One-pot synthesis of dynamically cross-linked polymers for serum-resistant nucleic acid delivery.

Cationic polymers used for nucleic acid delivery often suffer from complicated syntheses, undesired intracellular cargo release and low serum stability. Herein, a series of ternary polymers were synthesized via facile green chemistry to achieve efficient plasmid DNA and mRNA delivery in serum. During the one-pot synthesis of the ternary polymer, acetylphenylboric acid (APBA), polyphenol and low-molecular weight polyethyleneimine (PEI 1.8k) were dynamically cross-linked with each other due to formation of an imine between PEI 1.8k and APBA and formation of a boronate ester between APBA and polyphenol. Series of polyphenols, including ellagic acid (EA), epigallocatechin gallate (EGCG), nordihydroguaiaretic acid (NDGA), rutin (RT) and rosmarinic acid (RA), and APBA molecules, including 2-acetylphenylboric acid (2-APBA), 3-acetylphenylboric acid (3-APBA) and 4-acetylphenylboric acid (4-APBA), were screened and the best-performing ternary polymer, 2-PEI-RT, constructed from RT and 2-APBA, was identified. The ternary polymer featured efficient DNA condensation to favor cellular internalization, and the acidic environment in endolysosomes triggered effective degradation of the polymer to promote cargo release. Thus, 2-PEI-RT showed robust plasmid DNA transfection efficiencies in various tumor cells in serum, outperforming the commercial reagent PEI 25k by 1-3 orders of magnitude. Moreover, 2-PEI-RT mediated efficient cytosolic delivery of Cas9-mRNA/sgRNA to enable pronounced CRISPR-Cas9 genome editing in vitro. Such a facile and robust platform holds great potential for non-viral nucleic acid delivery and gene therapy.

Tailoring Hyperbranched Poly(ß-amino ester) as a Robust and Universal Platform for Cytosolic Protein Delivery.

Cytosolic protein delivery is a prerequisite for protein-based biotechnologies and therapeutics on intracellular targets. Polymers that can complex with proteins to form nano-assemblies represent one of the most important categories of materials, because of the ease of nano-fabrication, high protein loading efficiency, no need for purification, and maintenance of protein bioactivity. Stable protein encapsulation and efficient intracellular liberation are two critical yet opposite processes toward cytosolic delivery, and polymers that can resolve these two conflicting challenges are still lacking. Herein, hyperbranched poly(ß-amino ester) (HPAE) with backbone-embedded phenylboronic acid (PBA) is developed to synchronize these two processes, wherein PBA enhanced protein encapsulation via nitrogen-boronate (N-B) coordination while triggered polymer degradation and protein release upon oxidation by H2 O2 in cancer cells. Upon optimization of the branching degree, charge density, and PBA distribution, the best-performing A2-B3-C2-S2 -P2 is identified, which mediates robust delivery of various native proteins/peptides with distinct molecular weights (1.6-430 kDa) and isoelectric points (4.1-10.3) into cancer cells, including enzymes, toxins, antibodies, and CRISPR-Cas9 ribonucleoproteins (RNPs). Moreover, A2-B3-C2-S2 -P2 mediates effective cytosolic delivery of saporin both in vitro and in vivo to provoke remarkable anti-tumor efficacy. Such a potent and universal platform holds transformative potentials for protein pharmaceuticals.

Endothelial cell-targeting, ROS-ultrasensitive drug/siRNA co-delivery nanocomplexes mitigate early-stage neutrophil recruitment for the anti-inflammatory treatment of myocardial ischemia reperfusion injury.

