This study introduces a novel cross-linking strategy capable of successfully stabilizing CaP nanoparticles and stimuli-responsive small interfering RNA (siRNA) release. We synthesized a polysaccharide derivative thiolated hyaluronic acid (HA-SH), which was slightly modified but multifunctional and developed a smart redox-responsive delivery system. siRNA was efficaciously condensed by calcium phosphate (CaP) via electrostatic interaction to form a positively charged inner "core". Disulfide cross-linked HA (HA-ss-HA) was formed and played a role as an anionic outer "shell" to stabilize the CaP core. We demonstrated that the nanoparticles were stable both in the storage milieu and systemic circulation, thus overcoming the most serious disadvantage of CaP nanoparticles for gene delivery. Meanwhile, this smart system could selectively release siRNA into the cytosol by both a GSH-triggered disassembly and successful endosomal escape. Therefore, the hybrid delivery system achieved an 80% gene-silencing efficiency in vitro for both luciferase and Bcl2. Silencing of Bcl2 resulted in dramatic apoptosis of B16F10 cells. Besides, equipped with the tumor-targeting component HA, the nanoparticles significantly suppressed the growth of B16F10 xenograft tumor in mice. The anionic HA-ss-HA-equipped nanoparticles showed no apparent toxicity in vitro or in vivo, as well as showed a high transfection efficiency. Taken together, this redox-responsive, tumor-targeting smart anionic nanoparticle holds great promise for exploitation in functionalized siRNA delivery and tumor therapy.

Protein-based theranostic agents (PBTAs) exhibit superior performance in the diagnosis and therapy of cancers. However, the in vivo applications of PBTA are largely limited by undesired accumulation, penetration, or selectivity. Here, an ATP-supersensitive protein cluster is fabricated for promoting PBTA delivery and enhancing magnetic resonance imaging (MRI)-guided tumor photothermal therapy. Gd 3+ -and CuS-coloaded small bovine serum albumin nanoparticles (GdCuB) are synthesized as the model protein with a size of 9 nm and are encapsulated into charge switchable polycations (DEP) to form DEP/ GdCuB nanoclusters of 120 nm. In blood circulation, DEP/GdCuB significantly extends the half-lifetime and thereby enhances the tumor accumulation of GdCuB. When the clusters reach the tumor site, the extracellular adenosine triphosphate (ATP) can effectively trigger the release of GdCuB, resulting in tumoral deep penetration as well as the activation of T 1 -weighted MRI (r 1 value switched from 2.8 × 10 −3 to 11.8 × 10 −3 m −1 s −1 ). Furthermore, this delivery strategy also improves the tumoral photothermal therapy efficacy with the MRI-guided therapy. The study of ATP-activated nanoclusters develops a novel strategy for tumor deep penetration and on/off imaging of PBTA by size switchable technology, and reveals the potential for MRI-guided therapy of cancers.

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Cationic polyplex as commonly used nucleic acid carriers faced several shortcomings, such as high cytotoxicity, low serum stability, and slow cargo release at the target site. The traditional solution is covering a negative charged layer (e.g., hyaluronic acid, HA) via electrostatic interaction. However, it was far from satisfactory for the deshielding by physiological anions in circulation (e.g., serum proteins, phosphate). In this study, we proposed a new strategy of reversible covalent cross-linking to enhance stability in circulation and enable stimuli-disassembly of polyplexes in tumor cells. Here, 25k polyethylenimine (PEI) was chosen as model polycations for veriying the hypothesis. HA-PEI conjugation was formed by the cross-linking of adenosine triphosphate grafted HA (HA-ATP) with phenylboronic acid grafted PEI (PEI-PBA) via the chemical reaction between PBA and ATP. Compared with noncovalent polyplex by electrostatic interaction (HA/PEI), HA-PEI exhibited much better colloidal stability and serum stability. The covered HA-ATP layer on PEI-PBA could maintain stable in the absence of physiological anions, while HA layer on PEI in HA/PEI group showed obvious detachment after anion's competition. More importantly, the covalent cross-linking polyplex could selectively release siRNA in the ATP rich environment of cytosol and significantly improve siRNA silence. Besides, the covalent cross-linking with HA-ATP could effectively reduce the cytotoxicity of cationic polyplex, improve the uptake by B61F10 cells and promote the endosomal escape. Consequently, this strategy of HA-PEI conjugation significantly enhanced the siRNA transfection in the absence or presence of FBS (fetal bovine serum) on B16F10 cells and CHO cells. Taken together, the reversible covalent cross-linking approach shows obvious superiority compared with the noncovalent absorption strategy. It held great potential to be developed to polish up the performance of cationic polyplex on reducing the toxicity, enhancing the serum tolerance and achieving controlled release of siRNA at target site.

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Hydrophobic modification on polycations were commonly used to improve the stability and transfection efficiency of polyplexes. However, the improved stability often means undesired release of the encapsulated siRNA, limiting the application of cationic micelles for siRNA delivery. The current strategy of preparing bioresponsive micelles based on the cleavage of sensitive linkages between polycation and hydrophobic part was far from sufficient, owing to the siRNA binding of the separated polycations from micelles leading to the incomplete release of siRNA. In this study, we propose a new strategy by the combination of micelles disassembly and separated polycations charge reversal. FPBA (3-fluoro-4-carboxyphenylboronic acid) grafted PEI 1.8 k (polyethylenimine) as the polycations of PEI-FPBA and dopamine (with diol-containing moiety) conjugated with cholesterol as the hydrophobic part (Chol-Dopa). The PFCDM micelles was assembled by PEI-FPBA and Chol-Dopa, based on the FPBA-Dopa conjugation. The prepared PFCDM showed strong siRNA loading ability and superior stability in the presence of PBS or serum. Besides, the PFCDM exhibited excellent ATP sensibility. The intracellular ATP could effectively trigger the disassembly of micelles and charge reversal of PEI-FPBA, resulting in the burst release of siRNA in the cytosol. With the property of extracellular stability and intracellular instability, PFCDM displayed good performance on in vitro and in vivo luciferase silencing on 4T1 cells. It should also be noted that the assembly of low molecular weight PEI was relatively safe to cells compared with 25 k PEI. To sum up, the ATP-fueled assembly and charge reversible micelles gave examples for polyplexes to achieve better stability and on demand cargo release at the same time and shows potential to be used for in vitro and in vivo siRNA transfection.