TNF-α siRNA and DSP could be efficiently internalized by macrophages (~ 98%) and undergo redox-responsive membrane de-crosslinking to accelerate cargo release in the cytoplasm, thus inducing efficient gene silencing and anti-inflammatory effect. Intravenous injection of the co-delivery TKPR-RCP mediated potent and cooperative anti-inflammatory effect in inflamed colons of UC mice, and significantly prevented animals from colonic injury. This study therefore provides a promising approach for the co-delivery of hydrophilic drug/siRNA toward the treatment of inflammatory bowel diseases.