Neutrophils serve as a key contributor to the pathophysiology of myocardial ischemia reperfusion injury (MIRI), because the unregulated activation and infiltration of neutrophils lead to overwhelming inflammation in the myocardium to cause tissue damage. Herein, endothelial cell-targeting and  reactive oxygen species (ROS)-ultrasensitive nanocomplexes (NCs) were developed to mediate efficient co-delivery of VCAM-1 siRNA (siVCAM-1) and dexamethasone (DXM), which cooperatively inhibited neutrophil recruitment by impeding neutrophil migration and adhesion. RPPT was first synthesized via crosslinking of PEI 600 with ditellurium followed by modification with PEG and the endothelial cell-targeting peptide cRGD. RPPT was allowed to envelope the DXM-loaded PLGA nanoparticles and condense the siVCAM-1. After systemic administration in rats experiencing MIRI, the cRGD-modified NCs efficiently targeted and entered the inflamed endothelial cells, wherein RPPT was sensitively degraded by over-produced ROS to trigger intracellular siVCAM-1 release and potentiate the VCAM-1 silencing efficiency. As a consequence of the complementary function of DXM and siVCAM-1, the NCs notably mitigated neutrophil infiltration into ischemic myocardium, provoking potent anti-inflammatory efficacy to reduce MIRI and recover cardiac function. The present study offers an effective approach for the controlled co-delivery of siRNA and drug cargoes, and it also highlights the importance of multi-dimensional manipulation of neutrophils in anti-inflammatory treatment. STATEMENT OF SIGNIFICANCE: The unregulated activation and infiltration of neutrophils lead to overwhelming inflammation in the myocardium after myocardial ischemia reperfusion injury (MIRI). Here, endothelial cell-targeting and ROS-ultrasensitive nanocomplexes (NCs), comprised of PLGA NPs decorated with cRGD-poly(ethylene glycol) (PEG)-modified, ditellurium-crosslinked PEI (RPPT), were developed to mediate efficient co-delivery of VCAM-1 siRNA (siVCAM-1) and dexamethasone (DXM). DXM and siVCAM-1 with complementary functions inhibited both the migration and adhesion of neutrophils, efficiently interventing the neutrophil recruitment and interrupting the self-amplified inflammation cascade in the injured myocardium. The molecular design of RPPT renders an effective example for constructing polymeric materials with high ROS sensitivity, and it resolves the critical dilemma related to polycation-mediated siRNA delivery, such as siRNA encapsulation versus release, and transfection efficiency versus toxicity.

Reactive oxygen species-responsive branched poly (ß-amino ester) with robust efficiency for cytosolic protein delivery.

Protein therapy targeting the intracellular machinery holds great potentials for disease treatment, and therefore, effective cytosolic protein delivery technologies are highly demanded. Herein, we developed reactive oxygen species (ROS)-degradable, branched poly(ß-amino ester) (PBAE) with built-in phenylboronic acid (PBA) in the backbone and terminal-pendent arginine for the efficient cytosolic protein delivery. The PBAE could form stable and cell-ingestible nanocomplexes (NCs) with proteins via electrostatic interaction, nitrogen-boronate (N-B) coordination, and hydrogen bonding, while it can be degraded into small segments by the over-produced H2O2 in tumor cells to enable cytoplasmic protein release. As thus, PBAE exhibited high efficiency in delivering varieties of proteins with distinct molecular weights (12.4-430 kDa) and isoelectric points (4.7-10.5) into tumor cells, including enzymes, toxins, and antibodies. Moreover, PBAE mediated efficient delivery of saporin into tumor cells in vivo, provoking pronounced anti-tumor outcomes. This study provides a robust and versatile platform for cytosolic protein delivery, and the elaborately tailored PBAE may find promising applications for protein-based biological research and disease management. STATEMENT OF SIGNIFICANCE: Cytosolic delivery of native proteins holds great therapeutic potentials, which however, is limited by the lack of robust delivery carriers that can simultaneously feature strong protein encapsulation yet effective intracellular protein release. Herein, ROS-degradable, branched poly(ß-amino ester) (PBAE) with backbone-embedded phenylboronic acid (PBA) and terminal-pendent arginine was developed to synchronize these two processes. PBA and arginine moieties allowed PBAE to encapsulate proteins via N-B coordination, electrostatic interaction, hydrogen bonding, and salt bridging, while PBA could be oxidized by over-produced H2O2 inside cancer cells to trigger PBAE degradation and intracellular protein release. As thus, the top-performing PBAE mediated efficient cytosolic delivery of various proteins including enzymes, toxins, and antibodies. This study provides a powerful platform for cytosolic protein delivery, and may find promising utilities toward intracellular protein therapy against cancer and other diseases such as inflammation.

