Guangyu Rong

ConspectusThe cytosolic delivery of biomolecules such as genes, proteins, and peptides is of great importance for biotherapy but usually limited by multiple barriers during the process. Cell membrane with high hydrophobic character is one of the representative biological barriers for cytosolic delivery. The introduction of hydrophobic ligands such as aliphatic lipids onto materials or biomolecules could improve their membrane permeability. However, these ligands are lipophilic and tend to interact with the phospholipids in the membrane as well as serum proteins, which may hinder efficient intracellular delivery. To solve this issue, our research group proposed the use of fluorous ligands with both hydrophobicity and lipophobicity as ideal alternatives to aliphatic lipids to promote cytosolic delivery.In our first attempt, fluorous ligands were conjugated onto cationic polymers to increase their gene delivery efficacy. The fluorination dramatically increased the gene delivery performance at low polymer doses. In addition, the strategy greatly improved the serum tolerance of cationic polymers, which is critical for efficient gene delivery in vivo. Besides serum tolerance, mechanism studies revealed that fluorination increases multiple steps such as cellular uptake and endosomal escape. Fluorination also allowed the assembly of low-molecular-weight polymers and achieved highly efficient gene delivery with minimal material toxicity. The method showed robust efficiency for polymers, including linear polymers, branched polymers, dendrimers, bola amphiphilies, and dendronized polymers.Besides gene delivery, fluorinated polymers were also used for intracellular protein delivery via a coassembly strategy. For this purpose, two lead fluoropolymers were screened from a library of amphiphilic materials. The fluoropolymers are greatly superior to their nonfluorinated analogues conjugated with aliphatic lipids. The fluorous lipids are beneficial for polymer assembly and protein encapsulation, reduced protein denaturation, facilitated endocytosis, and decreased polymer toxicity compared to nonfluorinated lipids. The materials exhibited potent efficacy in therapeutic protein and peptide delivery to achieve cancer therapy and were able to fabricate a personalized nanovaccine for cancer immunotherapy. Finally, the fluorous lipids were directly conjugated to peptides via a disulfide bond for cytosolic peptide delivery. Fluorous lipids drive the assembly of cargo peptides into uniform nanoparticles with much improved proteolytic stability and promote their delivery into various types of cells. The delivery efficacy of this strategy is greatly superior to traditional techniques such as cell-penetrating peptides both in vitro and in vivo. Overall, the fluorination techniques provide efficient and promising strategies for the cytosolic delivery of biomolecules.

Cytosolic delivery of peptides remains a challenging task owing to their susceptibility to enzymatic degradation and the existence of multiple intracellular barriers. Here, we report a new strategy to address these issues by decoration of a fluorous tag on the terminal of cargo peptides. The fluorous-tagged peptides were assembled into nanostructures, efficiently internalized by cells via several endocytic pathways and released into the cytosol after endosomal escape. They were relatively stable against enzymatic degradation and showed much higher efficiency than nonfluorinated analogs and cell penetrant peptide-conjugated ones. The proposed strategy also efficiently delivered a proapoptotic peptide into specific sites in the cells and restored the function of cargo peptide after cytosolic delivery. The fluorous-tagged proapoptotic peptide efficiently inhibited tumor growth in vivo. This study provides an efficient fluorination strategy to promote the cytosolic delivery of peptides.

Tissue-specific delivery of oligonucleotide therapeutics beyond the liver remains a key challenge in nucleic acid drug development. To address this issue, exploiting exosomes as a novel carrier has emerged as a promising approach for efficient nucleic acid drug delivery. However, current exosome-based delivery systems still face multiple hurdles in their clinical applications. Herein, this work presents a strategy for constructing a hybrid exosome vehicle (HEV) through a DNA zipper-mediated membrane fusion approach for tissue-specific siRNA delivery. As a proof-of-concept, this work successfully fuses a liposome encapsulating anti-NFKBIZ siRNAs with corneal epithelium cell (CEC)-derived exosomes to form a HEV construct for the treatment of dry eye disease (DED). With homing characteristics inherited from exosomes, the siRNA-bearing HEV can target its parent cells and efficiently deliver the siRNA payloads to the cornea. Subsequently, the NFKBIZ gene silencing significantly reduces pro-inflammatory cytokine secretions from the ocular surface, reshapes its inflammatory microenvironment, and ultimately achieves an excellent therapeutic outcome in a DED mouse model. As a versatile platform, this hybrid exosome with targeting capability and designed therapeutic siRNAs may hold great potential in various disease treatments.

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a common respiratory critical syndrome that currently has no effective therapeutic interventions. Pulmonary macrophages play a principal role in the initiation and progression of the overwhelming inflammation in ALI/ARDS. Here, a type of fluorous-tagged bioactive peptide nanoparticle termed CFF13F is developed, which can be efficiently internalized by macrophages and suppress the excessive expression of cytokines and the overproduction of reactive oxygen species (ROS) triggered by lipopolysaccharide (LPS). The cytoprotective effect of CFF13F may be attributed to the lysosomal-stabilization property and regulation of the antioxidative system. Moreover, intratracheal pretreatment with CFF13F can effectively reduce local and systematic inflammation, and ameliorate pulmonary damage in an LPS-induced ALI murine model. The therapeutic efficacy of CFF13F is affected by the administration routes, and the local intratracheal injection is found to be the optimal choice for ALI treatment, with preferred biodistribution profiles. The present study provides solid evidence of the potent immunomodulatory bioactivity of the fluorous-tagged peptide nanoparticles CFF13F in vitro and in vivo, and sheds light on the development of novel efficient nanodrugs for ALI/ARDS.

Fluorinated polymers have attracted increasing attention in gene delivery and cytosolic protein delivery in recent years. In vivo tracking of fluorinated polymers will be of great importance to evaluate their biodistribution, clearance, and safety. However, tracking of polymeric carriers without changing their chemical structures remains a huge challenge. Herein, we reported a series of fluorinated poly-l-(lysine) (F-PLL) with high gene transfection efficiency and excellent biodegradation. Radionuclide 18F was radiolabeled on F-PLL by halogen replacement without chemical modification. The radiolabeling of F-PLL offers positron emission tomography (PET) imaging for in vivo tracking of the polymers. The biodistribution of F-PLL and the DNA complexes revealed by micro-PET imaging illustrated the rapid clearance of fluorinated polymers from liver and intestine after intravenous administration. The results demonstrated that the polymer F-PLL will not be accumulated in the liver and spleen when administrated as a gene carrier. This work presents a new strategy for in vivo tracking fluorinated polymers via PET imaging.