Guanidine-rich helical polypeptides bearing hydrophobic amino acid pendants for efficient gene delivery.

Non-viral gene delivery vectors with high transfection efficiency both in vitro and in vivo and low cytotoxicity are highly desirable for clinical applications. Herein, a series of guanidine-rich polypeptides bearing hydrophobic amino acid pendants was efficiently prepared via the 1,3-dipolar cycloaddition between azido decorated polypeptide and propargyl functionalized guanidinium and N-acetylamino acids. CD analysis indicated α-helical conformations of all resulting polypeptides in aqueous solution. The guanidine-rich polypeptide/DNA complexes showed significantly enhanced cellular internalization and high cell viability (>90%) in different mammalian cell lines (i.e., HeLa and RAW 264.7) at concentrations of the best performance. The top-performing guanidine-rich polypeptide containing 10% N-acetyl-l-valine pendants outperformed the commercial transfection reagent PEI by 400 times in vitro and 6 times in vivo. This study provides a new guidance for future molecular design of non-viral gene vectors with high delivery efficiency and low cytotoxicity.

A sulfonate-based polypeptide toward infection-resistant coatings.

Multifunctional coatings have gained significant attention for their promising potential to address the issue of medical device-related infections. However, they usually have multiple components in one layer which decreases the density of functional groups on surfaces and hence reduces the biological properties. Herein, we report a mono-component and sulfonate-based anionic polypeptide coating with on-demand antibacterial activity, antifouling property, and biocompatibility. The anionic polypeptide was prepared by ring-opening polymerization of L-cysteine-based N-carboxyanhydride (NCA) with allyl groups and a subsequent thiol-ene reaction to incorporate the sulfonate pendants. It adopted a 17.1-19.5% ß-sheet conformation and self-assembled into a spherical nanoparticle. The polypeptide coating showed excellent in vitro antibacterial activity against both Gram-positive (i.e., S. aureus) and Gram-negative bacteria (i.e., E. coli) with >99% killing efficacy after acidic solution treatment and prominent antifouling property and biocompatibility after weak base treatment. An in vivo study revealed that the sulfonate-based polypeptide-coated polydimethylsiloxane (PDMS) exhibited good anti-infection property and histocompatibility.

Light-assisted hierarchical intratumoral penetration and programmed antitumor therapy based on tumor microenvironment (TME)-amendatory and self-adaptive polymeric nanoclusters.

The anticancer performance of nanomedicine is largely impeded by insufficient intratumoral penetration. Herein, tumor microenvironment (TME)-amendatory and self-adaptive nanoclusters (NCs) capable of cancer-associated fibroblasts (CAFs) depletion and size/charge conversion were engineered to mediate light-assisted, hierarchical intratumoral penetration. Particularly, large-sized NCs (~50 nm) were prepared via self-assembly of FAP-α-targeting peptide-modified, 1O2-sensitive polymers, which were further used to envelope small-sized dendrimer (~5 nm) conjugated with Ce6 and loaded with DOX (DC/D). After systemic administration, the NCs efficiently targeted CAFs and generated lethal levels of 1O2 upon light irradiation, which depleted CAFs and concomitantly dissociated the NCs to liberate small-sized, positively charged DC/D. Such stroma attenuation and NCs transformation collectively facilitated the delivery of DC/D into deeper regions of CAF-rich tumors, where DOX and 1O2 provoked synergistic anti-cancer efficacies. This study provides an effective approach to facilitate the tumor penetration of nanomedicine by concurrently and spatiotemporally reconfiguring the nano-properties and remodeling the TME.